RNA-viruses. Polyovirus, Coxsackie and ЕСНО viruses. Rhinoviruses.

Aphthovirus and enterovirus lesions in the mouth cavity.

Orthomyxoviruses. Influenza viruses. Laboratory diagnosis of flu and prophylaxis.

Paramyxoviruses. Measles and Epidemic Parotitis Viruses. Respiratory syncytial virus. Laboratory diagnosis and prophylaxis of these diseases

 

 

 

Picornaviruses are small (20-30 nm) and nonenveloped and contain a single-stranded RNA genome (MW 2-3 x 10⁶).

The nucleocapsid has cubic sym­metry and contains 32 spherical subunits (capsomeres). Virus maturation takes place in the cytoplasm. Enteroviruses and rhinoviruses commonly infect hu­mans.

 

 

Enterovirus. Enteroviruses exist in many animals, including humans, cattle, pigs, and mice. Enteroviruses of human origin include the follow­ing:

 

(1) Polioviruses, types 1-3.

(2) Coxsackie viruses of group A, types 1-24.

(3) Coxsackie viruses of group B, types 1-6.

(4) Echoviruses, types 1-34.

(5) Aphthovirus.

(6) Hepatovirus.

(7) Rhinovirus

(5) Enteroviruses, types 68-70.

 

Enteroviruses are transient inhabitants of the human alimentary tract and may be isolated from the throat or lower intestine. Rhinoviruses, on the other hand, are isolated chiefly from the nose and throat. Among the enteroviruses that are cytopathogenic (polioviruses, echoviruses, and some coxsackievi-ruses), growth can be readily obtained at 36-37 °C in primary cultures of human and monkey kidney cells and certain cell lines (such as HeLa); in contrast, most rhinovirus strains can only be recovered in cells of human origin (embryonic human kidney or lung, human diploid cell strains) at 33 °C.

The enterovirus capsid is thought to be composed of 32 morphologic subunits, possibly in the form of a rhombic triacontahedron rather than a regular icosahedron. Rhinovirus capsid architecture appears to be similar. Infective nucleic acid has been extracted from several enteroviruses and rhinoviruses.

Enteroviruses are stable at acid pH (3.0-5.0) for 1-3 hours, whereas rhinoviruses are acid-labile. En­teroviruses and some rhinoviruses are stabilized by magnesium chloride against thermal inactivation.

Enteroviruses and rhinoviruses differ in buoyant density. Enteroviruses have a buoyant density in CsCI of about 1.34 g/mL; human rhinoviruses, about 1.40 g/mL.

Rhinovirus. Human rhinoviruses include more than 100 antigenic types. Rhinoviruses of other host species in­clude those of horses and cattle.

Other Genera. Other picomaviruses are foot-and-mouth disease of cattle (Aphthovirus) and encephalomyocarditis of rodents (Cardiovirus).

The host range of the picomaviruses varies greatly from one type to the next and even among strains of the same type. They may readily be induced, by laboratory manipulation, to yield variants that have host ranges and tissue tropisms different from those of certain wild strains; this has led to the development of attenuated poliovirus strains now used as vaccines.

 

Epidemic Poliomyelitis Virus

Poliomyelitis is an acute infectious disease that in its serious form affects the central nervous system. The destruction of motor neurons in the spinal cord results in flaccid paralysis. However, most poliovirus infec­tions are subclinical.

In spite of the fact that poliomyelitis is one of the most ancient conta­gious diseases, its infectious nature was ascertained only in 1905 by O. Wickman who investigated a poliomyelitis epidemic in Sweden.

In 1908-09, K. Landsteiner and E. Popper proved poliomyelitis to be of viral aetiology. They produced a febrile disease in monkeys by inject­ing an emulsion prepared from the spinal cord of a fatal case of polio­myelitis. The animals displayed typical manifestations of poliomyelitis accompanied with flaccid paralysis. The virus was isolated in tissue cul­ture in 1949 by J. Enders.

Morphology. The virus is 30 nm in size and forms intranuc­lear inclusions. The virion is icosahedral and consists of RNA and a protein capsid containing 32 spherical subunits (capsomeres). The poliomyelitis virus has neither an outer membrane nor lipids and is therefore not sensitive to the effect of ether and sodium desoxycholate.

The poliomyelitis virus has been obtained in crystalline form. The organism is devoid of fermentative systems, it is fully dependent on host cells and is an obligatory intracellular parasite.

