Adenoviruses. Laboratory diagnosis of diseases.

Herpesviruses. Laboratory diagnosis of herpesinfections

TOGAVIRUS ENCEPHALITIS

Pathogenesis and Pathology

The pathogenesis of the disease in humans has not been well studied, but the disease in experimental animals may afford a model for the human disease. The equine encephalitides in horses are diphasic. In the first phase (minor illness), the virus multiplies in non-neural tissue and is present in the blood 3 days before the first signs of involvement of the central nervous system. In the second phase (major illness), the virus multiplies in the brain, celts are injured and destroyed, and encephalitis becomes clinically apparent. The 2 phases may overlap. It is not known whether in humans there is a period of primary viral multiplication in the viscera with a secondary liberation of virus into the blood before its entry into the central nervous system. The viruses multiply in nonneural tissues of experimentally infected monkeys.

High concentrations of virus in brain tissue are necessary before the clinical disease becomes manifest. In mice, the level to which the vims multiplies in the brain is partly influenced by a genetic factor that behaves as a mendelian trait.

The primary encephalitides are characterized by lesions in all parts of the central nervous system, including the basal structures of the brain, the cerebral cortex, and the spinal cord. Small hemorrhages with perivascular cuffing and meningeal infiltration – chiefly with mononuclear cells—are common. Nerve cell degeneration associated with neuronophagia occurs. Purkinje's cells of the cerebellum may be destroyed. There are also patches of encephalomalacia; acellular plaques of spongy appearance in which medullary fibers, dendrites, and axons are destroyed; and focal microglial proliferation. Thus. not only the neurons but also the cells of the supporting structure of the central nervous system are attacked.

Widespread neuronal degeneration occurs with all arboviruses producing encephalitis, but some localization occurs.

Clinical Findings. Incubation periods of the encephalitides are between 4 and 21 days. There is a sudden onset with severe headache, chills and fever, nausea and vomiting, generalized pains, and malaise. Within 24-48 hours, marked drowsiness develops and the patient may become stuporous. Nuchal rigidity is common. Mental confusion, dysarthria, tremors, convulsions, and coma develop in severe cases. Fever lasts 4-10 days. The mortality rate in encephalitides varies (see Table 30-2). With JBE, the mortality rate in older age groups may be as high as 80%. Sequelae may include mental deterioration, personality changes, paralysis, aphasia, and cerebellar signs-Abortive infections simulate aseptic meningitis or nonparalytic poliomyelitis. Inapparent infections are common.

In California, where both WEE and SLE are prevalent, WEE has a predilection for children and infants. In the same area, SLE rarely occurs in infants, even though both viruses are transmitted by the same arthropod vector (Culex tarsalis}.

Laboratory Diagnosis

A. Recovery of Virus: The virus occurs in the blood only early in the infection, usually before the onset of symptoms. The virus is most often recovered from the brains of fatal cases by intracerebra! inoculation ofnewbom mice, and then it should be identified by serologic tests with known antisera,

B. Serology: Neutralising and hemagglutina-tion-inhibiling antibodies are detectable within a few days after the onset of illness. Complement-fixing antibodies appear later. The neutralizing and the hemagglutination-inhibiting antibodies endure for many years. The complement-fixing antibody may be lost within 2-5 years.

The HI test with newly hatched chick eryth-rocytes is the simplest diagnostic test, but it primarily identifies the group rather than the specific causative virus.

It is necessary to establish a rise in specific antibodies during infection in order to make the diagnosis. The first sample of serum should be taken as soon after the onset as possible and the second sample 2-3 weeks later. The paired specimens must be run in the same serologic test.

The cross-reactivity that takes place within group A or B arboviruses must be considered in making the diagnosis. Thus, following a single infection by one member of the group, antibodies to other members may also appear. These group-specific antibodies are usually of lower titer than the type-specific antibody. Serologic diagnosis becomes difficult when an epidemic caused by one member of the serologic group occurs in an area where another group member is endemic, or when an infected individual has been infected previously by a closely related arbovirus. Under these circumstances, a definite etiologic diagnosis may not be possible. Neutralizing, complement-fixing, and hemagglutination-inhibiting antibodies have a decreasing degree of specificity for the causative viral type (in the order listed).

Immunity

Immunity is believed to be permanent after a single infection. In endemic areas, the population may build up immunity as a result of inapparent infections; the proportion of persons with antibodies to the local arthropod-bome vims increases with age.

Because of antigens common to several members within a group, the response to immunization or to infection with one of the viruses of a group may be modified by prior exposure to another member of the same group. In general, the homologous response is greater than a cross-reacting one. This mechanism may be important in conferring protection on a community against an epidemic of another related agent (eg, no Japanese B encephalitis in areas endemic for West Nile fever).

Treatment

There is no specific treatment. In experimental animals, hyperimmune serum is ineffective if given after the onset of disease. However, if given 1-2 days after the invasion of the virus but before the signs of encephalitis are obvious, specific hyperimmiine serum can prevent a fatal outcome of the infection.

Epidemiology

In severe epidemics caused by the encephalitis viruses, the case rate is about 1:1000. In the large urban epidemic of St. Louis encephalitis that occurred in 1966 in Dallas (population 1 million), there were 545 reported cases, 145 (27%) laboratory-confirmed cases, and 15 deaths. The overall attack rate was 15 cases per 100,000, with a case fatality rate of 10%. All deaths were in persons age 45 years or older.

SLE is now appearing each year in the USA. In 1976, 372 cases with 17 deaths were reported in the USA.

The epidemiology of the arthropod-bome en-cephalitides must account for the maintenance and dissemination of the vimses in nature in the absence of humans. Most infections with the arboviruses occur in mammals or birds, with humans serving as an accidental host. The vims is transmitted from animal to animal through the bite of an arthropod vector. Viruses have been isolated from mosquitoes and ticks, which serve as reservoirs of infection. In ticks, the viruses may pass from generation to generation by the transovarian route, and in such instances the tick acts as a true reservoir of the virus as well as its vector. In tropical climates, where mosquito populations are present throughout the year. arboviruses cycle continuously between mosquitoes and reservoir animals.

It is not known whether in temperate climates the virus is reintroduced each year from the outside (eg, by birds migrating from tropical areas) or whether it somehow survives the winter in the local area. The overwintering mechanism is not known. Three possible mechanisms are (1) that hibernating mosquitoes at the time of their emergence could reinfect birds and thus reestablish a simple bird-mosquito-bird cycle; (2) that the virus could remain latent in winter within birds, mammals, or arthropods; and (3) that coldblooded vertebrates (snakes, turtles, lizards, alligators, frogs) may also act as winter reservoirs—eg, garter snakes experimentally infected with WEE virus can hibernate over the winter and circulate virus in high liters and for long periods the following spring. Normal mosquitoes can be infected by feeding on the emerged snakes and then can transmit the virus. Virus has been found in the blood of wild snakes.

C. Tick-Borne Encephalitis Complex:

1. Russian spring-summer encephalitis. This disease occurs chiefly in the early summer, particularly in humans exposed to the ticks Ixodes persulcatus and Ixodes ricinus in the uncleared forest. Ticks can become infected at any stage in their metamorphosis, and vims can be transmitted transovarially. The virus persists through the winter in hibernating ticks or in vertebrates such as hedgehogs or bats. Vims is se-

creted in the milk of infected goats for long periods, and infection may be transmitted to those who drink unpasteurized milk. Characteristics of the disease are involvement of the bulbar area or the cervical cord and the development of ascending paralysis or hemipa-resis. The mortality rate is about 30%.

2. Louping ill-This disease of sheep in Scotland and northern England is spread by the tick Ixodes ricinus. Humans are occasionally infected.

3. Tick-borne encephalitis (Central European or diphasic meningoencephalitis)-This virus is anti-genically related to Russian spring-summer encephalitis virus and louping ill virus. Typical cases have a diphasic course, the first phase being influenzalike and the second a meningoencephalitis with or without paralysis.

4. Kyasanur Forest disease is an Indian hemor-rhagic disease caused by a virus of the Russian spring-summer encephalitis complex. In addition to humans, langur (Presbylis entellus) and bonnet (Macaco. radiata) monkeys are naturally infected in southern India.

5. Powassan encephalitis-This tick-bome virus is the first member of the Russian spring-summer complex isolated in North America. Human infection is rare. Since 1959, when the original fatal case was reported from Canada, several additional cases have been confirmed in the northeastern portion of the USA.

Control

Biologic control of the natural vertebrate host is generally impractical, especially when the hosts are wild birds. The most effective method is arthropod control. Since the period of viremia in the vertebrate is of short duration (3-6 days for SLE infections of birds), any suppression of the vector for this period should break the transmission cycle. During the 1966 Dallas SLE epidemic, low-volume, high-concentration malathion mist was sprayed aerially over most of Dallas County. A striking decrease in the number and infectivity rate of the mosquito vectors occurred, demonstrating the effectiveness of the treatment.

Killed vaccines have not met with success. Several live attenuated encephalitis vaccines are being investigated. A live attenuated vaccine was successfully used to halt the severe epidemic of VEE in horses in Texas in 1971.

WEST NILE FEVER

West Nile fever is an acute, mild, febrile disease with lymphadenopathy and rash that occurs in the Middle East, tropical or subtropical Africa, and southwest Asia. It is caused by a group B arbovirus, a typical togavirus.

Clinical Findings. The virus is introduced through the bite of a Cuiex mosquito and produces viremia and a generalized systemic infection characterized by lymphadenopathy, sometimes with an accompanying maculopapular rash. Transitory meningeal involvement may occur during the acute stage. The virus may produce fatal encephalitis in older people, who have a delayed (and low) antibody response.

Laboratory Diagnosis. Virus can be recovered from blood taken in the acute stage of the infection. On paired serum specimens, CF, HI, and Nt titer rises may be diagnostic. Nt antibodies persist longer than CF antibodies. During convalescence, heterologous CF and Nt antibodies develop to JBE and SLE. The heterologous response is shorter and of lower titer than the homologous response.

Immunity. Only one antigenic type exists, and immunity is presumably permanent. Maternal antibodies are transferred from mother to offspring and disappear during the first 6 months of life.

Epidemiology and Control. West Nile fever appears to be limited to the Middle East. Antibodies to the virus have been found in Africa, India, and Korea, perhaps because of an antigenically related virus. In nonimmune populations, subclinical or clinical infections are common. In Cairo, 70% of persons over age 4 years have antibodies.

The disease is more common in summer and more prevalent in mral than urban areas. The virus has been isolated from Culex mosquitoes during epidemics, and experimentally infected mosquitoes can transmit the virus. Mosquito abatement appears to be a logical, if unproved, control measure.

YELLOW FEVER

Yellow fever (YF) is an acute, febrile, mos-quito-bome illness. Severe cases are characterized by jaundice, proteinuria, and hemorrhage. YF is a group B arbovirus, a typical togavirus. It multiplies in many different types of animals and in mosquitoes. It grows in embryonated chicks and in cell cultures made from chick embryos.

Strains freshly isolated from humans, monkeys, or mosquitoes are pantropic, ie, the virus invades all 3 embryonal layers. Fresh strains usually produce a severe (often fatal) infection with marked damage to the livers of monkeys after parenteral inoculation. After serial passage in the brains of monkeys or mice, such strains lose much of their viscerotropism; they cause encephalitis after intracerebral injection but only asymptomatic infection after subcutaneous injection. Cross-immunity exists between the pantropic and neurotropic strains of the virus.

During the serial passage of a pantropic strain of YF through tissue cultures, the relatively avirulent 17D strain was recovered. This strain lost its capacity to induce a viscerotropic or neurotropic disease in monkeys and in humans and is now used as a vaccine,

Hemagglutinins and complement-fixing antigens of this group B arbovirus may be prepared from infected tissues. Each antigen has 2 separable components: one is associated with the infectious particle; the other is probably a product of the action of YF virus on tissues it infects.

Pathogenesis and Pathology

Our understanding of the pathogenesis of YF is based on work with the experimental infection in monkeys. The virus enters through the skin and then spreads to the local lymph nodes, where it multiplies. From the lymph nodes, it enters the circulating blood and becomes localized in the liver, spleen, kidney, bone marrow, and lymph glands, where it may persist for days.

The lesions of YF are due to the localization and propagation of the virus in a particular organ. Death may result from the necrotic lesions in the liver and kidney, The most frequent site of hemorrhage is the mucosa at the pyloric end of the stomach.

The distribution of necrosis in the liver may be spotty but is most evident in the midzones of the lobules. The hyaline necrosis may be restricted to the cytoplasm; the hyaline masses are eosinophilic (Councilman bodies), Intranuclear eosinophilic inclusion bodies are also present and are of diagnostic value. During recovery, the parenchymatous cells are replaced, and the liver may be completely restored.

In the kidney, there is fatty degeneration of the tubular epithelium. Degenerative changes also occur in the spleen, lymph nodes, and heart. Intranuclear, acidophilic inclusion bodies may be present in the nerve and glial cells of the brain. Perivascular infiltrations with mononuclear cells also occur in the brain.

Clinical Findings

The incubation period is 3-6 days. At the onset, the patient has fever, chills, headache, and backache, followed by nausea and vomiting. A short period of remission often follows the prodrome. On about the fourth day, the period of intoxication begins with a slow pulse (90-100) relative to a high fever and moderate jaundice. In severe cases, marked proteinuria and hemorrhagic manifestations appear. The vomitus may be black with altered blood. Lymphopenia is present. When the disease progresses to the severe stage (black vomitus and jaundice), the mortality rate is high. On the other hand, the infection may be so mild as to go unrecognized. Regardless of severity, there are no sequelae; patients either die or recover completely.

Laboratory Diagnosis

A. Recovery of Virus: The vims may be recovered from the blood up to the fifth day of the disease by intracerebra! inoculation of mice. The isolated virus is identified by neutralization with specific antiserum.

B. Serology: Neutralizing antibodies develop early (by the fifth day) even in severe and fatal cases. In patients who survive the infection, circufating antibodies endure for life.

Complement-fixing antibodies are rarely found after mild infection or vaccination with the attenuated, live 17D strain. In severe infections, they appear later than the neutralizing antibodies and disappear more rapidly.

The serologic response in YF may be of 2 types. In primary infections of yellow fever, specific hemagglutination-inhibiting (HI) antibodies appear first, followed rapidly by antibodies to other group B viruses. The titers of homologous HI antibodies are usually higher than those of heterologous antibodies. CF and Nt antibodies rise slowly and are usually specific .

In secondary infections where YF occurs in a patient previously infected with another group B ar-bovirus, HI and CF antibodies appear rapidly and to high titers. There is no suggestion of specificity. The highest HI and CF antibodies are usually heterologous. Accurate diagnosis even by Nt test may be impossible.

Histopathologic examination of the liver in fatal cases is useful in those regions where the disease is endemic.

Immunity

Subtle antigenic differences exist between YF strains isolated in different locations and between pan-tropic and vaccine (17D) strains.

An infant born of an immune mother has antibodies at birth that are gradually lost during the first 6 months of life. Reacquisition of similar antibodies is dependent upon the individual's exposure to the virus under natural conditions or by vaccination.

Epidemiology

Two major epidemiologic cycles of YF are recognized: (1) classic (or urban) epidemic YF and (2) sylvan (or jungle) YF. Urban YF involves person-to-person transmission by domestic ^erfe^ mosquitoes. In the western hemisphere and West Africa, this species is primarily Aedes aegypti, which breeds in the accumulations of water that accompany human settlement. Mosquitoes remain close to houses and become infected by biting a viremic individual. Urban YF is perpetuated in areas where there is a constant influx of susceptible persons, cases of YF, and A aegypti. With the use of intensive measures for mosquito abatement, urban YF has been practically eliminated in South America.

Jungle YF is primarily a disease of monkeys. In South America and Africa, it is transmitted from monkey to monkey by arboreal mosquitoes (ie, Haemagogus, Aedes) that inhabit the moist forest canopy. The infection in animals may be severe or inapparent. Persons such as woodcutters, nut-pickers, or road-builders come in contact with these mosquitoes in the forest and become infected. Jungle YF may also occur when an infected monkey visits a human habitation and is bitten by A aegypll, which then transmits the virus to a human being.

The virus multiplies in mosquitoes, which remain infectious for life. After (he mosquito ingests a virus-containing blood meal, an interval of 12-14 days is required for it to become infectious. This interval is called the extrinsic incubation period.

All age groups are susceptible, but the disease in infants is milder than that in older groups. Large numbers of inapparent infections occur. The disease usually is milder in blacks. Yellow fever has never been reported in India or the Orient, even though the vector, A aegypti, is widely distributed there.

New outbreaks continue to occur. In Bolivia, 145 cases of jungle YF, with over 50% mortality, were reported in 1975. The disease occurred in nonimmune persons coming from distant places for the rice harvests . The rice fields, with the jungle adjacent to them, are located near towns. Most cases were in male agricultural workers. The virus had established itself in this area in reservoirs close to the towns. Jungle YF rarely affects the local population, which has developed immunity by having been in contact with the virus through previous minor infections and also by frequent vaccinations. The real number of cases and deaths from jungle YF in such areas is much higher than the reports indicate, as many patients do not go to the hospital but recover or die without any report being made.

Control

Vigorous mosquito abatement programs have virtually eliminated urban YF. The last reported outbreak of YF in the USA occurred in 1905. However, with the speed of modem air travel, wherever A aegypti is present, the threat of a YF outbreak exists. Most countries insist upon proper mosquito control on airplanes and vaccination of all persons at least 10 days before arrival in or from an endemic zone. The yellow fever vaccination requirement for travelers entering the USA was eliminated in 1972.

In 1978, a yellow fever outbreak occurred in Trinidad. Eight human cases and a number of infected forest monkeys were detected. The outbreak was quickly stopped by a mass immunization campaign and A aegypti control measures.

An excellent attenuated, live vaccine is available in the 17D strain. Vaccine is prepared in eggs and dispensed as a dried powder. It is a live virus and must be kept cold. It is rehydrated just before use and injected subcutaneously by skin scarification or by jet injector. A single dose produces a good antibody response in more than 95% of vaccinated persons that persists for at least 10 years. After vaccination, the virus multiplies and may be isolated from the blood before antibodies develop.

DENGUE (Breakbone Fever)

Dengue is a mosquito-borne infection characterized by fever, muscle and joint pain, lymphadenop-alhy, and rash and caused by a group B arbovirus, a togavirus (see above). Dengue and YF are antigenically related, but this does not result in significant cross-immunity.

Pathogenesis and Pathology

Viremia is present at the onset of fever and may persist for 3 days. The histopathologic lesion is in small blood vessels, with endothelial swelling, perivascular edema, and infiltration with mononuciear cells.

Clinical Findings

The onset of fever may be sudden or there may be prodromal symptoms of malaise, chills, and headache. Pains soon develop, especially in the back, joints, muscles, and eyeballs. The temperature returns to normal after 5-6 days or may subside on about the third day and rise again about 5-8 days after onset (' "saddle-back ' ' form). A rash (maculopapular or scar-latiniform) may appear on the third or fourth day and last for 24-72 hours, fading with desquamation. Lymph nodes are frequently enlarged. Leukopenia with a relative lymphocytosis is a regular occurrence. Convalescence may take weeks, although complications and death are rare. Especially in young children, dengue may occur as a mild febrile illness lasting 1-3 days.

A more severe syndrome — dengue hemorrhagic fever — may occur in individuals with passively acquired (as maternal antibody) or endogenously produced heterologous dengue antibody. Although initial symptoms simulate normal dengue, the patient's condition abruptly worsens and is associated with hy-poproteinemia, thrombocytopenia, prolonged bleeding time, and elevated prothrombin time. Dengue shock syndrome, characterized by shock and hemo-concentration, may supervene. These altered manifestations of dengue have been observed, often in epidemic form, in the Philippines, Southeast Asia, and India—regions in which several dengue serotypes are regularly present; the mortality rate is 5-10%. In studies of the dengue diseases in Southeast Asia, dengue hemorrhagic fever, with or without shock, has been found to occur more frequently when dengue type 2 is the secondary infecting virus and the patient is a female age 3 years or older. In 1981, over 40 type 2 dengue deaths occurred in Cuba as a result of hemorrhage and shock. Shock is probably a form of hy-persensitivity reaction. It is postulated that virus-antibody complexes are formed within a few days of the second dengue infection which activate the complement system and lead to the disseminated intravas-cular coagulation seen in the hemorrhagic fever syndrome.

Laboratory Diagnosis

Isolation of the virus is difficult. Injection of early fresh serum into mice rarely produces disease, but the animals may subsequently be immune to challenge. Dengue viruses often grow in cell cultures.

Nt and HI antibodies appear within 7 days of onset of dengue fever and CF antibodies somewhat later. Homotypic antibodies tend to reach higher liters than heterotypic ones.

Immunity

At least 4 antigenic types of the virus exist. Reinfection with a virus of a different serotype, 2-3 months after the primary attack, may give rise to a short, mild illness without a rash. Mosquitoes feeding on these reinfected patients can transmit the disease.

Epidemiology

The known geographic distribution of the dengue vimses today is India, the Far East, and the Hawaiian and Caribbean Islands. Dengue has occurred in the southern USA (1934) and in Australia. Most subtropical and tropical regions around the world where Aedes vectors exist are endemic areas or potential ones. For example, over 500,000 cases of dengue occurred in Colombia in 1972 following reinfestation of the Atlantic coastal areas by A aegypti. Over 100,000 cases occurred in 1981 in Cuba.

The infectious cycle is as follows:

Aedes ® Human ® Aedes ® Human

Aedes ® Monkey ® Aedes ® Monkey

A aegypti is a domestic mosquito; A edes albopic-tus exists in the bush or jungle and may be responsible for maintaining the infection among monkeys.

In urban communities, dengue epidemics are explosive and involve appreciable portions of the population. They often start during the rainy season, when the vector mosquito, A aegypti, is abundant. The mosquito has a short flight range, and urban spread of dengue is frequently house-to-house. The mosquito breeds in tropical or semitropical climates in artificial water-holding receptacles around human habitation or in tree holes or plants close to human dwellings. It apparently prefers the blood of humans to that of other animals. Since A aegypti is also the vector of yellow fever, the outbreak of dengue in the Caribbean serves as a warning of even more serious epidemics. Epidemics can be brought under control by aerial spraying with malathion to kill adult mosquitoes and by treatment of breeding sites to kill larvae,

/4 aegypfi is the only known vector mosquito for dengue in the western hemisphere. The female acquires the virus by feeding upon a viremic human. Mosquitoes are infective after a period of 8-14 days (extrinsic incubation time). In humans, clinical disease begins 2-15 days after an infective mosquito bite. Once infective, a mosquito probably remains so for the remainder of her life (1-3 months or more). Dengue virus is not passed from one generation of mosquitoes to the next. In the tropics, mosquito breeding throughout the year maintains the disease.

Epidemics of dengue are usually observed when the virus is newly introduced into an area or if susceptible persons move into an endemic area. The endemic dengue in the Caribbean is a constant threat to the USA, where A aegypti mosquitoes are prevalent in the summer months.

In 1977, a dengue type 1 virus was isolated from mosquitoes and from patients in Jamaica, from where it spread to the Bahamas, Trinidad, Cuba, and the USA. This was the first time type 1 virus had been isolated in the western hemisphere.

In 1979, an epidemic of dengue type 4 broke out in Tahiti, the first known appearance of type 4 outside of Southeast Asia. There were 6800 reported cases on the island (population 97,000).

Control

Control depends upon antimosquito measures, eg, elimination of breeding places and the use of insecticides. An experimental attenuated virus vaccine has been produced but not tested.

Additional materials about diagnosis of arboviral infections

According to the current data, there are over 430 types of arbo-viruses, with some 80 of them being pathogenic to humans.

Arboviral infections include systemic arboviral fevers (Plilebotomus, dengue), arboviral haemorrhagic fevers (yellow, dengue, Chikungunya, Crimean haemorrhagic. Omsk haemorrhagic, Kyasanur Forest disease), arbovirai encephalitides and encephalomyelites (tick-borne encephalitis, American West and East, and Venezuelan equine encephalitides, St. Louis encephalitis, Murray Valley encephalitis, West Nile, Japanese, and African encephalitides), etc.

Many arboviruses cause similar clinical manifestations and sub-clinical forms of the disease. These peculiarities of arbovira] infections, as well as simultaneous spread in endemic foci of clinically similar diseases of the viral and bacterial nature caused by adenoviruses, enteroviruses, Rickettsia, and spirochaetes. hinder their clinical recognition. Of a decisive significance in this situation becomes the laboratory diagnosis of arboviral infections, which should be made with special precautions. Examination of arboviruses should be carried out by qualified virologists in laboratories specially designed for work with particularly dangerous infections.

Virological and serological examination aimed al detecting natural foci of arboviral infections involves collection of the Arthropoda, withdrawal of blood from domestic and wild animals, collection of pieces of the internal organs (brain, liver, kidneys, spleen, lungs) from dead domestic animals and from wild vertebral animals with manifestations of the disease.

The Arthropoda are sorted out by species and placed into test tubes which are tightly stoppered with cotton plugs and sent to the laboratory. Into the test tube put a label carrying information on the number assigned to an animal, from which these ectoparasitos have been collected, and its species, as well as the place and date of material collection.

To study natural foci of arboviral infections, one can also use the method of sentinel annuals. According to this technique, sensitive animals (suckling mice, rabbils, guinea pigs, chicken, etc.) are kept in cages in the focus where they are liable to be attacked by infected Arthropoda vectors, liiood sample? are periodically collected from these animals and examined for the presence of the virus or antiviral antibodies.

Laboratory diagnosis of arboviral infection in humans is based on rapid methods, isolation of the virus, and determination of an increase in the antibody titre in paired sera (Table 5).

Material to be studied includes blood, cerebrospinal fluid, washings off the nasal portion of the throat, urine, and pleural fluiu which are taken during the first 3-4 days of tlio acute period. Pieces of The post-mortem hrain, liver, spleen, lungs, and kidneys should be preferably taken within 3-4 days after death. Isolation of The virus is most probable when the material studied is taken from people who have died during the lirst week of the acute period of the disease.

Sera for serological examination are obtained on the first-third day of the disease and then 2-3 weeks later.

Material collected for virological examination should preferably be utilized on the same day. When the material is to be kept for a short period of time (1-2 days), adequate storage is ensured at -4 °C.

Laboratory Diagnosis ot Arboviral Infection

Type of examination	Purpose of examination	Material	Inoculation	Detection	Identification
Rapid diagnosis	Recovery and identification of virus-specific antigen and anti-virus antibodies	Blood, tissues of inter- nal organs, blood serum	–	IF, RIHA, ELISA	IF, RIHA, ELISA
Virological	Isolation and typing of the virus	Blood, cerebrospinal fluid (in encephalitides), tissues of the internal organs (in haemorrhagic fevers), washings off the nasal portion of the throat (rarely)	Suckling mice are infect- ed intracerebrally and subcutaneously; young mice, intracerebrally and intraperitoneally Chicken embryos are inoculated into the am- nion, yolk sac, onto the CAM, and into the body (of the embryo) Cell cultures of the chick-en embryo, kidneys of the swine embryo, BHK-21, Vero, SPEV, PEC	Tremor, ataxia, convulsions, pa- ralyses, death of the animal, HA Retarded move- ments of the em- bryo, death, HA Cytopathic effect, appearance of plaques, HA, Hads	N, HAT, CF (less commonly), PG, RIHA, ELISA
Serological	Detection of a four-fold or greater increase in the titre of antibodies and the appearance of antibodies (seroconversion)	Paired sera obtained at the beginning of the disease and 2-3 weeks after its onset	–		Simultaneous conduction of such tests as HAI, CF, N, less frequently IF, PG, ELISA, RIA, RIHA

Long-term storage, however, requires freezing at -80 to -180 °C (pieces of organs may also be stored in 50 per cent glycerol at 4 °C). Only a single freezing and thawing of the infective material is possible.

Serum is kept in small vials at 4 °C (for a short period of time) or ill a frozen state. When bacteria! contamination is suspected, 1 per cent sodium azide is added to the serum (final concentration).

From the material studied prepare under sterile conditions its 10 per cent suspension in isotonic sodium chloride solution, Hanks' solution (pH 7,4-7,6) or in nutrient media obtained through inoculation of cell cultures. Add 0.75-2.0 per cent of bovine albumin or 10-25 per cent of normal rabbit serum and centrifuge for 20 min at 1500-2000 X g and 4 °C.

To prevent bacterial contamination, (the material is treated witli penicillin (200 U/ml) and streptomycin (100 mg/ml). Part of the material is kept at temperatures below -20 °C to reisolale the virus.

Rapid melhods are aimed at defecting the antigen by direct examination of (the palient's material. For this purpose, one can use such tests as IF, IHA, ELISA, and their modifications which make possible the detection and typing of the virus-specific antigen in cells.

The immunoftuorescence lest is employed to demonstrate the virua-specific antigen showing a stable link with blood leucocytes in dengue fever, Crimean haemorrhagic and Colorado fevers. One should also take into account the possibility of false positive results owing to autofluorescence of leucocytes. In Colorado fever, the virus antigen persists in leucocytes for more than three months.

The immunofluorescence test is also employed for detecting the virus antigen in the salivary glands of vectors and in the haemo-lymph of ticks.

In Japanese encephalitis the virus-specific antigen in the section material may be revealed by the immunoenzymatic technique.

The RIHA test with an erythrocyte antibody diagnosticum also allows demonstration of the virus antigen in the material studied This reaction is used for the rapid diagnosis of Crimean haemorrhagic fever and lymphocytic choriomeningitis. Detection and typing of the virus-specific antigen can also be done, using ELISA and RIHA on a solid basis. Positive results point to the presence of the corresponding antigens.

To isolate the virus, the material tested (undiluted and diluted 1:10 and 1:50) is inoculated into newborn white mice, cell cultures, and, less frequently, into chicken embryos (Table 5). This excludes the possible impact on the virus that may be exerted by antibodies or interferon. Newborn mice are infected intracerebrally (0.03 ml), intraperitoneally (0.05 ml), or subcutaneously (0.03 ml) (if arenaviruses have been detected, only adult white mice are infected). The infected rodents are observed for 2-3 weeks; animals which have developed the disease, are dissected and a 10 per cent suspension of the brain is prepared. Animals, which die within 1-3 days after the disease onset, are discounted.

Low capacity of the virus of dengue fever to adapt to the cerebral tissue of mice means that to ensure its isolation one should make 6-7 blind passages through the animal brain.

To isolate viruses, one employs such primary cell cultures as chicken and duck fibroblasts, cells of the kidneys of the swine embryo continuous cell lines BHK-21, SPEV, PEC, Vero, as well as tissue cultures from Arthropoda (ticks, mosquitoes).

To infect chicken embryos, the inoculum is introduced into the body, amnion, yolk sac, and on the chorio-allantoic membrane.

Demonstration of viruses is based on the haemagglutination test, cytopathic effect, plaque formation, and death of mice and chicken embryos. Goose red blood cells are commonly employed for tlic haemagglutination test.

To identify the isolated viruses, the neutralization test is used as the most specific one. The HAI and CF tests are less specific: they only provide the possibility to identify general antigen characteristic of definite groups of arboviruses. I mmuno fluorescence and radioimmuno-assay are utilized less commonly.

In cases where the isolated virus eludes identification, it is expedient to study its physico-chemical properties: namely, the size of the virions, type of nucleic acid, and the presence of lipids. These indirect indicators help to carry out preliminary "pre-serological" identification of arboviruses (Table 6). Comprehensive investigation of the above properties permits a preliminary conclusion whether or not the causative agent studied belongs to the arbovirus group. Study of the biological attributes of the isolated causative agent includes the determination of its pathogenicity for laboratory animals and tissue cultures. For the differential diagnosis, the immune sera or immune ascitic fluids against the isolated virus are obtained and studied in cross serological reactions.

The virological examination allowing to make a retrospective diagnosis has such a serious drawback as long duration (up to three weeks).

Serological examination includes the investigation of paired sera. In selecting the antigen, the epidemiological situation arid clinical data are taken into consideration. To detect an increase in the titre of antibodies of haemagglutinating arboviruses, the HAI test is utilized. As to recovering other arboviruses. the CF and N reactions are used for this purpose. The difficulty of the diagnosis lies in the fact that circulation in the area invaded by some virus may be attended by the production of group antibodies toward antigen-homogeneous viruses. The employment of the complement-fixation and neutralization reactions, which are more specific than the HAI test, is advisable in such cases. Of diagnostic significance is a four-fold or greater increase in the titre of antibodies in one of the above mentioned reactions. It is necessary to remember that complement-fix ating antibodies have a short life span.

For the serological diagnosis of arboviruses the IF, IHA, RIHA, RH, ELISA, and RIA tests are also employed.

HEMORRHAGIC FEVERS

Hemorrhagic fever has been reported from Africa, Siberia, Central and Southeast Asia, Eastern and Northern Europe, and South America.

Four categories have been suggested for hemor-rhagic fevers: (1) tick-borne, which includes some members of the Russian spring-summer encephalitis complex (Omsk hemorrhagic fever and Kyasanur Forest disease), and the Crimean-Congo hemorrhagic fever group; (2) mosquito-borne, which includes the dengue viruses (see above), Chikungunya virus, and yellow fever virus; (3) zoonotic, which includes the viruses of hemorrhagic fever with renal syndrome (Korean hemorrhagic fever), Argentinian hemorrhagic fever (Junin), Bolivian hemorrhagic fever (Machupo), and Lassa fever; and (4) African hemorrhagic fever. The latter is represented by Marburg and Ebola viruses (see p 464).

Common clinical features of the epidemic hemorrhagic fevers include fever; petechiae or purpura; gas-trointestinal, nasal, and uterine bleeding; leukopenia; hypotension; shock; proteinuria; thrombocytopenia; and central nervous system signs, often ending in death.

Machupo virus was recovered from a patient in Bolivia who died of hemorrhagic fever in 1963. The virus has been isolated from the mouse Calomys callosits. The systematic extermination of this field mouse has been successful in controlling the spread of the disease in Bolivia.

Marburg virus, which was first associated with a 1967 outbreak of hemorrhagic fever in Germany among persons who came in contact with vervet monkeys from Uganda, has an unknown route of transmission.

Outbreaks involving hundreds of cases of African hemorrhagic fever caused by Ebola virus were reported in Sudan and Zaire in 1976-1977. The incubation period was 4-16 days. The mortality rate was as high as 50% in some outbreaks. Transmission required close contact, especially with blood or secretions containing blood. Cases were common among hospital staff members. Barrier nursing and protective clothing permitted containment.

The disease is suspected of being a zoonosis, with rodents or bats as the animal reservoir. It is assumed that "Jungle" cases of the virus infection occur in humans from time to time but that the disease dies out spontaneously before reaching epidemic proportions. Exceptionally, as in 1976, nosocomial transmission creates an amplifying cycle of African hemorrhagic fever.

The virus grows in cultures of monkey cells and is infectious for guinea pigs. Virus can be recovered during the incubation period and for several days after onset of illness.

LASSA FEVER

The first recognized cases of this disease occurred in 1969 among Americans stationed in the Nigerian village of Lassa. The causative virus is extremely virulent, with a mortality rate of 36-67% in 4 epidemics in West Africa involving about 100 cases. Transmission can occur by human-to-human contact, presenting a hazard to hospital personnel. Nine of 20 medical workers have died from infections. Lassa fever can involve almost all the organ systems, although symptoms may vary in the individual patient. The disease is characterized by very high fever, mouth ulcers, severe muscle aches, skin rash with hemorrhages, pneumonia, and heart and kidney damage. Benign, febrile cases do occur. The virus can be isolated from the patient's blood in Vero monkey cell cultures.