 

 

Animal Susceptibility and Growth of Virus. Polioviruses have a very restricted host range. Most strains will infect monkeys by direct inoculation into the brain or spinal cord. Chimpanzees and cynomolgus monkeys can also be infected by the oral route; in chimpanzees, the infection thus produced is usually asymptomatic. The animals become intestinal carriers of the virus; they also develop a viremia that is quenched by the appearance of antibodies in the cir­culating blood. Unusual strains have been transmitted to mice or chick embryos.

Most strains can be grown in primary or continu­ous cell line cultures derived from a variety of human tissues or from monkey kidney, testis, or muscle, but not in cells of lower animals. Spherical bodies which are 0.2 mcm in size and stain light-blue or violet with the Romanowsky-Giemsa stain are found in the cells of tissue cultures.

The poliomyelitis virus is also cultivated on kidney cells of green Afri­can monkeys and on diploid human cells devoid of latent SV40 viruses. The cytopathic effect is attended by destruction and the formation of granules in the infected cells.

                              Normal cell culture

                                                                               

 

Poliovirus requires a primate-specific membrane receptor for infection, and the absence of this receptor on the surface of nonprimate cells makes them virus-resistant. This restriction can be overcome by introduc­ing poliovirus into resistant cells by means of synthetic lipid vesicles called liposomes. Once inside the cell, poliovirus replicates normally.

Virus Replication. After attaching to virus receptors (which seem to be controlled in humans by genes on chromosome 19). Poliovirus RNA serves both as its own messenger RNA and as the source of the genetic information. Viral protein is synthesized on polysomes held together by viral RNA.

Guanidine in concentrations greater than 1 mM and 2-(alpha-hydroxybenzyl)-benzimidazole inhibit poliovirus multiplication in tissue culture. Guanidine acts by inhibiting the release of newly made viral RNA from the replicative complex.

Antigenic Properties. There are 3 antigenic types. Type I viruses include the strains, which are pathogenic for man and monkeys. They are identical as regards immunological properties. Type II viruses include the strains, which are responsible for diseases not only in man and monkeys, but also in rodents (cotton rats, white and grey mice, field voles, hamsters, etc.). They do not produce immunity to strains of other types of poliomyelitis virus. Type III viruses are patho­genic only for man and monkeys and differ from the first two types in their immunological properties.

During epidemic outbreaks, type I is most frequently isolated (in 65-95 per cent of cases) while types II and III account for the remaining 5-35 per cent of cases.

Paralytic forms of the disease are more frequently produced by the type I organism. It causes production of virus-neutralizing and comple­ment-fixing antibodies in human and animal bodies.

Resistance. The virus is extremely resistant to photodynamic inactivation. It survives in sterile water at room temperature for a period of more than 100 days, in milk for 90 days, in faeces in the cold for more than 6 months, and in sewage for several months. It withstands expo­sure to 0.5-1 per cent phenol solutions and remains viable for several weeks at pH 3.8-8.5.

The poliomyelitis virus is sensitive to calcium chlorate lime, chloramine, formalin, potassium permanganate, and hydrogen peroxide solu­tions. It is rapidly killed on boiling.

Pathogenesis & Pathology. The mouth is the portal of entry of the virus, and primary multiplication takes place in the oropharynx or intestine. The virus is regularly present in the throat and in the stools before the onset of illness. One week after onset there is little virus in the throat, but virus continues to be excreted in the stools for several weeks, even though high antibody levels are present in the blood.

The virus may be found in the blood of patients with abortive and nonparalytic poliomyelitis and in orally infected monkeys and chimpanzees in the pre-paralytic phase of the disease. Antibodies to the virus appear early in the disease, usually before paralysis occurs-

Viremia is also associated with type 2 oral vacci­nation. Free virus is usually present in the blood be­tween days 2 and 5 after vaccination, and virus is bound to antibody for an additional few days. Boundvirusis detected by acid treatment, which inactivates the antibody and liberates active virus.

These findings have led to the view that the virus first multiplies in the tonsils, the lymph nodes of the neck, Peyer’s patches, and the small intestine. The central nervous system may then be invaded by way of the circulating blood. In monkeys infected by the oral route, small amounts of antibody prevent the paralytic disease, whereas large amounts are necessary to pre­vent passage of the virus along nerve fibers. In humans also, antibody in the form of pooled human gamma globulin may prevent paralysis if given before expo­sure to the virus.

Poliovirus can spread along axons of peripheral nerves to the central nervous system, and there it continues to progress along the fibers of the lower motor neurons to increasingly involve the spinal cord or the brain. This may occur in children after tonsillec-tomy. Poliovirus present in the oropharynx may enter nerve fibers exposed during the surgical procedure and spread to the central nervous system.

Poliovirus invades certain types of nerve cells, and in the process of its intracellular multiplication it may damage or completely destroy these cells. The anterior horn cells of the spinal cord are most promi­nently involved, but in severe cases the intermediate gray ganglia and even the posterior horn and dorsal root ganglia are often involved. In the brain, the reticu-lar formation, vestibular nuclei, and deep cerebellar nuclei are most often affected. The cortex is virtually spared, with the exception of the motor cortex along the precentral gyms.

Poliovirus does not multiply in muscle in vivo. The changes that occur in peripheral nerves and volun­tary muscles are secondary to the destruction of nerve cells. Changes occur rapidly ierve cells, from mild chromatolysis to neuronophagia and complete destruc­tion. Cells that lose their function may recover com­pletely. Inflammation occurs secondary to the attack on the nerve cells; the focal and perivascular infiltra­tions are chiefly lymphocytes, with some polymor-phonuclear cells, plasma cells, and microglia.

In addition to pathologic changes in the nervous system, there may be myocarditis, lymphatic hyperplasia, ulceration of Peyer’s patches, prominence of follicles, and enlargement of lymph nodes,

Clinical Findings. When an individual susceptible to infection is exposed to the virus, one of the following responses may occur: (1) inapparent infection without symp­toms, (2) mild illness, (3) aseptic meningitis, (4) paralytic poliomyelitis. As the disease progresses, one response may merge with a more severe form, often resulting in a biphasic course: a minor illness, followed first by a few days free of symptoms and then by the major, severe illness. Only about 1% of infections are recognized clinically.

The incubation period is usually 7-14 days, but it may range from 3 to 35 days.

Abortive Poliomyelitis. This is the com­monest form of the disease. The patient has only the minor illness, characterized by fever, malaise, drowsiness, headache, nausea, vomiting, constipation, and sore throat in various combinations. The patient recov­ers in a few days. The diagnosis of abortive poliomyelitis can be made only when the virus is isolated or antibody development is measured.

Nonparalytic Poliomyelitis (Aseptic Meningitis). In addition to the above symptoms and signs, the patient with the nonparalytic form presents stiffness and pain in the back and neck. The disease lasts 2-10 days, and recovery is rapid and complete. In a small percentage of cases, the disease advances to paralysis. Poliovirus is only one of many viruses that produce aseptic meningitis.

Paralytic Poliomyelitis. The major illness usually follows the minor illness described above, but it may occur without the antecedent first phase. The predominating complaint is flaccid paralysis resulting from lower motor neuron damage. However, incoordination secondary to brain stem invasion and painful spasms of nonparalyzed muscles may also occur. The amount of damage varies greatly. Muscle involvement is usually maximal within a few days after the paralytic phase begins. The maximal recovery usually occurs within 6 months, with residual paralysis lasting much longer.

Laboratory Diagnosis

Cerebrospinal Fluid. The cerebrospinal fluid contains an increased number of leukocytes—usually 10-200/mcL, seldom more than 500/mcL. In the early stage of the disease, the ratio of polymorphonuclear cells to lymphocytes is high, but within a few days the ratio is reversed. The total cell count slowly subsides to normal levels. The protein content of the cerebrospinal fluid is elevated (average, about 40-50 mg/dL), but high levels may occur and persist for weeks. The glucose content is normal.

Recovery of Virus. Cultures of human or monkey cells may be used. The virus may be recovered from throat swabs taken soon after onset of illness and from rectal swabs or feces collected for longer periods. The virus has been found in about 80% of patients during the first 2 weeks of illness but in only 25% during the third 2-week period. No permanent carriers are known. Recovery of poliovirus from the cerebro­spinal fluid is uncommon, unlike that of the coxsackieviruses or echoviruses.

In fatal cases, the virus should be sought in the cervical and lumbar enlargements of the spinal cord, in the medulla, and in the colon contents. Histologic examination of the spinal cord and parts of the brain should be made. If paralysis has lasted 4-5 days, it is difficult to recover the virus from the cord.

Specimens should be kept frozen during transit to the laboratory. After treatment with antibiotics, cell cultures are inoculated, incubated, and observed. Cytopathogenic effects appear in 3-6 days. An iso­lated virus is identified and typed by neutralization with specific antiserum.

Serology. Paired serum specimens are re­quired to show a rise in antibody titer.

During poliomyelitis infection, complement-fixing H antibodies form before N antibodies. The level of H an­tibodies declines first. Early acute stage sera thus con­tain H antibodies only; 1-2 weeks later, both N and H antibodies are present; in late convalescent sera, only N antibodies are present. Only first infection with poliovirus produces strictly type-specific complement fixation responses. Subsequent infections with heterotypic polioviruses recall or induce antibodies, mostly against the heat-stable group antigen shared by all 3 types of poliovirus.