Lassa virus is an arenavirus. Four arenaviruses cause human disease—Lassa, lymphocytic choriomeningitis, Junin, and Machupo. They can be distinguished by immunofluorescent antibody tests.

Lassa virus seems to be transmitted by human contact and also to have a nonhuman cycle. During an epidemic in Sierra Leone in 1972, Lassa virus was isolated from a house rat (Mastomys natalensis). When the virus spreads within a hospital, human contact is the mode of transmission.

The only available therapy for Lassa fever has employed hyperimmune semm from recovered patients. Interferon is being considered now. Rodent control may limit the natural cycle of the virus.

RUBELLA (German Measles)

Rubella is an acute febrile illness characterized by a rash and posterior auricular and suboccipital lymphadenopathy that affects children and young adults. Infection in early pregnancy may result in serious abnormalities of the fetus.

Properties of the Virus

The virus is RNA-containing, ether-sensitive, and about 60 nm in diameter. It contains a 30-nm internal nucleocapsid with a double membrane and forms by budding from the endoplasmic reticulum into intracytoplasmic vesicles and at the marginal cell membrane. Projections of the virion, 6 nm long, possess hemagglutinin for some avian erythrocytes. Receptor-destroying enzyme has no effect, and there is no spontaneous elution after hemagglutination.

Rubella virus can be propagated in cell culture. In some cultures, eg, human amnion cells, rabbit kidney cells, and a line of monkey kidney (VERO) cells, the virus produces detectable cytopathologic changes. In other cell cultures, rubella virus replicates without causing a cytopathic effect; however, interference is induced that protects the cells against the cytopathic effect of other viruses. One method of isolating rubella virus consists of inoculating green monkey kidney cells with the specimen and, after 7-10 days of incubation, challenging the cultures with echovirus 11. If echovirus cytopathic effect develops, the specimen is considered negative for rubella vims; conversely, the absence of echovirus cytopathic effect implies the presence of rubella vims in the original specimen.

1. POSTNATAL RUBELLA

Pathogenesis

Infection occurs through the mucosa of the upper respiratory tract. The vims probably replicates primarily in the cervical lymph nodes. After a period of 7 days, viremia develops that lasts until me appearance of antibody on about day 12-14. The development of antibody coincides with the appearance of the rash, suggesting an immunologic basis for the rash. After the rash appears, the vims remains detectable only in the nasopharynx.

Clinical Features

Rubella usually begins with malaise, low-grade fever, and a morbilliform rash appearing on the same day. Less often, systemic symptoms may precede the rash by 1 or 2 days, or the rash and lymphadenopathy may occur without systemic symptoms. The rash starts on the face, extends over the trunk and extremities, and rarely lasts more than 3 days. Posterior auricular and suboccipital lymphadenopathy are present. Transient arthralgia and arthritis are commonly seen in adult females. Rare complications include thrombocytopenia and encephalitis.

Unless an epidemic occurs, the disease is difficult to diagnose clinically, since the rash caused by other viruses such as the enteroviruses is similar. However, rubella has a peak occurrence in the spring, whereas enterovirus infections occur mainly in the summer and tall.

Immunity

Rubella antibodies appear in the serum of patients as the rash fades, and the titer of antibody rises rapidly over the next 1 -3 weeks (Fig. 1). Much of the initial antibody consists of IgM. IgM mbella antibodies found in a single serum obtained 2 weeks after the rash give evidence of recent rubella infection.

One attack of the disease confers lifelong immunity, as only one antigenic type of the virus exists. A history of rubella is not a reliable index of immunity. The presence of antibody at a 1:8 dilution implies immunity. Immune mothers transfer antibodies to their offspring, who are then protected for 4-6 months.

Treatment

No specific treatment is given unless the patient is pregnant. Rubellalike illness in the first trimester of pregnancy should be substantiated by isolation of the virus from the throat or by demonstrating a 4-fold rise in antibody titer to the virus by means of the HI, CF, or Nt test. Laboratory-proved rubella in the first 10 weeks of pregnancy is almost uniformly associated with fetal infection. Therapeutic abortion is strongly recommended in laboratory-proved cases to avoid the risk of malformed infants. It should be noted that gamma globulin injected into the mother does not protect the fetus against rubella infection.

2. CONGENITAL RUBELLA SYNDROME

Pathogenesis

Rubella infection during pregnancy may result in infection of the placenta and fetus. A limited number of cells of Ihe fetus become infected. Although the virus does not destroy the cells, the growth rate of the infected cells is reduced, which results in fewer than normal numbers of cells in the organs at birth. The earlier in pregnancy infection occurs, the greater the chance of extensive involvement, with the birth of an infant afflicted with severe anomalies. Infection in the first month of pregnancy results in abnormalities in about 80% of cases, whereas detectable defects are found in about 15% of infants acquiring the disease during the third month of gestation. The intrauterine infection is associated with chronic persistence of the virus in the newborn, which may last for 12-18 months after birth.

Figure. Virus and antibody dynamics in rubella. HI = hemaggiutination-inhibiting antibody; FA = fluorescent antibody; CF = complement-fixing antibody.

Clinical Findings

Infants with congenital rubella syndrome may have one or more abnormalities, which include defects of the heart and great vessels (patent ductusarteriosus, pulmonary artery stenosis, pulmonary valvular stenosis, ventricular septal defect, and atrial septal defect), eye defects (cataracts, glaucoma, and chorioretinitis), and neurosensory deafness. Infants may also display growth retardation, failure to thrive, hepatosplenomegaly, thrombocytopenia with purpura, anemia, osteitis, and an encephalitic syndrome leading to cerebral palsy. The infants often have increased susceptibility to infection and abnormal immunoglob-ulins, most commonly elevated IgM with low levels of IgG and IgA.

There is a 20% mortality rate among congenially vims-infected infants symptomatic at birth. Some virus-infected infants appearing normal at birth may manifest abnormalities at a later date. Severely affected infants may require institutional ization.

The spectrum of neurologic and neurosensory involvement in surviving infants is wide. Among 100 patients with congenital rubella infection, neurologic manifestations were found in 81 at some time between birth and 18 months. Sequelae include hearing impairment, visual impairment, growth disturbance, microcephaly, mental retardation, and cerebral dysfunction. Problems with balance and motor skills develop in preschool children. Psychiatric disorders and behavioral manifestations occur in preschool and school age children.

In one study. the neurologic course of congenital rubella syndrome was traced in nonretarded children. During the first 2 years, manifestations involved abnormal tone and reflexes (69%), motor delays (66%), feeding difficulties (48%), and abnormal clinical behavior (45%). Hearing loss was documented in 76%. At 3-7 years, poor balance, motor incoordination (69%), and behavioral disturbances (66%) predominated. Hearing losses increased to 86%. At 9-12 years, the following were noted: residua that included learning deficits (52%), behavioral disturbances (48%), poor balance (61%), muscle weakness (54%), and deficits in tactile perception (41%). Thus, the encephalitic manifestations of congenital rubella syndrome are persistent and diverse.

Immunity. While maternal rubella antibody in the form of IgG is transferred to the infant with congenital rubella, the infant also produces IgM antibodies. Nonaffected infants lose maternal antibody.

Epidemiology. The virus has been recovered from the nasopharynx, throat, blood, cerebrospinal fluid, and urine. The infection is spread by respiratory pathways (droplets).

Infants continue to be infectious, with virus found in the throat for up to 18 months after birth. Virus has been recovered from many tissues tested postmortem.

Congenitally infected infants who appear normal but who shed virus are capable of transmitting rubella to susceptible contacts such as nurses and physicians caring for the infants. This represents a serious hazard to women in the first trimester of pregnancy, who should avoid contact with these babies.

Rubella without rash is of importance because inapparent rubella infection (with viremia) acquired during pregnancy has the same deleterious effect on the fetus as rubella with the typical rash.

3. CONTROL OF RUBELLA. In the 20th century, epidemics of rubella have occurred every 6-9 years. After each epidemic, cases declined for the next 5 years, then increased to epidemic levels 6-9 years after the last major outbreak. In the 1964 epidemic, more than 20,000 infants were born with severe manifestations of congenital rubella.

In the USA, the control of rubella is being attempted by routine vaccination of children age 1-12 years and selected immunization of adolescents and women of childbearing age. Before vaccine became available in 1969, about 50,000 cases were being reported annually. Tn the next decade, about 100 million doses of vaccine were administered, which resulted in a 70% decrease in rubella incidence. However, the decrease occurred primarily in children. Persons 15 years of age and older experienced only a small decrease in incidence and now account for over 70% of cases. (Before 1969, they accounted for only 20%.) Since vaccine-induced antibodies persist for at least 10 years, the changing pattern may not be due to vaccine failure as much as to failure to adequately vaccinate susceptible adults.

Since the introduction of vaccine, scattered outbreaks have been reported, chiefly among nonvacci-nated adolescents in high school and college who had not received vaccine in the routine immunization program. The changing age incidence of rubella since introduction of vaccine is similar to the changing epi-demiologic pattern with measles.

In postpubertal females, the vaccine produces self-limited arthralgia and arthritis in about one-third of the vaccinees. Since mbella virus vaccine may infect the placenta, the vaccine should not be given to a postpubertal female unless she is not pregnant, is susceptible (ie, serologically negative), understands that she should not become pregnant for at least 3 months after vaccination, and is adequately warned of the complications of arthralgia. Nevertheless, since rubella vaccination is an effective way of preventing birth defects, it should be vigorously encouraged in women of childbearing age.

In children, the vaccine may also produce mild febrile episodes with arthralgia, often several months after vaccination, but without any permanent residual effects. Vaccinated children are not infectious and do not transmit the virus to contacts at home, even to mothers who are susceptible and pregnant. In contrast, nonimmunized children can bring home wild virus and spread it to susceptible family contacts.

Opinions have been expressed that vaccination of children cannot prevent future infection of pregnant women exposed to wild virus. Therefore, vaccination of prepubertal girls and women in the immediate post-partum period has also been proposed. It would seem wise for all pregnant women to undergo a serum antibody test for rubella and, if found to be susceptible, receive a vaccination immediately after delivery. Conception in the 6-8 weeks after delivery is rare, so the risk of harming a fetus would be minimal.

There is conflicting evidence on the nature and duration of postvaccination immunity with the first (HPV77) rubella vaccine, the risk of super-infection with wild virus, and the subsequent spread of such virus to pregnant women.

In 1979, the second rubella vaccine, RA23/3, grown in human diploid cells, was licensed, and this is the vaccine of choice. It produces much higher antibody liters and a more enduring and more solid immunity than HPV77, and there is evidence that it largely prevents subclinical superinfecrion with wild vims. This vaccine is available as a single antigen or combined with measles and mumps vaccine. It may effectively produce IgA antibody in the respiratory tract and thus interfere with infection by wild virus,

RABIES

Rabies is an acute infection of the central nervous system that is almost always fatal. The virus is usually transmitted to humans from the bite of a rabid animal.

Properties of the Virus

A. Structure: Rabies virus is a rhabdovirus with morphologic and biochemical properties in common with vesicular stomatitis virus of cattle and several animal, plant, and insect viruses. The rhabdoviruses are rod- or bullet-shaped particles measuring 60-400 nm x 60-85 nm. The particles are surrounded by a membranous envelope with protruding spikes 10 nm long. Inside the envelope is a ribonucleocapsid. The genome is single-stranded RNA (MW 3-5 x 10⁶) that is not infectious and does not serve as a messenger. Virions contain an RNA-dependent RNA polymerase.

B. Reactions to Physical and Chemical Agents: Rabies virus survives storage at 4 "C for weeks but is inactivated by CO₂. On dry ice, therefore, it must be stored in glass-sealed vials. Rabies virus is killed rapidly by exposure to ultraviolet radiation or sunlight, by heat (1 hour at 50 °C), by lipid solvents (ether, 0.1% sodium deoxycholate), and by trypsin.

C. Animal Susceptibility and Growth of Virus: Rabies virus has a wide host range. All warmblooded animals, including humans, are susceptible. The virus is widely distributed in infected animals, especially in the nervous system, saliva, urine, lymph, milk, and blood. Recovery from infection is rare except in certain bats, where the virus has become peculiarly adapted to the salivary glands. Vampire bats may transmit the virus for months without themselves ever showing any signs of disease.

When freshly isolated in the laboratory, the strains are referred to as street virus. Such strains show long and variable incubation periods (usually 21-60 days in dogs) and regularly produce intracytoplasmic inclusion bodies- Inoculated animals may exhibit long periods of excitement and viciousness. The virus may invade the salivary glands as well as the central nervous system.

Serial brain-to-brain passage in rabbits yields a "fixed" virus that no longer multiplies in extraneural tissues. This fixed virus multiplies rapidly, and the

incubation period is shortened to 4-6 days. At this stage, inclusion bodies are found only with difficulty.

The virus may be propagated in chick embryos, baby hamster kidney cells, and human diploid cell cultures. One strain (Flury), after serial passage in chick embryos, has been modified so that it fails to produce disease in animals injected extraneurally. This attenuated virus is used for vaccination of animals.

The replication of rabies virus is similar to that of the most studied rhabdovirus, vesicular stomatitis virus. The single-stranded RNA genome of molecular weight 4.6 x 10⁶ is transcribed by the virion-associated RNA polymerase to 5 mRNA species that are complementary to parts of the genome. These mRNAs code for the 5 virion proteins. The genome is a template for a replicative intermediate responsible for the generation of progeny RNA. After encapsidation, the bullet-shaped particles acquire the envelope by budding through the cytoplasmic membrane.

D. Antigenic Properties: The purified spikes elicit neutralizing antibody in animals. Antiserum prepared against the purified nucleocapsid is used in diagnostic immunofiuorescence.

Pathogenesis and Pathology. Rabies virus multiplies in muscle or connective tissue and is propagated through the endoneurium of the Schwann cells or associated tissue spaces of the sensory nerves to the central nervous system. It multiplies there and may then spread through peripheral nerves to the salivary glands and other tissues. Rabies virus has not been isolated from the blood of infected persons.

The incubation period may depend on the amount of inoculum, severity of lacerations, and distance the virus has to travel from its point of entry to the brain. There is a higher attack rate and shorter incubation period in persons bitten on the face or head.

There are hyperemia and nerve cell destruction in the cortex, midbrain, basal ganglia, pons, and especially in the medulla. Demyelinization occurs in the white matter, and degeneration of axons and myelin sheaths is common. In the spinal cord, the posterior horns are most severely involved, with neuronophagia and cellular infiltrates (mononuclear, perivascular, and perineural).

Rabies virus produces a specific cytoplasmic inclusion, the Negri body, in infected nerve cells. The presence of such inclusions is pathognomonic of rabies but may not be observed in all cases. The inclusions are eosinophilic, sharply demarcated, and more or less spherical, with diameters of 2-10 mcm. Several may be found in the cytoplasm of large neurons. They occur throughout the brain and spinal cord but are most frequent in Ammon's horn. Negri bodies contain rabies virus antigens and can be demonstrated by immunofluorescence.

Rabies virus multiplies outside the central nervous system and may produce cellular infiltrates and necrosis in salivary and other glands, in the cornea, and elsewhere.

The post-rabies vaccine reaction is an allergic encephalomyelitis.

Clinical Findings. The usual incubation period in dogs ranges from 3 to 8 weeks, but it may be as short as 10 days. Clinically, the disease in dogs is divided into 3 phases: prodromal, excitative, and paralytic. The prodromal phase is characterized by fever and a sudden change in the temperament of the animal; docile animals may become snappy and irritable, whereas aggressive animals may become more affectionate. The excitative phase lasts 3-7 days, during which the dog shows symptoms of irritability, restlessness, nervousness, and exaggerated response to sudden light and sound stimuli. At this stage the animal is most dangerous because of its tendency to bite. The animal has difficulty in swallowing, suffers from convulsive seizures, and enters into a paralytic stage with paralysis of the whole body, coma, and death. Sometimes the animal goes into the paralytic stage without passing through the excitative stage.

The incubation period in humans varies from 2 to 16 weeks or more, but in many cases it is only 2-3 weeks. It is usually shorter in children than in adults. The clinical spectrum can be divided into 4 phases: a short prodromal phase, a sensory phase, a period of excitement, and a paralytic or depressive phase. The prodrome, lasting 2-4 days, may show any of the following: malaise, anorexia, headache, nausea and vomiting, sore throat, and fever. Usually there is an abnormal sensation around the site of infection. The patient may show increasing nervousness and apprehension. General sympathetic overactivity is observed, including lacrimation, pupillary dilatation, and increased salivation and perspiration. The act of swallowing precipitates a spasm of the throat muscles; a patient may allow saliva to drool from the mouth simply to avoid swallowing and the associated painful spasms. (Because of the patient's apparent fear of water, the disease has been known as hydrophobia since ancient days.) This phase is followed by convulsive seizures or coma and death, usually 3-5 days following onset. Progressive paralytic symptoms may develop before death.

Hysteria may simulate certain features of rabies, particularly in persons who have been near a rabid animal or have been bitten by a nonrabid one.

Clinical features of rabies in humans

The incubation period is between 20 and 90 days and more than two-thirds of cases, with an extreme range of 4 days to more than 20 years. In some animals, latent infections can be reactivated by corticosteroids and stress, providing a possible explanation for the rare authentic reports of very long incubation periods in humans. Facial and severe multiple bites, transmission by corneal transplant, and accidental inoculation of live virus (rage de laboratoire) are associated with relatively short incubation periods. A few days of prodromal symptoms may precede the development of definite signs of rabies encephalomyelitis. These may consist of fever, changes of mood, and nonspecific “flulike” symptoms, but in more than one-third of cases itching, neuritic pain, or paresthesia at the site of the healed bite wound suggests impending rabies. The existence of two distinct clinical patterns of rabies, furious (agitated) and paralytic (“dumb”, “rage mue” or “rage muette”), depends on whether the brain or spinal cord is predominantly infected and may reflect differences in the infecting strain of rabies virus or in the host's immune response.

Furious rabies, the more common presentation in humans except those infected by vampire bats, is characterized by hydrophobia, aerophobia, and episodic generalized arousal interspersed with lucid intervals of normal cerebration. Hydrophobia is a reflex series of forceful jerky inspiratory muscle spasms provoked by attempts to drink water and associated with an inexplicable terror. A draft of air on the skin produces a similar reflex response, “aerophobia”. Initially, the spasms affect the diaphragm, sternomastoids, and other accessory muscles of inspiration, but a generalized extension response may be produced ending in opisthotonos and generalized convulsions with cardiac or respiratory arrest. Without supportive care, about one-third of patients with furious rabies die during a hydrophobic spasm in the first few days of their illness. There is hyperesthesia and periods of generalized excitation during which the patient becomes hallucinated, wild, and sometimes aggressive. These grotesque symptoms are explained by a selective encephalitis involving the brain stem and limbic system. In rabies, unlike most other encephalitides, patients may remain intermittently conscious and rational. Hypersalivation, lacrimation, sweating, and fluctuating blood pressure and body temperature result from disturbances of hypothalamic or autonomic nervous system function (Fig.2). Conventional neurologic examination may fail to disclose any abnormality unless a hydrophobic spasm is observed. Physical findings include meningism, cranial nerve and upper motor neuron lesions, muscle fasciculation, and involuntary movements. Increased libido, priapism, and frequent spontaneous orgasms may be the presenting symptom in some patients, suggesting involvement of the amygdaloid nuclei. Furious rabies naturally progresses to coma and death within a week, but some patients have been kept alive for several months in intensive care units.

Fig.2. Clinical features of rabies

Paralytic rabies is apparently much less common than the furious form in humans but is frequently undiagnosed. All reported cases of rabies transmitted by vampire bats in Latin America and the Caribbean are of this type. The paralytic form of rabies was also seen in patients with postvaccinal rabies and in the two patients who inhaled fixed virus. It seems more likely to develop in patients who have received antirabies vaccine. After the prodromal symptoms (see above), paralysis, fasciculation, pain, and paresthesia start in the bitten limb and ascend symmetrically or asymmetrically. There is progression to paraplegia with sphincter involvement, quadriparesis, and finally paralysis ofbulbar and respiratory muscles (Fig.3). Hydrophobia is usually absent. Patients with paralytic rabies may survive for several weeks even without intensive care.

Fig.3. Paralytic rabies

Differential diagnosis

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Laboratory Diagnosis.

A. Microscopy: Tissues infected with rabies virus are currently identified most rapidly and accurately by means of direct immunofluorescence using antirabies hamster serum. Impression preparations of brain or cornea tissue are often used.

A definitive pathologic diagnosis of rabies is based on the finding of Negri bodies in the brain (especially Ammon's horn) or the spinal cord. Negri bodies are found in impression preparations or histologic sections. They are sharply demarcated, more or less spherical, and 2-10 mcm in diameter, and they have a distinctive internal structure with basophilic granules in an eosinophilic matrix. Negri bodies (and rabies antigen) can usually be found in animals or humans suffering from rabies or dead from the infection, but they are rarely found in bats.

B. Virus Isolation: Available tissue (or saliva) is inoculated intracerebrally into mice. Infection in mice results in flaccid paralysis of legs, encephalitis, and death. The central nervous system of the inoculated animal is examined for Negri bodies and rabies antigen. In specialized laboratories, hamster and mouse cell lines can be inoculated for rapid (2-4 day) growth of rabies virus; this is much faster than growth in mice. An isolated virus is identified by neutralization tests with specific antiserum.

C. Serology: Antibodies to rabies can be detected by immunofluorescence, complement fixation, or neutralization, Such antibodies may develop in infected persons or animals during progression of the disease.

All animals considered "rabid or suspected rabid" (Table 1) should be sacrificed immediately for laboratory examination of tissues. Other animals, if available, should be held for observation for 10 days. If they show any signs of encephalitis, rabies, or unusual behavior, they should be killed humanely and the tissues examined in the laboratory. On the other hand, if they appear normal after 10 days, decisions must be made on an individual basis in consultation with public health officials.

Immunity & Prevention

Only one antigenic type of rabies virus is known. More than 99% of infections in humans and mammals who develop symptoms end fatally. Survival after proved rabies infection is extremely rare. It is therefore essential that individuals at high risk receive preventive immunization, that the nature and risk of any exposure be evaluated (Table 1), and that individuals be given postexposure prophylaxis if their exposure is believed to have been dangerous.

A. Pathophysiology of Rabies Prevention by Vaccine: It is likely that rabies virus remains latent in tissues for some time after virus is introduced from a bite. If immunogenic vaccine or antibody can be administered promptly, the virus can be prevented from invading the central nervous system. The action of passively administered antibody is to provide additional time for a vaccine to stimulate active antibody production before the central nervous system is invaded.

The following recommendations are only a guide. In applying them, take into account the animal species involved, the circumstances of the bite or other exposure, the vaccination status of the animal, and presence of rabies in the region. Local or state public health officials should tie consulted if Questions arise about the need for rabies prophylaxis.

Animal Species	Condition of Animal at Time of Attack	Treatment of Exposed Person*
Domestic Dog and cat	Healthy and available for 10 days of observation	None, unless animal develops rabies**
	Rabid or suspected rabid	RIG*** and HDCV****
	Unknown (escaped)	Consult public health officials, If treatment is indicated, give RIG*** and HDCV****
Wild Skunk, bat, fox, coyote, raccoon, bobcat, and other carnivores	Regard as rabid unless proved negative by laboratory tests*****	RIG*** and HDCV****
Other Livestock, rodents, and lagomorphs (rabbits and hares)	Consider individually. Local and state public health officials should be consulted on questions about the need for rabies prophylaxis. Bites of squirrels, hamsters, guinea pigs. gerbils, chipmunks, rats, mice, other rodents, rabbits, and hares almost never call for antirabies prophylaxis.

* All bites and wounds should immediately be thoroughly cleansed with soap and water. If antirabies treatment is indicated, both rabies immune globulin (RIG) and human diploid cell rabies vaccine (HDCV) should be given as soon as possible, regardless of the interval from exposure.

** During the usual holding period of 10 days, begin treatment with RIG and vaccine (preferably HDCV) at first sign of rabies in a dog or cat that has bitten someone. The symptomatic animal should be killed immediately and tested.

*** If RIG is not available, use antirabies serum, equine (ARS). Do not use more than the recommended dosage.

**** If HDCV is not available, use duck embryo vaccine (DEV). Local reactions to vaccines are common and do not contraindicate continuing treatment. Discontinue vaccine if fluorescent antibody (FA) tests of the animal are negative.

***** The animal should be killed and tested as soon as possible. Holding for observation is not recommended.

B. Types of Vaccines: All vaccines for human use contain only inactivated rabies virus.

1. Nerve tissue vaccine-This is made from infected sheep, goat, or mouse brains and used in many parts of the world including Asia, Africa, and South America. It causes sensitization to nerve tissue and results in postvaccinal encephalitis (an allergic disease) with substantial frequency (0.05%). It has not been used in the USA for several decades. Estimates of its efficacy in persons bitten by rabid animals vary from 5% to 50%.

2. Duck embryo vaccine-This was developed to minimize the problem of postvaccinal encephalitis. The rabies virus is grown in embryonated duck eggs, but the head is removed before the vaccine is prepared so as to remove nervous tissue and avoid allergic encephalitis. It produces local reactions regularly and systemic reactions (fever, malaise, myalgia) in one-third of recipients. Neuroparalytic (< 0.001%) and anaphylactic (< 1%) reactions are infrequent, but the antigenicity of the vaccine is low. Consequently, many (16-25) doses have to be given to obtain a satisfactory postexposure antibody response. This was the vaccine used in the USA in the recent past.

3. Human diploid cell vaccine (HDCV)-To obtain a rabies virus suspension free from nervous system and foreign proteins, rabies virus was adapted to growth in the WI-38 human normal fibroblast cell line. The rabies virus harvest is concentrated by ultrafillration and inactivated with beta propiolactone or tri-N-butyl phosphate. This material is sufficiently antigenic that only 4-6 doses of virus (Table 2) need to be given to obtain a substantial antibody response in most recipients. Local reactions (erythema, itching, swelling at the injection site) occur in 25% of recipients, and mild systemic reactions (headache, nausea, myalgia, dizziness) occur in about one-fifth of recipients. No serious anaphylactic, neuroparalytic, or encephalitic reactions have been reported. This vaccine has been used in the USA since 1979 and is the immunizing agent of choice.

4. Live attenuated viruses adapted to growth in chick embryos (eg, Flury strain) are used for animals but not for humans. Occasionally, such vaccines can cause death from rabies in injected cats or dogs. Rabies viruses grown in various animal cell cultures have also been used as vaccines for domestic animals.

C. Types of Available Rabies Antibody:

1. Rabies immune globulin, human (RIG)-This is a gamma globulin prepared by cold ethanol fractionation from the plasma of hyperimmunized humans. The neutralizing antibody content is standardized to contain 150 lU/mL. The dose is 20 lU/kg. half given around the bite wound, half intramuscularly.

Preexposurg: Preexposure rabies prophylaxis for persons with special risks of exposure to rabies, such as animal-care and control personnel and selected laboratory workers, consists of immunization with either human diploid cell rabies vaccine (HDCV) or duck embryo vaccine (DEV), according to the following schedule.

Rabies Vaccine	Number of 1-mL Doses	Route of Administration	Intervals Between Doses	If No Antibody Response to Primary Series, Give *
HDCV	3	Intramuscular	One week between 1st and 2nd; 2-3 weeks between 2nd and 3rd t	One booster dose**
DEV	3 or 4	Subcutaneous	One month between 1st and 2nd; 6—7 months between 2nd and 3rd** or One week between 1 st, 2nd, and 3rd; 3 months between 3rd and 4th**	Two booster doses, ** 1 week apart

Postexposure: Postexposure rabies prophylaxis for persons exposed to rabies consists of the immediate, thorough cleansing of all wounds with soap and water, administration of rabies immune globulin (RIG) or, if RIG is not available, antirabies serum, equine (ARS), and the initiation of either HDCV or DEV, according to the following schedule^

HDCV	5****	Intramuscular	Doses to be given on days 0, 3, 7, 14, and 28**	An additional booster dose**
DEV	23	Subcutaneous	Twenty-one daily doses followed by a booster on d 31 and another on day41** or Two daily doses in the first 7 days, followed by 7 daily doses. Then one booster on day 24 and another on day 34**	Three doses of HDCV at weekly intervals**

* If no antibody response is documented after the recommended additional booster dose(s), consult the state health department or CDC.

**Serum for rabies antibody testing should be collected 2-3 weeks after the last dose.

***The postexposure regimen is greatly modified for someone with previously demonstrated rabies antibody.

****The World Health Organization recommends a sixth dose 90 days after the first dose.

2. Antirabies serum» equine (ARS)-This Is concentrated serum from horses hyperimmunized with rabies virus. The neutralizing antibody content is standardized to contain 1000 IU per vial (approximately 5 mL). The dose is 40 lU/kg.

D. Choice of Rabies Immunizing Products:

This is an application of the risk/benefit ratio, as far as known for each product. HDCV has the greatest efficacy among known vaccines in stimulating antibody production, and few adverse effects are associated with it. There are fewer reactions to RIG (especially rare serum sickness, anaphylaxis) than to ARS, and RIG has a much longer half-life, since it is protein homologous for the human recipient.

ROTAVIRUSES (Infantile Gastroenteritis)

The rotaviruses are closely related to reoviruses. They are a major cause of diarrheal illness in human infants and young animals, including calves, mice, piglets, and many others. Among rotaviruses are the agents of human infantile diarrhea, Nebraska calf diarrhea, and epizootic diarrhea of infant mice and SA11 virus of monkeys.

Properties of the Viruses

A. Structure: The name rotavirus (Latin rota "wheel") is based on the electron microscopic appearance of the outer capsid margin as the rim of a wheel surrounding radiating spokes from the inner hublike core. The particles have a double-shelled capsid and are about 60-75 nm in diameter. Single-shelled viral particles that lack the outer capsid exhibit rough outer edges and are 50-60 nm in diameter. The inner core of the particles is 33-40 nm in diameter. The virus particle contains 11 segments of double-stranded RNA (total MW 10 x 10⁶). Virions contain an RNA-dependent RNA polymerase that can be activated by chelating agents and a poly A polymerase. The double-shelled particle is the infectious form of the virus. Infectivity of the virions is enhanced by treatment with proteolytic enzymes, eg, trypsin, and this is used in virus isolation in cell culture.

B. Animal Susceptibility and Pathogenesis:

Cross-species infections can occur in experimental inoculations , but it is not clear if they occur in nature. In experimental studies, human rotavirus can induce diarrheal illness in newborn colostrum-deprived animals (eg, piglets, calves). Homologous infections may have a wider age range. Swine rotavirus infects both newborn and weanling piglets. Newborn often exhibit subclinical infection due perhaps to the presence of maternal antibody, while overt disease is more common in weanling animals.

Rotaviruses infect cells in the villi of the small intestine. They multiply in the cytoplasm of these enterocytes and damage their transport mechanisms. Damaged cells may slough into the lumen of the intestine and release large quantities of virus, which appear in the stool. The diarrhea caused by rotaviruses may be due to impaired sodium and glucose absorption as the damaged cells on villi are replaced by nonabsorbing immature crypt cells

C. Virus Replication: Human rotaviruses have not been regularly cultured in vitro, although one human rotavirus strain and several hybrids containing both human and animal rotavirus genes are now cultivable. Rotaviruses from calves, pigs. and monkeys have been grown in cell culture and adapted to laboratory cultivation. This required treatment with proteolytic enzymes (trypsin, pancreatin). Such cultivated viruses now serve as antigens for serologic testing.

In vitro, rotavirus growth is maximal at 18-20 hours. Viral antigens are detected within 4-S hours in the cytoplasm of infected cells stained by the immunofluorescence technique, where they appear initially as distinct perinuclear granules. Later, antigen is present throughout the cytoplasm. Different types of cell culture manifest great differences in susceptibility to rotavirus infection.

D. Antigenic Properties: The rotaviruses possess common antigens located on the inner shell. These can be detected by immunofluorescence, immune electron microscopy, and many other methods. Typespecific antigens are located on the outer capsid layers. These type-specific antigens differentiate among rotaviruses from different species and are demonstrable by imrnunofluorescence and neutralization tests. At least 4 serotypes have been serologicaliy identified among human rotaviruses, but more may exist.

Molecular epidemiologic studies have analyzed the number of human strains based on differences in the migration of the 11 genome segments following electrophuresisoftheRNAinpolyacrylamidegels- At least 17 electropherotypes of human virus were observed in one 6-year study, suggesting extensive genome heterogeneity. It remains to be detemiined whether these differences in electropherotypes reflect changes in serotypes.

Clinical Findings and Laboratory Diagnosis

Rotaviruses cause the major portion of diarrhoea illness in infants and children but not in adults. Typical symptoms include diarrhoea, fever, abdominal pain, and vomiting, leading to dehydration.

Adult contacts may be infected, as evidenced by seroconversion, but they rarely exhibit symptoms, and virus is infrequently detected in their stool. However, epidemics of clinical disease have occurred in institutionalized adult populations and in adults in nonimmune isolated communities.

In infants and children, severe loss of electrolytes and fluids may be fatal unless treated. Patients with milder cases have symptoms for 3-5 days, then recover completely. Asymptomatic infections, with seroconversion, occur.

Laboratory diagnosis rests on demonstration of virus in stool collected early in the illness and on a rise in antibody liter. Virus in stool is demonstrated by immune electron microscopy, immunodiffusion, and other methods. Many serologic tests can be used to detect an antibody titer rise, particularly CF and ELISA.

Epidemiology and Immunity. Epidemiologic studies on the prevalence of rotavirus infections have shown these ubiquitous agents to be a major cause of gastroenteritis in children. Typically, 50-60% of the cases of acute gastroenteritis of hospitalised children throughout the world are caused by rotaviruses. Rotavirus infections usually predominate during the winter season, with an incubation period of 2-4 days. Symptomatic infections are most common in children between ages 6 months and 12 years, and transmission appears to be by the fecal-oral route. Nosocomial infections are frequent.

Rotaviruses are ubiquitous. By age 6, 60-90% of children have serum antibodies to one or more types. Both humans and animals can become infected even in the presence of antibodies. Local immune factors, such as secretory IgA or interferon, may be important in protection against rotavirus infection. Alternatively, reinfection in the presence of circulating antibody could reflect the presence of multiple serotypes of virus. Asymptomatic infections are common in infants before age 6 months, the time during which protective maternal antibody acquired passively by newborns should be present. Breast-fed babies excrete fewer virus particles per gram of feces than bottle-fed babies, although both groups can become infected. Rotavirus antibody has been detected in colostrum for up to 9 months postpartum.

Treatment. Treatment of gastroenteritis is supportive, to correct the loss of water and electrolytes, which may lead to dehydration, acidosis, shock, and death. Management consists of replacement of fluids and restoration of electrolyte balance either intravenously or orally, as feasible.

The multiplicity of rotavirus serotypes and other factors make the development of vaccines uncertain. In view of the probable fecal-oral route of transmission, waste-water treatment and sanitation are significant control measures.

CORONAVIRUSES

The coronaviruses include human strains from the respiratory tract, avian infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), an enteritis virus of swine, and others. The human coronaviruses cause common colds. Coronaviruses of lower animals establish persistent infections in their natural hosts. Because the murine infection can result in a high incidence of subacute to chronic demyelinating disease, it is being studied as a model for multiple sclerosis in humans.

Properties of the Viruses. Coronaviruses are enveloped, 80- to 130-nm particles that contain an unsegmented genome of single-stranded RNA (MW 7 x 10⁶). The helical nucleocapsid is 7-9 nm in diameter; it matures in the cytoplasm by budding into cytoplasmic vesicles. There are 20-nm-long club-shaped or petal-shaped projections that are widely spaced on the outer surface of the envelope, resembling a solar corona. The 3 chief virus proteins include a 60K phosphorylated nucleocapsid protein, a 90K glycoprotein making up the petal-shaped structures, and a 23K glycoprotein embedded in the envelope lipid bilayer. Viral antigens are found only in the cytoplasm of infected cells.

Growth of Virus. The human coronaviruses are difficult to grow in cell cultures. Some strains require human embryonic tracheal and nasal organ cultures; others will grow in human embryonic intestine or kidney cell cultures. The optimal temperature for growth is 33-35 °C.

Antigenic Properties. The human prototype strain is 229E. Some human isolates are closely related; others are not. Cross-reactions occur between some human and some animal strains, but avian IBV appears to be unrelated to human agents. All or most strains have CF antigens; some have hemagglutinins.

Clinical Features andLaboratory Diagnosis. The human coronaviruses produce "colds, "usually afebrile, in adults. If virus is isolated, diagnosis can be confirmed by demonstrating a significant rise in CF or Nt antibody titer in paired serum specimens.

In the absence of virus isolation, serologic diagnosis can be made on the basis of significantly increased antibody liters. The CF test is a more sensitive index .of human coronavirus infections than is virus isolation with cell and organ culture methods available at present. Serologic diagnosis of infections with strain 229E is now possible by means of passive hemagglutination test. Red cells coated with coronavirus antigen are agglutinated by antibody-containing sera. The test is type-specific, as sensitive as the Nt test, rapid, and convenient.

Epidemiology. As indicated in the foregoing, the coronaviruses are a major cause of respiratory illness in adults during some winter months when the incidence of colds is high but the isolation of rhinoviruses or other respiratory viruses is low. These viruses are a common cause of virus-induced exacerbations in patients with chronic bronchitis.

The apparent infrequency of coronavirus infections in children may be a result of the type of test used: initial infections with strain 229E are accompanied by only a transient CF antibody response, whereas in reinfections in adults, the CF response is enhanced and the Nt antibody response is diminished. Therefore, the Nt test should be the procedure of choice for infants and children and the CF test more sensitive for adults.

Coronaviruses of lower animals can establish long-term infections in their natural hosts (pigs, chickens, mice). They may also set up inapparent persistent infections in humans.

Arenaviruses

Pleomorphic particles contain a segmented single negative strand RNA genome (MW 3-5 x 10⁶), are surrounded by an envelope, and measure 50-300 nm. They contain granules believed to be ribosomes. Several hemorrhagic fever viruses that are antigenically related fall into this group. Most have a rodent host in their natural cycle.

Lassa fever

The first recognised cases of this disease occurred in 1969 among Americans stationed in the Nigerian village of Lassa. The causative virus is extremely virulent, with a mortality rate of 36-67% in 4 epidemics in West Africa involving about 100 cases. Transmission can occur by human-to-human contact, presenting a hazard to hospital personnel. Nine of 20 medical workers have died from infections. Lassa fever can involve almost all the organ systems, although symptoms may vary in the individual patient. The disease is characterized by very high fever, mouth ulcers, severe muscle aches, skin rash with hemorrhages, pneumonia, and heart and kidney damage. Benign, febrile cases do occur. The virus can be isolated from the patient's blood in Vero monkey cell cultures.

Lassa virus is an arenavirus. Four arenaviruses cause human disease—Lassa, lymphocytic choriomeningitis, Junin, and Machupo. They can be distinguished by immunofluorescent antibody tests.

Lassa virus seems to be transmitted by human contact and also to have a nonhuman cycle. During an epidemic in Sierra Leone in 1972, Lassa virus was isolated from a house rat (Mastomys natalensis). When the virus spreads within a hospital, human contact is the mode of transmission.

The only available therapy for Lassa fever has employed hyperimmune serum from recovered patients. Interferon is being considered now. Rodent control may limit the natural cycle of the virus.

Lymphocytic choriomeningitis

Lymphocytic choriomeningitis (LCM) is an acute disease with aseptic meningitis or a mild systemic influenzalike illness. Occasionally there is a severe encephalomyelitis or a fatal systemic disease. The incubation period is usually 18-21 days but may be as short as 1-3 days. The mild systemic form is rarely recognized clinically. There may be fever, malaise, generalized muscle aches and pains, weakness, sore throat, and cough. The fever lasts for 3-14 days.

LCM is an RNA-containing arenavirus 50-150 nm in diameter.

Diagnosis. Specific diagnosis can be made by the isolation of virus from spinal fluid or blood during the acute phase and by tests demonstrating a rise in antibody titer between acute and convalescent serum specimens. Complement-fixing antibodies rise to diagnostic levels in 3-4 weeks, then fall gradually and reach normal levels after several months. Neutralizing antibodies appear later and reach diagnostic levels 7-8 weeks after onset; they may persist for 4-5 years.

Laboratory Findings. In the prodromal period (or mild systemic form), leukopenia with relative lymphocytosis is frequently present. In the meningitic form, there is pleocytosis in the spinal fluid (100-3000 cells/mcL), with a predominance of lymphocytes. The glucose is normal and the protein content slightly elevated.

Epidemiology and Control. The disease is endemic in mice and other animals (dogs, monkeys, guinea pigs) and is occasionally transmitted to humans. One large epidemic in the USA was caused by infected pet hamsters. There is no evidence of person-to-person spread.

Infected grey house mice, probably the most common source of human infection, excrete the virus in urine and feces. The virus may be harboured by mice throughout their lives, and females transmit it to their offspring, which In turn become healthy carriers. Mice inoculated as adults develop a rapidly fatal generalised infection. In contrast, congenitally or neonatally infected mice do not become acutely ill, but 10-12 months later many develop a fatal debilitating disease involving the central nervous system. The animals exhibit chronic glomerulonephritis and hypergamma-globulinemia; the glomerular lesions are due to deposition of antigen-antibody complexes, and the infection in mice is considered an immune complex disease (see Slow Virus Diseases, below). The mode of transmission from mice to humans is uncertain. Mice and their droppings should be controlled.

Additional material about laboratory diagnosis

Rabies

The virus of rabies belongs to the genus Lassavirus, family Rhabdoviridae. Rabies is characterized by involvement of the central nervous system, so the diagnosis is based on detection in the brain tissue of Babes-Negri bodies (around the hippocampus), viral antigen, or virus. Examination of the animal that has bitten the patient involves recovery of the virus or viral antigen in the tissue of the salivary gland by the IF or biological tests.

To isolate the virus at the earliest stages of the disease, the cere-brospinal fluid, sputum, urine and sublingual saliva are studied. The material is collected with a cotton swab which is rinsed in several millilitres of sterile isotonic saline and squeezed dry.

If the material is to be examined within the first 24 hrs, the specimens are kept at 4 °C. When the material has to be transported, it is frozen and sent in containers with dry ice. In cases of prolonged storage (at —60 °C), a 20 per cent suspension of the material is prepared.

Rapid diagnosis. The most widely spread method of examination is detection of Babes-Negri bodies in preparations of the brain tissue under a light microscope. For this purpose, the hippocampus tissue and the cortex of the cerebrum and cerebellum are examined. A cover slip is slightly pressed onto the surface of the section, the impression is stained by the Romanowsky-Giemsa technique or by the method proposed by Turevich or Muromtsev, dried, and examined microscopically (the preparation should be handled as an infective material). Babes-Negri bodies are seen in the cytoplasm of large neurons and appear as spherical or oblong pink-violet formations 2-10 mcm in diameter, with a visible internal structure.

The virus is detected in brain preparations by electron microscopy. The viral antigen in the brain tissue, impressions of the submaxillary salivary glands, and oral mucosa is demonstrated by immunofluorescence with the use of hyperimmune sera of animals.

Isolation of the virus. To isolate the virus, infect white mice (preferably 1-2-day-old ones) with a suspension of the tissue from the brain and other organs obtained from people who have died of the infection and also with the cerebrospinal fluid and saliva of living patients.

If the result is positive, mice develop muscular tremor, motor discoordination, excitation, or paralysis. As a rule, the animals die within five days.

To confirm the diagnosis, the brain of sick animals is examined, using the immunofiuorescence test, for the presence of Babes-Negri bodies or for the antigen of the rabies virus. The virus is identified by the neutralization reaction in mice.

Serological examination. To recover antibodies in patients' blood serum, such tests as neutralization on mice, HAI, CF, IF, and ELISA are utilized. The above methods are also used for determining the level of immunity in humans and animals after vaccination.

Laboratory diagnosis of rotaviral gastroenteritis

It is known at present that rotaviruses (the reovirus family) are responsible for 50 per cent of all cases of gastroenteritis in childhood. The aetiological role of these viruses in gastroenteritis in animals has also been elucidated.

The material to be examined is the patient's faeces.

Rotaviruses are typically recovered from faeces during the first 6-8 days of the disease. Their concentrations peak is on the 3rd-5th day of the first clinical manifestations of the disease when their number reaches 10¹⁰–10¹¹ viral particles in 1 g of faeces.

Faeces are collected in sterile vials which are filled to one-third of their volume, closed with sterile stoppers, and transported in containers with melting ice. Samples are either treated immediately after their arrival at the laboratory or kept frozen.

Rapid diagnosis. The concentration of viral particles in faeces specimens collected during the first days of the disease amounts to 10⁶-10⁸ and over per 1 g, which makes possible their detection by electron microscopy. To perform electron microscopic and virological examination, prepare 10-20 per cent suspension of faeces in Hanks' solution. Centrifuge the suspension for 30 min at 3000 X g to remove gross particles, transfer the supernatant fraction to a sterile vial, add penicillin (1000 U/ml) and streptomycin (500 mg/ml), and let the mixture stand at 4 °C for 10-12 hrs. Following centrifugation of the faecal extract, a drop of the supernatant is contrasted with 2 per cent phosphotungstic acid (pH 6.5); the preparation is made and examined microscopically (50 000 X).

Immunoelectron microscopic examination has gained the widest application for this purpose owing to the fact that it makes possible both to detect the virus in faeces and to identify it.

Figure. Rotaviruses in a faecal sample from a patient with gastroenteritis (immunoelectron microscopy)

To carry out this examination, 0.1 ml of immune serum diluted 1 to 5 is mixed with 0.4 ml of 10 per cent faecal suspension which has already been centrifuged. The mixture is allowed to stand for 1 h at room temperature and for 12 hrs at 4 °C; then, it is centrifuged for 90 min at 15 000 X g. The pellet is resuspended in several drops of distilled water, contrasted with 2 per cent phosphotungstic acid (pH 6.5), and examined under the electron microscope for the aggregates of virions with typical morphology.

Immune precipitation involving staining of precipitates with fluorescent antibodies is also employed for the rapid diagnosis. Prepare a 2 per cent faecal suspension, then centrifuge it and filter through a millipore filter with the pores measuring 1.2 mcm in diameter. Mix the suspension (0.2 ml) with 0.2 ml of diluted immune serum, let the mixture stand at 37 °C for 1 h, and then centrifuge it at 12 000 X g for 1 h. Resuspend the sediment in 0.2 ml of phosphate buffer and add 0.2 ml of fluorescent immimoglobulin. Following a 10-min incubation, centrifuge the mixture at 2000 X g for 10 min, resuspend it in phosphate buffer, place on a glass slide, cover with a cover slip, and examine under the oil-immersion microscope with a fluorescent attachment.

RIA and ELISA tests are the most sensitive for demonstrating the rotaviral antigen. To run ELISA, place 0.5-ml portions of 2 per cent faecal suspension into wells on polysterene panels treated with immune globulin, add 0.25 ml of 2 per cent calf embryonic serum with 0.1 per cent of Twin-20 into each well, incubate for 1 h at 37 °C, then wash (three times) the wells by phosphate buffer witli Twin-20, and introduce 10 ml of conjugated rotaviral antiserum. Following 1-hour incubation at 37 °C, wash the wells once again and add the substrate. The results of the reaction are read by the intensity of substrate staining.

Reversed indirect haemagglutination and haemadsorption on a solid medium are finding an increasingly wider application in the rapid diagnosis of rotaviral infections.

Isolation of the virus from faeces is complicated by the fact that there are no cell cultures or animals sensitive to the rotavirus.

Serological examination is aimed at detecting specific antibodies in the paired blood sera taken within the first 3-4 days of the disease and then 12-14 days after the disease outset. The presence of antibodies is established by immunoelectron microscopic examination, using as an antigen a faecal preparation with a high level of rotaviral particles or a preparation of animal rotaviruses in passaged tissue cultures.

The complement-fixation test, in which the antigen used consists of previously selected suspensions of faeces from patients with gastroenteritis or rotaviruses of animals, has also found wide application. Other tests employed for this purpose include HAI with animal rotaviruses serving as an antigen, IF with a cell culture infected with animal rotaviruses (typically with the virus of diarrhoea of Nebraska calves), RIA, and ELISA.

Adenovirus Family

Adenoviruses consist of 2 genera, one that infects birds and another that infects mammals. Human adenoviruses are divided into 5 groups (A-E) based on their physical, chemical, and biologic properties. There are at least 37 antigenic types of human adenoviruses that may produce subclinical infection, respiratory tract or eye diseases, and occasionally other disorders. A few types serve as models for cancer induction in animals.

Properties of the Virus. Structure: Infective virions, 70-90 nm in diameter, are icosahedrons with capsids composed of 252 capsomeres. Three structural proteins, produced in large excess, constitute "soluble antigens" A, B, and C (Table). There is no envelope. The DNA is linear and double-stranded (MW 20-30 x 10⁶). The guanine-cytosine (G + C) content of the DNA is lowest (48-49%) in group A (types 12, 18, and 31), which are the most strongly oncogenic types. The DNA can be isolated in an infectious form capable of transforming cells in culture.

Table.

Comparative data on adenovirus type 2 morphologic and antigenic subunits and protein components

Dodecon: Hemagglutinin made up of 12 pentons

with their fibers.

Animal Susceptibility and Transformation of Cells. Most laboratory animals are not readily infected with adenoviruses. Newborn hamsters sustain a fatal infection with type 5 and develop malignant tumours when inoculated with any of 8 or more types, including types 12, 18, and 31. Adenovirus cannot be recovered from these tumours, but in the tumour a new antigen can be detected by complement fixation or immunofluorescence. This tumour, or T, antigen also develops in hamster cells that are infected or transformed by oncogenic adenovirus types. Transformed cells produce tumours when inoculated into adult hamsters but do not contain infectious virus. Only a small part (< 10%) of the adenovirus genome is present in many transformed cells. This explains the inability to recover infectious virus from such cells.

Adenovirus messenger RNA (mRNA) can be detected in transformed or tumour cells. Different types of adenovirus result in different mRNA in transformed cells.

In human tumours, adenovirus DNA or mRNA has never been found.

Antigenic Properties. All adenoviruses contain a common complement-fixing antigen mat persists in suspensions of virus treated with heat or formalin to inactivate infectivity. At least 47 antigenic types have been isolated from humans and many additional ones from various animals. They are typed by cross-neutralization tests or hemagglutination-inhibition.

The major antigens, their size, and their structural position in the virion are shown in Table 1. Group-reactive complement-fixing antigens are hexons that form a majority of capsomeres and are 8 nm in diameter. Pentons have a similar size, occur at the 12 vertices of the capsid, and have a fiber protruding from them. The penton base carries a toxinlike activity that results in detachment of cells from the surface on which they are growing. Pentons and fibers are associated with hemagglutinating activity.

Group B adenoviruses (types 3,7,11, 14, 16,21, 34, 35) clump rhesus but not rat erythrocytes: group D (types 8, 9, 10, 13, 15, 17, 19. 22, 23, 24, 26, 27, 29, 30, 32, 33, 36, 37) clump rat but not rhesus erythrocytes; groups C (types 1, 2, 5, 6) and D (type 4) only partly clump rat cells. Types 20, 25, and 28 are atypical in that they have the physical and chemical properties of group D but agglutinate only rhesus cells. Group A (oncogenic types 12, 18, and 31) adenoviruses usually fail to hemagglutinate. Inhibition of hemagglutination by type-specific sera can be used for typing isolates. Some cross-reactions, however, do occur.

Virus Growth in Cell Culture. Adenoviruses are cytopathic for human cell cultures, particularly primary kidney and continuous epithelial cells. Growth of virus in tissue culture is associated with a stimulation of acid production (increased glycolysis) in the early stages of infection. The cytopathic effect usually consists of marked rounding and aggregation of affected cells into grapelike clusters. The infected cells do not lyse even though they round up and leave the glass surface on which they have been grown.

In HeLa cells infected with adenovirus types 3,4, and 7, rounded intranuclear inclusions containing DNA are seen. The virus particles develop in the nucleus and frequently exhibit crystalline arrangement. Many cells infected with type 5 virus also contain crystals, but these crystals are composed of a protein that has not been clearly identified.

During adenovirus replication in cultures of human cells, at least 12 virus-specific polypeptides are synthesized. These peptides are cataloged and their relationship to the virus structure is shown in Table 1.

Adenovirus DNA replication occurs in the nucleus and requires host cell DNA polymerase. Adenovirus mRNA is also made in the nucleus in a complex sequence that requires first the synthesis of larger molecules which are broken up and some sections of which are respliced by special enzymes. The spliced mRNA is translated into virus proteins.

Adenovirus-specific proteins are synthesized in the cytoplasm of infected cells and then move rapidly into the nucleus, where viral maturation occurs. In the adenovirus growth cycle in human epithelial cells, new virus particles can be detected about 16-20 hours after inoculation and continue to be formed at a uniform rate for the next 24 hours. About 7000 virus particles are produced per infected cell, and most of them remain intracellular. Particles having a density of 1.34 are infectious (one particle in 5 is infectious), whereas those having densities of less than 1.30 are noninfectious, since they lack the DNA core. Crude infected cell lysates show huge quantities of capsomeres, sometimes partially assembled into viral components.

When infecting cells derived from species other than humans, the human adenoviruses undergo an abortive replication cycle. Adenovirus tumour antigen, mRNA, and DNA are all synthesized, but no capsid proteins or infectious progeny are produced.

Adenovirus-SV40 "Hybrids": Certain adenoviruses grown in monkey kidney cell cultures have become "contaminated" with the monkey virus SV40. While some of it was free in the mixture, other SV40 genomes became covalently linked to the adenovirus, so that stable "hybrids" were formed. Two types of hybrids have been identified. One is a defective adenovirus-SV40 genome encased in an adenovirus capsid. The other consists of nondefective (i.e., self-replicating) adenovirus type 2 that carries 5-40% of the SV40 genome. These hybrids have been used in genetic analysis but have no manifest medical relevance.

Adenoassociated Virus (AAV): In some adenovirus preparations, small 20-nm particles were found. These proved to be small viruses that could not replicate unless adenovirus (or sometimes herpesvirus) was present as a helper. AAV contains single-stranded DNA (MW 1.6 x 10⁶) and is serologically unrelated to adenovirus. Four antigenic types of AAV are known, 3 of which infect humans but do not seem to produce disease. AAV can infect cells in the absence of an adenovirus helper and induce a latent infection. AAV enters the cell nucleus and is uncoated there, but no mRNA synthesis occurs. Upon addition of an adenovirus, AAV is "rescued" and replication occurs

Pathogenesis. Adenoviruses infect epithelial cells of mucous membranes, the cornea, and other organ systems. They can be isolated from such structures during acute illness and may persist for long periods. Types 1,2,5 and 6 can be isolated from surgically removed adenoids or tonsils of most children by growing the epithelium in vitro. Gradual removal of antibody during long culture in vitro permits the viruses to grow, as they cannot be isolated directly from suspensions of such tissues.

Most human adenoviruses grow in intestinal epithelium after ingestion but usually do not produce symptoms or lesions.

Clinical Findings. Adenovirus diseases include syndromes designated as undifferentiated acute respiratory disease, pharyngoconjunctival fever, nonstreptococcal exudative pharyngitis, and primary atypical pneumonia not associated with the development of cold agglutinins.

Pharyngoconjunctival fever may be caused by several adenovirus types. It is characterized by fever, conjunctivitis, pharyngitis, malaise, and cervical lymphadenopathy. The conjunctivitis is readily reproduced when any adenovirus is swabbed onto the eyes of volunteers, However, under natural conditions, only types 3 and 7 regularly cause outbreaks in which conjunctivitis is a predominant symptom. Types 1, 2, 5, 6, 37, and many others have produced sporadic cases of conjunctivitis.

Types 8 and 19 cause epidemic keratoconjunctivitis (shipyard eye). The disease is characterized by an acute conjunctivitis, with enlarged, tender preauricular nodes, followed by keratitis that leaves round, subepithelial opacities in the cornea for up to 2 years. Type 8 infections have been characterized by their lack of associated systemic symptoms except in infants. Intussusception of infancy has been ascribed to adenoviruses 1, 2, 3, and 5.

Types 11 and 21 may be a cause of acute hemorrhagic cystitis in children. Virus commonly occurs in the urine of such patients- Type 37 occurs in cervical lesions and in male urethritis and may be sexually transmitted.

A newly discovered serotype has been associated with infantile gastroenteritis. The virus is abundantly present in stools but has not been grown in cell culture.

Laboratory Diagnosis. Recovery of Virus. The viruses are isolated by inoculation of tissue cultures of human cells in which characteristic cytopathic changes are produced.

The viruses have been recovered from throat swabs, conjunctival swabs, rectal swabs, stools of patients with acute pharyngitis and conjunctivitis, and urine of patients with acute hemorrhagic cystitis. Virus isolations from the eye are obtained mainly from patients with conjunctivitis.

A new serotype that has not been isolated in cell cultures can be detected by direct examination of fecal extracts by electron microscopy or by enzyme-linked immunosorbent assay.

Serology. In most cases, the neutralizing antibody titer of infected persons shows a 4-fold or greater rise against the type recovered from the patient and a lesser response to other types. Neutralizing antibodies are measured in human cell cultures using the cytopathic end point in tube cultures or the colour test in panel cups. The latter test depends upon the phenomenon that adenovirus growing in HeLa cell cultures produces an excess of acid over that of uninfected control cultures. This viral lowering of pH can be prevented by immune serum. The pH is measured by incorporating phenol red into the medium and observing the colour changes after 3 days of incubation. Serum and cell control cultures reach a pH of 7.4; virus activity is indicated by a pH of 7.0; and neutralization is presumed to have occurred when the pH is 0.2 unit above that of the virus control.

Infection of humans with any adenovirus type stimulates a rise in complement-fixing antibodies to adenovirus antigens of all types. The CF test, using the common antigen, is an easily applied method for detecting infection by any member of the group.

A sensitive radioimmunoassay can measure serum antibody to type 5 fiber antigen. In response to vaccination with the fiber subunit, volunteers exhibited a 54-fold increase in antifiber antibody.

Immunity. Studies in volunteers revealed that type-specific neutralizing antibodies protect against the disease but not always against reinfection. Infections with the viruses were frequently induced without the production of overt illness.

Neutralizing antibodies against one or more types may be present in over 50% of infants 6-11 months old. Normal healthy adults generally have antibodies to several types. Neutralizing antibodies to types 1 and 2 occur in 55-70% of individuals age 6-15, but antibodies to types 3 and 4 are less prevalent. Neutralizing antibodies probably persist for life.

Infants are usually born without complement-fixing antibodies but develop these by age 6 months. Older individuals with neutralizing antibodies to 4 or more strains frequently give completely negative complement fixation reactions. For military recruits, the incidence of infection (especially due to types 3 and 4) was not influenced by the presence of group complement-fixing antibodies.

Epidemiology. Adenoviruses can readily spread from person to person. Type 1, 2, 5, and 6 infections occur chiefly during the first years of life and are associated with fever and pharyngitis or asymptomatic infection. These are the types most frequently obtained from the adenoids and tonsils.

In children and young adults, types 3 and 7 commonly cause upper respiratory illness, pharyngitis, and conjunctivitis. While the illness is usually mild, occasionally there is high fever, cervical lymphadenitis, and even pneumonitis. Sometimes enteric infection produces gastroenteritis, but more commonly it is asymptomatic. Types 11 and 21 can produce acute hemorrhagic cystitis in children.

In adolescents and young adults, eg, college populations, only 2-5% of respiratory illness is caused by adenoviruses. In sharp contrast, respiratory disease due to types 3, 4, 7, 14, and 21 is common among military recruits. Adenovirus disease causes great morbidity when large numbers of persons are being inducted into the armed forces; consequently, its greatest impact is during periods of mobilization. During a 1-year study, 10% of recruits in basic training were hospitalized for a respiratory illness caused by an adenovirus. During the winter, adenovirus accounted for 72% of all the respiratory illness. However, adenovirus disease is not a problem in seasoned troops.

The follicular conjunctivitis caused by many adenovirus types resembles chlamydial conjunctivitis and is self-limited.

Epidemic keratoconjunctivitis caused by type 8 spread in 1941 from Australia via the Hawaiian Islands to the Pacific Coast. There it spread rapidly through the shipyards and other industries, thence to the East Coast, and finally to the Midwest. A large outbreak caused by type 8 occurred in 1977 in Georgia among patients subjected to invasive eye procedures by one ophthalmologist. The initial case was a nurse who returned from a vacation in Korea with severe keratoconjunctivitis. In the USA, the incidence of neutralizing antibody to type 8 adenovirus in the general population has been about 1 %, whereas in Japan it has been over 30%. In Japan, type 8 spreads via the respiratory route in children. Since 1973, adenovirus type 19 has also caused epidemics of typical epidemic keratoconjunctivitis.

Canine hepatitis virus is an adenovirus. Therefore, humans infected with adenoviruses develop group complement-fixing antibodies that also react with canine hepatitis virus.

In prospective family studies, adenovirus infections have been found to be predominantly enteric; they may be abortive or invasive and followed by persistent intermittent excretion of virus. Such excretion is most characteristic of types 1,2,3, and 5, which are usually endemic. Infection rates are highest among infants, but siblings who introduce the infection into a household are more effective in spreading the disease than are infants; similarly, duration of excretion is more important than the mode. In the families studied, neutralizing antibodies provided immunity (85% protective) against homotypic but not heterotypic infection. The contribution of adenoviruses to all infectious illness in the families, based on virus-positive infections only, was 5% in infants and 3% in the 2- to 4-year-old age group.

Prevention & Control. A trivalent vaccine was prepared by growing type 3,4, and 7 viruses in monkey kidney cultures and then inactivating the viruses with formalin. However, when it was found that the vaccine strains were contaminated genetically with SV40 tumour virus determinants, this vaccine was withdrawn from use. Subsequently, it was found that most adenovirus strains do not replicate in monkey cells unless SV40 is present as a helper virus. Thus, a vaccine had to be made from noncontaminated live virus that could be grown in human diploid cells, The vaccine is given orally in a coated capsule to liberate the virus into the intestine. By this route, the live vaccine produces a subclinical infection that confers a high degree of immunity against wild strains. It does not spread from a vaccinated person to contacts. Such live virus vaccines against type 4 and type 7 are licensed and recommended for immunization of military populations. When both are administered simultaneously, vaccines respond with neutralizing antibodies against both virus types.

Rigid asepsis during eye examination is essential in the control of epidemic keratoconjunctivitis.

Herpesvirus Family

All herpesviruses have a core of double-stranded DNA surrounded by a protein coat that exhibits icosahedral symmetry and has 162 capsomeres. The nucleocapsid is surrounded by an envelope. The enveloped form measures 150-200 nm; the "naked" virion, 100 nm. The double-stranded DNA (MW 85-150 x lO⁶) has a wide range of guanine + cytosine content in different herpesviruses, There is little DNA homology among different herpesviruses, except herpes simplex types 1 and 2.

Various classifications for herpesviruses have been proposed, but individual virus names are generally used. Common and important herpesviruses of humans include herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr (EB) virus, and cytomegalovirus. They have a propensity for subclinical infection, latency following the primary infection, and reactivation thereafter.

Herpesviruses that infect lower animals are B virus of Old World monkeys; herpesviruses saimiri, aotus, and ateles; marmoset herpesvirus of New World monkeys, pseudorabies virus of pigs; virus III of rabbits; infectious bovine rhinotracheitis virus; and many others. Herpesviruses are also known for birds, fish, fungi, and oysters, although the only link between some of these viruses is their appearance in the electron microscope.

Herpesviruses have been linked with malignant diseases in humans and lower animals: herpes simplex virus type 2 with cervical and vulvar carcinoma; EB virus with Burkilt 's lymphoma of African children and with nasopharyngeal carcinoma; Lucke virus with renal adenocarcinomas of the frog; Marek's disease virus with a lymphoma of chickens; Hinze virus with a lymphoma of rabbits; and a number of New World primate herpesviruses with reticulum cell sarcomas and lymphomas in these animals.

HERPES SIMPLEX (Human Herpesvirus 1 & 2) (Herpes Labialis, Herpes Genitalis, & Many Other Syndromes).

Infection with herpes simplex virus (herpesvirus hominis) may take several clinical forms. The infection is most often inapparent. The usual clinical manifestation is a vesicular eruption of the skin or mucous membranes. Infection is sometimes seen as severe keratitis, meningoencephalitis, and a disseminated illness of the newborn.

Properties of the Virus. Morphologically and chemically, herpes simplex virus has been studied in great detail (see Fig. 1). The envelope is derived from the nuclear membrane of the infected cell. It contains lipids, carbohydrate, and protein and is removed by ether treatment. The double-stranded DNA genome is linear (MW 85-106 x 10⁶). Types 1 and 2 show 50% sequence homology. Treatment with restriction endonucleases yields characteristically different cleavage patterns for type 1 and 2 viruses and even for different strains of each type. This "fingerprinting" of strains allows epidemiologic tracing of a given strain, whereas in the past, the ubiquitousness of herpes simplex virus made such investigations impossible.

Animal Susceptibility and Growth of Virus. The virus has a wide host range and can infect rabbits, guinea pigs, mice, hamsters, rats, and the chorioallantois of the embryonated egg.

In rabbits, herpesvirus produces a vesicular eruption in the skin of the inoculated area, sometimes progressing to fatal encephalitis. Corneal inoculation results in dendritic keratitis, which may progress to encephalitis, The virus may remain latent in the brains of survivors, and anaphylactic shock can precipitate an acute relapse of encephalomyelitis. Herpetic keratitis heals, but infective herpesvirus may be recovered from the eye intermittently with or without clinical activity. The virus remains latent in the trigeminal ganglion.

In the chorioallantoic membrane of embryonated eggs the lesions are raised white plaques, each induced by one infectious virus particle. The plaques produced by herpesvirus type 2 are larger than the tiny plaques produced by type 1 virus. The virus grows readily and produces plaques in almost any cell culture. Infected cells develop inclusion bodies and then undergo necrosis (cytopathic effect).

In Chinese hamster cells, which contain 22 chromosomes, the virus causes breaks in region 7 of chromosome No. 1 and in region 3 of the X chromosome. The Y chromosome is unaffected.

Virus Replication. The virus enters the cell either by fusion with the cell membrane or by pinocytosis. It is then uncoated, and the DNA becomes associated with the nucleus. Normal cellular DNA and protein synthesis virtually stop as virus replication begins. The virus induces a number of enzymes, at least 2 of which — thymidine kinase and DNA polymerase — are virus-coded. Thymidine kinases produced by different herpesviruses are serologically different from each other and different from the enzyme in uninfected cells. Phosphonoacetic acid specifically inhibits herpesvirus replication by inhibiting viral DNA polymerase.

Viral proteins are made in a controlled sequence that must proceed stepwise. They are made in the cytoplasm and most are transported to the nucleus, where they take part in virus DNA synthesis and the assembly of nucleocapsids, Maturation occurs by budding of nucleocapsids through the altered inner nuclear membrane. Enveloped virus particles are then released from the cell through tubular structures that are continuous with the outside of the cell or from vacuoles that release their contents at the cell surface.

Defective Interfering Herpesvirions: Serial passage of undiluted herpes simplex virus results in cyclic production of infectious and defective virions. The DNA in defective virions is made up of reiterated sequences of small fragments of the virus DNA. Defective virions interfere with the replication of standard virus and stimulate overproduction of a large polypeptide, which may have a regulatory function. The biologic role of the defective virions is not known.

Antigenic Properties. There are 7-12 precipitating antigens that represent structural and non-structural viral proteins. Some of these antigens are common to both types 1 and 2 and some are specific for one type. A number of tests, eg, fluorescent antibody, complement fixation, virus neutralization, and radioimmunoassay, have been used to detect herpesvirus antigens.

Differentiation of Types 1 and 2. Herpes simplex virus types 1 and 2 cross-react serologically but may be distinguished by a number of tests: (1) The use of type-specific antiserum prepared by adsorption of the viral antiserum with heterotypically infected cells or by inoculation of rabbits with individual type-specific proteins. (2) The greater temperature sensitivity of type 2 infectivity. (3) Preferential growth in different cell species. (4) Restriction enzyme patterns of virus DNA molecules. (5) Differences in the polypeptides produced by type 1 and type 2.

Oncogenic Properties: After inactivation of their lytic capabilities by ultraviolet irradiation or other means, herpesvirus types 1 and 2 can cause transformation of cultured hamster cells, which may induce tumours when inoculated into newborn hamsters. Viral genetic information can be demonstrated in the tumour cells.

Pathogenesis & Pathology. The lesion in the skin involves proliferation, ballooning degeneration, and intranuclear acidophilic inclusions. In fatal cases of herpes encephalitis, there are meningitis, perivascular infiltration, and nerve cell destruction, especially in the cortex. Neonatal generalized herpes infection causes areas of focal necrosis with a mononuclear reaction and formation of intranuclear inclusion bodies in all organs. Survivors may sustain permanent damage.

The fully formed early inclusion (Cowdry type A inclusion body) is rich in DNA and virtually fills the nucleus, compressing the chromatin to the nuclear margin. Later, the inclusion loses its DNA and is separated by a halo from the chromatin at the nuclear margin.

Clinical Findings. Herpesvirus may cause many clinical entities, and the infections may be primary or recurrent. Primary infections occur in persons without antibodies and often result in the virus assuming a latent state in sensory ganglia of the host. Latent infections persist in persons with antibodies, and recurrent lesions are common (eg, recurrent herpes labialis). The primary infection in most individuals is clinically inapparent but is invariably accompanied by antibody production.

The recurrent attacks, in the presence of viral neutralizing antibody, follow non-specific stimuli such as exposure to excess sunlight, fever, menstruation, or emotional stresses.

Herpesvirus Type 1. The clinical entities attributable to herpesvirus type 1 include the following:

1. Acute herpetic gingivostomatitis (aphthous stomatitis, Vincent's stomatitis). This is the most common clinical entity caused by primary infections with type 1 herpesvirus. It occurs most frequently in small children (1-3 years of age) and includes extensive vesiculoulcerative lesions of the mucous membranes of the mouth, fever, irritability, and local lymphadenopathy. The incubation period is short (about 3-5 days), and the lesions heal in 2-3 weeks.

2. Eczema herpeticum (Kaposi's varicelliform eruption). This is a primary infection, usually with herpesvirus type 1, in a person with chronic eczema. In this illness, there may be extensive vesiculation of the skin over much of the body and high fever. In rare instances, the illness may be fatal.

3. Keratoconjunctivitis. The initial infection with herpesvirus may be in the eye, producing severe keratoconjunctivitis. Recurrent lesions of the eye appear as dendritic keratitis or corneal ulcers or as vesicles on the eyelids. With recurrent keratitis, there may be progressive involvement of the corneal stroma, with permanent opacification and blindness.

4. Encephalitis. A severe form of encephalitis may be produced by herpesvirus. In adults, the neurologic manifestations suggest a lesion in the temporal lobe. Pleocytosis (chiefly of lymphocytes) is present in the cerebrospinal fluid; however, definite diagnosis during the illness can usually be made only by isolation of the virus (or by demonstrating viral antigens by immunofluorescence) from brain tissue obtained by biopsy or at post-mortem. The disease carries a high mortality rate, and those who survive often have residual neurologic defects.

5. Herpes labialis (cold sores, herpes febrilis). This is the most common recurrent disease produced by type 1. Clusters of localized vesicles occur, usually at the mucocutaneous junction of the lips. The vesicle ruptures, leaving a painful ulcer that heals without scarring. The lesions may recur, repeatedly and at various intervals of time, in the same location. The permanent site of latent herpes simplex virus is the trigeminal ganglion.

Herpesvirus Type 2. The clinical entities associated with herpesvirus type 2 include the following:

1. Genital herpes (herpes progenitalis). Genital herpes is characterized by vesiculoulcerative lesions of the penis of the male or the cervix, vulva, vagina, and perineum of the female. The lesions are more severe during primary infection and may be associated with fever, malaise, and inguinal lymphadenopathy. In women with herpesvirus antibodies, only the cervix or vagina may be involved, and the disease may therefore be asymptomatic. Recurrence of the lesions is common. Type 2 virus remains latent in lumbar and sacral ganglia. Changing patterns of sexual behaviour are reflected by an increasing number of type 1 virus isolations from genital lesions and of type 2 from facial lesions, presumably as a result of oral-genital sexual activity.

2. Neonatal herpes. Herpesvirus type 2 may be transmitted to the newborn during birth by contact with herpetic lesions in the birth canal. The spectrum of illness produced in the newborn appears to vary from subclinical or local to severe generalized disease with a fatal outcome. Severely affected infants who survive may have permanent brain damage. To avoid infection, delivery by cesarean section has been used in pregnant women with genital herpes lesions. To be effective, cesarean section must be performed before rupture of the membranes.

Severe generalized disease of the newborn can be acquired postnatally by exposure to either type 1 or 2. Efforts should be made to prevent exposure to active lesions among family and especially among hospital personnel.

Transplacental infection of the fetus with types 1 and 2 herpes simplex virus may cause congenital malformations, but this phenomenon is rare.

Miscellaneous. Localized lesions of the skin caused by type 1 or 2 may occur in abrasions that become contaminated with the virus (traumatic herpes), These lesions are seen on the fingers of dentists, hospital personnel (herpetic whitlow), or persons with genital lesions and on the bodies of wrestlers.

Primary and recurrent herpes can occur in the nose (acute herpetic rhinitis).

Mild aseptic meningitis has been attributed to the virus, and recurrent episodes of meningeal irritation have been observed.

Epidemiologic evidence has demonstrated that in most geographic areas, patients with cervical and vulvar cancer have a high frequency of type 2 antibodies. In addition, herpesvirus type 2 non-structural antigens have been detected by immunofluorescence in biopsies of cervical and vulvar carcinomas.

Laboratory Diagnosis

Recovery of Virus. The virus may be isolated from herpetic lesions (skin, cornea, or brain). It may also be found in the throat, saliva, and stools, both during primary infection and during asymptomatic periods. Therefore, the isolation of herpesvirus is not in itself sufficient evidence to indicate that this virus is the causative agent of a disease under investigation.

inoculation of tissue cultures is used for virus isolation. The appearance of typical cytopathic effects in cell culture suggests the presence of herpesvirus in 18-36 hours. The agent is then identified by neutralization test or immunofluorescence staining with specific antiserum.

Scrapings or swabs from the base of early herpetic lesions contain multinucleated giant cells.

Serology. Antibodies may be measured quantitatively by neutralization tests in cell cultures. In the early stage of the primary immune response, neutralizing antibody appears that is detectable only in the presence of fresh complement. This antibody soon is replaced by neutralizing antibody that can function without complement.

Since the only hope for treatment of herpes simplex virus encephalitis lies in early diagnosis, a rapid means of diagnosis is needed. The fluorescent antibody test using brain biopsy material is the method of choice. Passive hemagglutinating antibodies in the cerebrospinal fluid are a better indicator of the presence of infectious virus than are antibody titters in serum.

A soluble complement-fixing antigen of much smaller size than the virus can be prepared from infected chorioallantoic membranes or from tissue culture. This soluble antigen of herpesvirus can detect dermal hypersensitivity in previously infected persons. There is a good correlation between dermal hypersensitivity and the presence of serum antibodies.

Antibodies appear in 4-7 days; can be measured by neutralization, complement fixation, radioimmunoassay, or immunofluorescence; and reach a peak in 2-4 weeks. They persist with minor fluctuations for the life of the host. The majority of adults have antibodies in their blood at all times.

After a primary type I infection, the IgM neutralizing antibody response is type-specific, but after a primary type 2 infection the IgM that develops neutralizes both type 1 and type 2 virus. Subsequently, IgG antibodies react with both type 1 and type 2 antigens, albeit in varying ratios.

There is also some cross-stimulation between herpes simplex and varicella-zoster antigens in patients with pre-existing antibody to the other virus.

Immunity. Many newborns have passively transferred maternal antibodies. This antibody is lost during the first 6 months of life, and the period of greatest susceptibility to primary herpes infection occurs between ages 6 months and 2 years. Type 1 antibodies begin to appear in the population in early childhood; by adolescence they are present in most persons. Antibodies to type 2 (genital herpesvirus) rise during the age of adolescence and sexual activity.

After recovery from a primary infection (inapparent, mild, or severe), the virus is usually carried in a latent state, in the presence of antibodies.

Treatment. Topically applied idoxuridine (5-iodo-2'-deoxy-uridine, IUDR), trifluorothymidine, vidarabine (ade-nine arabinoside, ara-A), acyclovir, and other inhibitors of viral DNA synthesis are effective in herpetic keratitis. These drugs inhibit herpesvirus replication and may suppress clinical manifestations. However, the virus remains latent in the sensory ganglia, and the rate of relapse is similar in drug-treated and untreated individuals. Some drug-resistant virus strains have emerged. Most strains of type 2 herpesvirus are suppressed less effectively than type 1.

For systemic administration, vidarabine (15 mg/kg/d intravenously) is accepted in herpes encephalitis diagnosed by biopsy. Best results are obtained if treatment is begun early in the disease, before coma sets in. Vidarabine also has some effect in disseminated herpes simplex.

Other drugs, especially acyclovir, are undergoing clinical trial, and many new antiherpes compounds are being developed. Acyclovir has low toxicity and has been administered systemically to suppress the activation of a latent herpes infection in immunosuppressed patients.

Epidemiology. The epidemiology of type 1 and type 2 herpesvirus differs. Herpesvirus type 1 is probably more constantly present in humans than any other virus. Primary infection occurs early in life and is often asymptomatic or produces acute gingivostomatitis. Antibodies develop, but the virus is not eliminated from the body; a carrier state is established that lasts throughout life and is punctuated by transient attacks of herpes. If primary infection is avoided in childhood, it may not occur in later life, perhaps because the thicker adult epithelium is less susceptible or because the opportunity for contact with the virus is diminished (less contact with saliva of infected persons).

The highest incidence of type I virus carriage in the oropharynx of healthy persons occurs among children 6 months to 3 years of age. By adulthood, 70-90% of persons have type 1 antibodies.

Type 1 virus is transmitted more readily in families of lower socioeconomic groups; the most obvious explanation is their more crowded living conditions and lower hygienic standards. The virus is spread by direct contact (saliva) or through utensils contaminated with the saliva of a virus shedder. The source of infection for children is usually a parent with an active herpetic lesion.

Type 2 is usually acquired as a sexually transmitted disease, and the age distribution of primary infection is a function of sexual activity. The neonate may acquire type 2 infection from an active lesion in the mother's birth canal.

Control. Neonates and persons with eczema should be protected from evident active herpetic lesions. Although certain drugs are effective in treatment of herpesvirus infections, once a latent infection is established there has been no known treatment that would prevent recurrences until the recent successful results with acyclovir in immunosuppressed patients.

Little Is known about vaccines. Herpes recurs in the presence of circulating antibody, so a vaccine would be of little use in a person who already had a primary infection. A vaccine currently made in Europe has not been adequately tested.

VARICELLA-ZOSTER VIRUS (Human Herpesvirus 3)

VARICELLA (Chickenpox) ZOSTER (Herpes Zoster, Shingles, Zona)

Varicella (chickenpox) is a mild, highly infectious disease, chiefly of children, characterized clinically by a vesicular eruption of the skin and mucous membranes. However, in immunocompromised children the disease may be severe. The causative agent is indistinguishable from the virus of zoster.

Zoster (shingles) is a sporadic, incapacitating disease of adults (rare in children) that is characterized by an inflammatory reaction of the posterior nerve roots and ganglia, accompanied by crops of vesicles (like those of varicella) over the skin supplied by the affected sensory nerves.

Both diseases are caused by the same virus. Varicella is the acute disease that follows primary contact with the virus, whereas zoster is the response of the partially immune host to a reactivation of varicella virus present in latent form in sensory ganglia.

Properties of the Virus. Varicella-zoster virus is morphologically identical with herpes simplex virus. The virus propagates in cultures of human embryonic tissue and produces typical intranuclear inclusion bodies. Supernatant fluids from such infected cultures contain a complement-fixing antigen but no infective virus. Infectious virus is easily transmitted by infected cells. The virus has not been propagated in laboratory animals. Virus can be isolated from the vesicles of chickenpox or zoster patients or from the cerebrospinal fluid in cases of zoster aseptic meningitis.

Inoculation of vesicle fluid of zoster into children produces vesicles at the site of inoculation in about 10 days. This may be followed by generalized skin lesions of varicella. Generalized varicella may occur in such inoculated children without local vesicle formation. Contacts of such children develop typical varicella after a 2-week incubation period. Children who have recovered from zoster virus-induced infection are resistant to varicella, and those who have had varicella are no longer susceptible to primary zoster virus.

Antibody to varicella-zoster virus can be measured by CF, gel precipitation, neutralization, or indirect immunofluorescence to virus-induced membrane antigens.

The virus has a colchicinelike effect on human cells. Arrest in metaphase, overcontracted chromosomes, chromosome breaks, and formation of micronuclei are often seen.

Pathogenesis & Pathology. Varicella: The route of infection is probably the mucosa of the upper respiratory tract. The virus probably circulates in the blood and localizes in the skin. Swelling of epithelial cells, ballooning degeneration, and the accumulation of tissue fluids result in vesicle formation. In nuclei of infected cells, particularly in the early stages, eosinophilic inclusion bodies are found.

Zoster: In addition to skin lesions — histopathologically identical with those of varicella — there is an inflammatory reaction of the dorsal nerve roots and sensory ganglia. Often only a single ganglion may be involved. As a rule, the distribution of lesions in the skin corresponds closely to the areas of innervation from an individual dorsal root ganglion. There is cellular infiltration, necrosis of nerve cells, and inflammation of the ganglion sheath.

Varicella virus seems able to enter and remain within dorsal root ganglia for long periods. Years later, various insults (eg, pressure on a nerve) may cause a flare-up of the virus along posterior root fibers, whereupon zoster vesicles appear. Thus, varicella-zoster and herpes simplex viruses are similar in their ability to induce latent infections with clinical recurrence of disease in humans. However, zoster rarely occurs more than once.

Clinical Findings. Varicella: The incubation period is usually 14-21 days. Malaise and fever are the earliest symptoms, soon followed by the rash, first on the trunk and then on the face, the limbs, and the buccal and pharyngeal mucosa. Successive fresh vesicles appear in crops during the next 3-4 days, so that all stages of papules, vesicles, and crusts may be seen at one time. The eruption is found together with the fever and is proportionate to its severity. Complications are rare, although encephalitis does at times occur about 5-10 days after the rash. The mortality rate is much less than 1% in uncomplicated cases. In neonatal varicella (contracted from the mother just before or just after birth), the mortality rate may be 20%. In varicella encephalitis, the mortality rate is about 10%, and another 10% are left with permanent injury to the central nervous system. Primary varicella pneumonia is rare in children but may occur in about 20-30% of adult cases, may produce severe hypoxia, and may be fatal.

Children with immune deficiency disease or those receiving immunosuppressant or cytotoxic drugs are at high risk of development of very severe and sometimes fatal varicella or disseminated zoster.

Zoster: The incubation period is unknown. The disease starts with malaise and fever that are soon followed by severe pain in the area of skin or mucosa supplied by one or more groups of sensory nerves and ganglia. Within a few days after onset, a crop of vesicles appears over the skin supplied by the affected nerves. The eruption is usually unilateral; the trunk, head, and neck are most commonly involved. Lymphocytic pleocytosis in the cerebrospinal fluid may be present.

In patients with localized zoster and no underlying disease, vesicle interferon levels peak early during infection (by the sixth day), whereas those in patients with disseminated infection peak later. Peak interferon levels are followed by clinical improvement within 48 hours. Vesicles pustulate and crust, and dissemination is halted.

Zoster tends to disseminate when there is an underlying disease, especially if the patient is taking immunosuppressive drugs or has lymphoma treated by irradiation.

Laboratory Diagnosis. In stained smears of scrapings or swabs of the base of vesicles, multinucleated giant cells are seen. In similar smears, intracellular viral antigens can be demonstrated by immunofluorescence staining.

Virus can be isolated in cultures of human or other fibroblastic cells in 3-5 days. It does not grow in epithelial cells, in contrast to herpes simplex, and does not infect laboratory animals or eggs. An isolate in fibroblasts is identified by immunofluorescence or neutralization tests with specific antisera.

Herpesviruses can be differentiated from pox-viruses by (1) the morphologic appearance of particles in vesicular fluids examined by electron microscopy, and by (2) the presence of antigen in vesicle fluid or in an extract of crusts as determined by gel diffusion tests with specific antisera to herpes, varicella, or vaccinia viruses, which give visible precipitation lines in 24-48 hours.

A rise in specific antibody titer can be detected in the patient's serum by CF, Nt (in cell culture), indirect immunofluorescence tests, or enzyme immunoassay, Zoster can occur in the presence of relatively high neutralizing antibody in the blood just prior to onset. The role of cell-mediated immunity is unknown.

Immunity. Varicella and zoster viruses are identical, the 2 diseases being the result of differing host responses. Previous infection with varicella leaves the patient with enduring immunity to varicella. However, zoster may occur in persons who have contracted varicella earlier. This is a reactivation of a varicella virus infection that has been latent for years.

Prophylaxis & Treatment. Gamma globulin of high specific antibody titer prepared from pooled plasma of patients convalescing from herpes zoster (zoster immune globulin) can be used to prevent the development of the illness in immunocompromised children who have been exposed to varicella. Standard immune serum globulin is without value because of the low titer of varicella antibodies.

Zoster immune globulin is available from the American Red Cross Blood Services (through 13 regional blood centres) for prophylaxis of varicella in exposed high-risk immunodeficient or immunosuppressed children. It has no therapeutic value once varicella has started.

Idoxuridine and cytarabine inhibit replication of the viruses in vitro but are not an effective treatment for patients.

Adenine arabinoside (vidarabine, ara-A) has been beneficial in adults with severe varicella pneumonia, immunocompromised children with varicella, and adults with disseminated zoster. Human leukocyte interferon in large doses appears to be similarly beneficial.

Epidemiology. Zoster occurs sporadically, chiefly in adults and without seasonal prevalence. In contrast, varicella is a common epidemic disease of childhood (peak incidence is in children age 2-6 years, although adult cases do occur). It is much more common in winter and spring than in summer. Almost 200,000 cases are reported annually in the USA.

Varicella readily spreads, presumably by droplets as well as by contact with skin. Contact infection is rare in zoster, perhaps because the virus is absent in the upper respiratory tract.

Zoster, whether in children or adults, can be the source of varicella in children and can initiate large outbreaks.

Control. None is available for the general population. Varicella may spread rapidly among patients, especially among children with immunologic dysfunctions or leukemia or in those receiving corticosteroids or cytotoxic drugs. Varicella in such children poses the threat of pneumonia, encephalitis, or death. Efforts should be made to prevent their exposure to varicella. Zoster immune globulin may be used to modify the disease in such children who have been exposed to varicella.

A live attenuated varicella vaccine has been developed in Japan and tested in hospitalized immune-suppressed children who were exposed to varicella. It appeared to prevent spread of chickenpox. The vaccine is being used experimentally for similar high-risk children in the USA.

A number of problems are envisioned for the use of such a vaccine for the general population as opposed to high-risk patients. The vaccine would need to confer immunity comparable to that of natural infections. A short-lasting immunity might result in an increased number of susceptible adults, in whom the disease is more severe. Furthermore, any such vaccine would need to be evaluated for later morbidity due to zoster as compared to that following natural childhood infections with varicella virus.

CYTOMECALOVIRUS (Human Herpesvirus 5) (Cytomegalic Inclusion Disease)

Cytomegalic inclusion disease is a generalized infection of infants caused by intrauterine or early postnatal infection with the cytomegaloviruses. The disease causes severe congenital anomalies in about 10,000 infants in the USA per year. Cytomegalovirus can be found in the cervix of up to 10% of healthy women. Cytomegalic inclusion disease is characterized by large intranuclear inclusions that occur in the salivary glands, lungs, liver, pancreas, kidneys, endocrine glands, and. occasionally, the brain. Most fatalities occur in children under 2 years of age. Inapparent infection is common during childhood and adolescence. Severe cytomegalovirus infections are frequently found in adults receiving immunosuppressive therapy.

Properties of the Virus. Morphologically, cytomegalovirus is indistinguishable from herpes simplex or varicella-zoster virus.

In infected human fibroblasts, virus particles are assembled in the nucleus. The envelope of the virus is derived from the inner nuclear membrane. The growth cycle of the virus is slower, and infectious virus is more cell-associated than herpes simplex virus.

Animal Susceptibility. All attempts to infect animals with human cytomegalovirus have failed. A number of animal cytomegaloviruses exist, all of them species-specific in rats, hamsters, moles, rabbits, and monkeys. The virus isolated from monkeys propagates in cultures of monkey as well as human cells.

Human cytomegalovirus replicates in vitro only in human fibroblasts. although the virus is often isolated from epithelial cells of the host. The virus can transform human and hamster cells in culture, but whether it is oncogenic in vivo is unknown.

Pathogenesis & Pathology. In infants, Cytomegalic inclusion disease is con-genitally acquired, probably as a result of primary infection of the mother during pregnancy. The virus can be isolated from the urine of the mother at the time of birth of the infected baby, and typical cytomegalic cells, 25-40 mcm in size, occur in the chorionic villi of the infected placenta.

Foci of cytomegalic cells are found in fatal cases in the epithelial tissues of the liver, lungs, kidneys, gastrointestinal tract, parotid gland, pancreas, thymus, thyroid, adrenals, and other regions. The cells can be found also in the urine or adenoid tissue of healthy children. The route of infection in older infants, children, and adults is not known.

The isolation of the virus from urine and from tissue cultures of adenoids of healthy children suggests subclinical infections at a young age. The virus may persist in various organs for long periods in a latent state or as a chronic infection. Virus is not recovered from the mouths of adults. Disseminated inclusions in adults occur in association with other severe diseases.

Clinical Findings. Congenital infection may result in death of the fetus in utero or may produce the clinical syndrome of cytomegalic inclusion disease, with signs of prematurity, jaundice with hepatosplenomegaly, thrombocytopenic purpura, pneumonitis, and central nervous system damage (microcephaly, periventricular calcification, chorioretinitis, optic atrophy, and mental or motor retardation).

Infants born with congenital cytomegalic inclusion disease may appear well and live for many years. It has been estimated that one in every 1000 babies born in the USA is seriously retarded as a result of this congenital infection.

Inapparent intrauterine infection seems to occur frequently. Elevated IgM antibody to cytomegalovirus or isolation of the virus from the urine occurs in up to 2% of apparently normal newborns. This high rate occurs in spite of the fact that women may already have cytomegalovirus antibody before becoming pregnant. Such intrauterine infections have been implicated as possible causes of mental retardation and hearing loss.

Many women who have been infected naturally with cytomegalovirus at some time prior to pregnancy begin to excrete the virus from the cervix during the last trimester of pregnancy. At the time of delivery, infants pass through the infected birth canal and become infected, although they possess high titters of maternal antibody acquired transplacentally. These infants begin to excrete the virus in their urine at about 8-12 weeks of age. They continue to excrete the virus for several years but remain healthy.

Acquired infection with cytomegalovirus is common and usually inapparent, In children, acquired infection may result in hepatitis, interstitial pneumonitis, or acquired hemolytic anemia. The virus is shed in the saliva and urine of infected individuals for weeks or months.

Cytomegalovirus can cause an infectious mononucleosis-like disease without heterophil antibodies. "Cytomegalovirus mononucleosis" occurs either spontaneously or after transfusions of fresh blood during surgery ("postperfusion syndrome"). The incubation period is about 30-40 days. There is cytomegaloviruria and a rise of cytomegalovirus antibody. Cytomegalovirus has been isolated from the peripheral blood leukocytes of such patients. Perhaps the postperfusion syndrome is caused by cytomegalovirus harboured in the leukocytes of the blood donors.

Patients with malignancies or immunologic defects or those undergoing immunosuppressive therapy for organ transplantation may develop cytomegalo-virus pneumonitis or hepatitis and occasionally generalized disease. In such patients a latent infection may be reactivated when host susceptibility to infection is increased by immunosuppression. In seronegative patients without evidence of previous cytomegalovirus infection, the virus may be transmitted exogenously. Eighty-three percent of seronegative patients who received kidneys from seropositive transplant donors developed infection. Thus, the kidneys seemed to be the source of virus.

Laboratory Diagnosis. Recovery of Virus: The virus can be recovered from mouth swabs, urine, liver, adenoids, kidneys, and peripheral blood leukocytes by inoculation of human fibroblastic cell cultures. In cultures, 1-2 weeks are usually needed for cytotogic changes consisting of small foci of swollen, rounded, translucent cells with large intranuclear inclusions. Cell degeneration progresses slowly, and the virus concentration is much higher within the cell than in the fluid. Prolonged serial propagation is needed before the virus reaches high titters.

Rapid diagnosis of cytomegalovirus infection in infants is possible by detection of inclusion-bearing "owl cells" in the urine. These are desquamated cells from infected kidney tubules.

Serology. Antibodies may be detected by neutralization, complement fixation, or immunofluorescence tests. Such tests may be useful in detecting congenitally infected infants with no clinical manifestations of disease.

Immunity. Complement-fixing and neutralizing antibodies occur in most human sera. In young children possessing CF antibodies, virus may be detected in the mouth and in the urine for many months.

Virus may occur in the urine of children even though serum-neutralizing antibody is present. This suggests that the virus propagates in the urinary tract rather than being filtered from the bloodstream. Virus is not found in young children who lack antibody.

Intrauterine infection may produce a serious disease in the newborn. Infants infected during fetal life may be born with antibody that continues to rise after birth in the presence of persistent virus excretion. (This is similar to the situation in congenital rubella infection.)

Most infants infected with cytomegalovirus in the perinatal period are asymptomatic, and infection continues in the presence of high antibody titters.

Treatment. There is no specific treatment. Neither immune gamma globulin nor DNA virus-inhibitory drugs have any effect.

Epidemiology. The mechanism of virus transmission in the population remains unknown except in congenital infections and those acquired by organ transplantation, blood transfusion, and reactivation of latent virus. Infection with cytomegaloviruses is widespread. Antibody is found in 80% of individuals over 35 years of age. The prolonged shedding of virus in urine and saliva suggests a urine-hand-oral route of infection. Cytomegalovirus can also be transmitted by sexual contact.

Control. Specific control measures are not available. Isolation of newborns with generalized cytomegalic inclusion disease from other neonates is advisable.

Screening of transplant donors and recipients for cytomegalovirus antibody may prevent some transmissions of primary cytomegalovirus. The cytomegalovirus-seronegative transplant recipient population represents a high-risk group for cytomegalovirus infections as well as other lethal superinfections and would be a target population for a vaccine.

A live cytomegalovirus "vaccine" has been developed and has had some preliminary clinical trials, Since cytomegalovirus, like other herpesviruses, causes latent persistent infection, there is doubt that such a "vaccine" would be useful for the population at large. The possible benefits and dangers of a vaccine program for prevention of cytomegalovirus congenital infections require further study.

EB HERPESVIRUS (Human Herpesvirus 4).

(Infectious Mononucleosis, Burkitt's Lymphoma, Nasopharyngeal Carcinoma).

EB (Epstein-Barr) virus is the causative agent of infectious mononucleosis and has been associated with Burkitt's lymphoma and nasopharyngeal carcinoma. The virus is an antigenically distinct herpesvirus.

Properties of the Virus. Morphology: EB virus is indistinguishable in size and structure from other herpesviruses.

Antigenic Properties: EB virus is distinct from all other human herpesviruses. Many different EB virus antigens can be detected by CF, immunodiffusion, or immunofluorescence tests. A lymphocyte-detected membrane antigen (LYDMA) is the earliest-detected virus-determined antigen. EBNA is a complement-fixing nuclear antigen. Early antigen (EA) is formed in the presence of DNA inhibitors and membrane antigen (MA), the neutralizing antigen, is a cell surface antigen. The virus capsid antigen (VCA) is a late antigen representing virions and structural antigen.

C. Virus Growth: Human blood B lymphocytes infected in vitro with EB virus have resulted in the establishment of continuous cell lines, suggesting that these cells have been transformed by the virus.

This transformation by EB virus enables B lymphocytes to multiply continuously, and all cells contain many EB virus genomes and express EBNA. Some EB virus cell lines express certain antigens but produce no virus particles or VCA; others produce virus particles. EB virus is carried in lymphoid cell lines derived from patients with African Burkitt's lymphoma, nasopharyngeal carcinoma, or infectious mononucleosis. Non-virus-producing B lymphocyte cell lines can be established in vitro from the blood of patients with infectious mononucleosis. Such lines represent a latent state of the virus; the cells contain EB virus genomes but express only the earliest antigen (LYDMA) and possibly EBNA.

Owl monkeys and marmosets inoculated with cell-free EB virus can develop fatal malignant lymphomas. Lymphoblastoid cells from such monkeys cultured as continuous cell lines give positive reactions with EB virus antisera by immunofluorescence.

Immunity. The most widely used and most sensitive serologic procedure for detection of EB virus infection is the indirect immunofluorescence test with acetone-fixed smears of cultured Burkitt's lymphoma cells. The cells containing the EB virus exhibit fluorescence after treatment with fluorescent antibody. Detectable levels of antibody persist for many years.

Early in acute disease, a transient rise in IgM antibodies to VCA occurs, replaced within 2 weeks by IgG antibodies to VCA, which persist for life. Slightly later, antibodies to MA and to EBNA arise and persist throughout life.

Epidemiology. Seroepidemiologic studies using the immunofluorescence technique and CF reaction indicate that infection with EB virus is common in different parts of the world and that it occurs early in life. In some areas, including urban parts of the USA, about 50% of children 1 year old. 80-90% of children over age 4, and 90% of adults have antibody to EB virus.

In groups at a low socioeconomic level, EB virus infection occurs in early childhood without any recognizable disease. These inapparent infections result in permanent seroconversion and total immunity to infectious mononucleosis. In groups living in comfortable social circumstances, infection is often postponed until adolescence and young adulthood. Again, the majority of these adult infections are asymptomatic, but In almost half of cases the infection is manifested by heterophil-positive infectious mononucleosis.

Antibody to EB virus is also present in nonhuman primates.

EB Virus & Human Disease. Most EB virus infections are clinically inapparent. The virus causes infectious mononucleosis and is strongly associated with Burkitt's lymphoma and nasopharyngeal carcinoma.

Infectious mononucleosis (glandular fever) is a disease of children and young adults characterized by fever and enlarged lymph nodes and spleen. The total white blood count may range from 10,000/mcL to 80,000/mc, with a predominance of lymphocytes. Many of these are-large "atypical" cells with vacuo-lated cytoplasm and nucleus. These atypical lymphocytes, probably T cells, are diagnostically important. During mononucleosis, there often are signs of hepatitis.

During the course of infection, the majority of patients develop heterophil antibodies, detected by sheep cell agglutination or the mononucleosis spot test.

Although the pathogenesis of infectious mononucleosis is still not understood, infectious EB virus can be recovered from throat washings and saliva of patients ("kissing disease"). Infectious virus is produced by B lymphocytes in the oropharynx and perhaps in special epithelial cells of this region. Virus cannot be recovered from blood, but EB virus genome-containing B lymphocytes are present in up to 0.05% of me circulating mononuclear leukocytes as demonstrated by me establishment of cell lines. These EB virus genome-containing cells express the earliest antigen, LYDMA, which is specifically recognized by killer T cells.

These T cells reach large numbers and can lyse EB virus genome-positive but not EB virus genome-negative target cells. Part of the infectious mononucleosis syndrome may reflect a rejection reaction against virally converted lymphocytes.

Patients with infectious mononucleosis develop antibodies against EB virus, as measured by immunofluorescence with virus-bearing cells. Antibodies appear early in the acute disease, rise to peak levels within a few weeks, and remain high during convalescence. Unlike the short-lived heterophil antibodies, those against EB virus persist for years.

The role that EB virus may play in Burkitt's lymphoma (a tumour of the jaw in African children and young adults) and nasopharyngeal carcinoma (common in males of Chinese origin) is less well established. The association with EB virus is based primarily on the finding that the prevalence of antibody is greater and the antibody titters are higher among patients with Burkitt's lymphoma and nasopharyngealcarcinoma than in healthy matched controls or individuals with other types of malignancies. The significance of these associations is uncertain at present. All cells from Burkitt's lymphoma of African origin and from nasopharyngeal carcinoma carry multiple copies of the EB virus genome and express the antigen EBNA.

Students Practical activities

1. To inoculate the pig embryo kidneys cell culture by blood of the patient with suspicion on a tick-borne encephalitis.

There is pig embryo kidneys cell culture the sterile bottle. It is on the side of bottle opposite to vertical line. In sterile conditions it is necessary to pour out the medium and to fill in the bottle 1,5 ml of the defibrinated patient’s blood. To close the bottle and to put it on a horizontal surface by the line upwards for 1 h at 37 °C for adsorption of the viruses on the cells surface. After that sterilely to add in the bottle 10,0 ml of medium 199.

In 72-96 hours material is inoculating into the brain of newborn white mice for the identification of viruses.

2. To carry out neutralization test with type specific sera in the pig embryo kidneys cell culture.

The scheme of the neutralization test for viruses identification

Ingredients	Tubes
	1	2
Patient’s defibrinated blood or serum (virus-containing specimen)	0,5 ml	0,5 ml
Tick-borne encephalitis viruses antiserum	0,5 ml	–
Japanese encephalitis viruses antiserum	–	0,5 ml
Incubation 1 h, temperature 18-20 °C
Pig embryo kidneys cell culture	5,0 ml	5,0 ml
Incubation 4-7 days, temperature 37 °C
Results

3. To carry out Complement fixation test with paired sera for serological diagnosis tick-borne encephalitis.

The scheme of the Complement fixation test

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8
Isotonic sodium chloride solution	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2
Patient’s serum diluted 1:5
I	0,2	®	®	®	®	¯	–	0,2
II	0,2	®	®	®	®	¯	–	0,2
Serum dilution	1:10	1:20	1:40	1:80	1:160	1:320	–	–
Viral diagnosticum (tick-borne encephalitis viruses)	0,2	0,2	0,2	0,2	0,2	0,2	0,2	–
Complement	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature
Hemolytic system (Hemolytic serum and 3 % sheep erythrocytes suspension )	0,4	0,4	0,4	0,4	0,4	0,4	0,4	0,4
Incubation for 30-60 min, temperature 37 °C
Results
Patient’s serum
I
II

In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).

The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.

4. To carry out put Complement fixation test with specific serum against Crimean-Congo hemorrhagic fever viruses.

The scheme of Complement fixation test for laboratory diagnosis of

Crimean-Congo hemorrhagic fever

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8
Dilution of antigen	1:4	1:8	1:16	1:32	1:4	1:4	1:8	1:8
Investigated antigen	0,1	0,1	0,1	0,1	0,1	0,1	0,1	0,1
Specific serum	0,1	0,1	0,1	0,1	–	–	–	–
Non-specific serum	–	–	–	–	0,1	–	0,1	–
Isotonic sodium chloride solution	–	–	–	–	–	0,1	–	0,1
Complement (2 U)	0,1	0,1	0,1	0,1	0,1	0,1	0,1	0,1
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature
Hemolytic system	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2
Incubation for 30-60 min, temperature 37 °C
Results

In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).

5. To carry out Hemagglutination inhibition test with paired sera for diagnosis of rubella.

The scheme of Hemagglutination inhibition test for serological diagnosis of rubella

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8
Dextrose-gelatin-veronal buffer with 0,4 % of bovine albumin	0,1	0,1	0,1	0,1	0,1	0,1	0,1	0,1
Patient’s serum diluted 1:5
I	0,1	®	®	®	®	¯	0,1	–
II	0,1	®	®	®	®	¯	0,1	–
Dilution	1:10	1:20	1:40	1:80	1:160	1:320	–	–
Viral diagnosticum (Rubella virus, 4 HAU/ml)	0,1	0,1	0,1	0,1	0,1	0,1	–	0,1
Incubation for 30 min, temperature 18-20 °C
1 % suspension of chicken erythrocytes	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2
Incubation for 45 min, temperature 18-20 °C
Result: Serum I
II

Test results are assessed after complete erythrocyte sedimentation in control (7 well). In the experimental well a markedly localized erythrocytes sediment (“rouleaus”), and in the control well (8) the rapid erythrocytes agglutination with star-like, marginally festooned sediment (“umbrella”) on the bottom are observed. The titer of serum is its biggest dilution, which inhibits hemagglutination. The growth of patient’s antiviral antibodies titers at least in 4 times testifies about disease.

6. To do Complement fixation test for serological diagnosis of rotavirus infection.

The scheme of the Complement fixation test

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8
Isotonic sodium chloride solution	0,1	0,1	0,1	0,1	0,1	0,1	0,1	0,1
Patient’s serum diluted 1:5 I	0,1	®	®	®	®	¯	–	0,1
II	0,1	®	®	®	®	¯	–	0,1
Serum dilution	1:10	1:20	1:40	1:80	1:160	1:320	–	–
Viral diagnosticum (rotavirus)	0,1	0,1	0,1	0,1	0,1	0,1	0,1	–
Complement	0,1	0,1	0,1	0,1	0,1	0,1	0,1	0,1
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature
Hemolytic system	0,2	0,2	0,2	0,2	0,2	0,2	0,2	0,2
Incubation for 30-60 min, temperature 37 °C
Result : Serum I
II

In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood). The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.

7. To carry out complement fixation test with patient’s paired sera for serological diagnosis of Herpes simplex.

The scheme of Complement fixation test

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8
Isotonic sodium chloride solution	0,5	0,5	0,5	0,5	0,5	0,5	0,5	0,5
Patient’s serum diluted 1:5
I	0,5	®	®	®	®	¯	–	0,5
II	0,5	®	®	®	®	¯	–	0,5
Serum dilution	1:10	1:20	1:40	1:80	1:160	1:320	–	–
Viral diagnosticum	0,5	0,5	0,5	0,5	0,5	0,5	0,5	–
Incubation for 45 min, temperature 37 °C
Hemolytic system (Hemolytic serum and 3 % sheep erythrocytes suspension )	1,0	1,0	1,0	1,0	1,0	1,0	1,0	1,0
Incubation for 30-60 min, temperature 37 °C
Results
Patient’s serum
I
II

In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (+++ , ++), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).

The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.

The titer of antibody in the second serum must increase in 4 times as compared with the first one.

References:

1. Ronald M. Atlas. Microbiology in our World, 1995.

2. Handbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 199-204, 210-214.

3. Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P. 221-234, 244-249, 255-256.

4. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P.361-390, 433-440, 441-458, 488-498