Neutralizing antibodies appear early and are usu­ally already detectable at the time of hospitalization. If the first specimen is taken sufficiently early, a rise in titer can be demonstrated during the course of the disease.

Type-specific, short-lived virus-precipitating an­tibodies develop in convalescence. The microprecipitation test is less useful than the CF and Nt tests.

 

Immunity

Immunity is permanent to the type causing the infection. There may be a low degree of heterotypic resistance induced by infection, especially between type 1 and type 2 polioviruses.

Passive immunity is transferred from mother to offspring. The maternal antibodies gradually disappear during the first 6 months of life. Passively administered antibody lasts only 3-5 weeks.

Virus neutralizing antibody forms soon after ex­posure to the virus, often before the onset of illness, and apparently persists for life. Its formation early in the disease implies that viral multiplication occurs in the body before the invasion of the nervous system. As the virus in the brain and spinal cord is not influenced by high titers of antibodies in the blood (which are found in the preparalytic stage of the disease), im­munization is of value only if it precedes the onset of symptoms referable to the nervous system.

Operations on the oropharynx and tonsillectomy enhance the likelihood of central nervous system in­volvement during prevalence of polioviruses in the community. This may be attributable to the access of cut nerve fibers to virus in the pharynx or to the removal of immunologically active lymphoid tissue.

 

Treatment

There is no specific treatment.Treatment involves reduction of pain and muscle spasm and maintenance of respiration and hydration. When the fever subsides, early mobilization and active exercise are begun. There is no role for antiserum. Early injections of gamma-globulin, blood transfusion, wide use of vitamins C and B,-, amino acids (leucine, glutamic acid), analgesics (analgine, amidopyrine, pantopon, etc.), mediators, and stimulants (proserine, galanthamine, dibazol, etc.) are recommended. An orthopaedic regimen is set up from the first day that paralysis develops to prevent contractures and deformations, and exercise therapy is carried out during the rehabilitation period. An apparatus for artificial respiration is employed when there are respi­ration disturbances.

 

Epidemiology

Poliomyelitis occurs world-wide — year-round in the tropics and during summer and fall in the temperate zones. Winter outbreaks are rare.

The disease occurs in all age groups, but children are usually more susceptible than adults because of the acquired immunity of the adult population. In isolated populations (Arctic Eskimos), poliomyelitis attacks all ages equally. In underdeveloped areas, where condi­tions favour the wide dissemination of virus, poliomye­litis continues to be a disease of infancy. In developed countries, before the onset of vaccination, the age distribution shifted so that most patients were over age 5 and 25% were over age 15 years. With rising levels of hygiene and sanitation, a similar trend is now occur­ring in developing countries. Since poliomyelitis in older persons is more likely to be a clinically manifest infection rather than subclinical one, the reported inci­dence of clinical disease is actually rising in areas where vaccination is not widespread, and outbreaks of poliomyelitis are being recorded in some such areas.

The case fatality rate is variable. It is highest in the oldest patients and may reach 5-10%.

Humans are the only known reservoir of infec­tion. Under conditions of poor hygiene and sanitation in warm areas, where almost all children become im­mune early in life, polioviruses maintain themselves by continuously infecting a small part of the popula­tion. In temperate zones with high levels of hygiene, epidemics have been followed by periods of little spread of virus, until sufficient numbers of susceptible children have grown up to provide a pool for transmis­sion in the area. Warm weather favours the spread of virus by increasing human contacts, the susceptibility of the host, or the dissemination of virus by extrahuman sources. Virus can be recovered from the pharynx and intestine of patients and healthy carriers. The prevalence of infection is highest among household contacts. When the first case is recognized in a family, all susceptibles in the family are already infected, the result of rapid dissemination of virus,

During periods of wide prevalence of poliovirus in an area, flies become contaminated and may distribute virus to food. The role of flies in disease transmission is unsettled. Virus is present in sewage during such periods and can serve as a source of con­tamination of flies or water used for drinking, bathing, or irrigation.

In temperate climates, infection with enteroviruses, including polio, occurs mainly during the summer. There is a direct correlation between poor hygiene, sanitation, and crowding and the acquisition of infection and antibodies at an early age.

 

Prevention & Control

Both live and killed virus vaccines are available. Formalinized vaccine (Salk) is prepared from virus grown in monkey kidney cultures. At least 4 inocula­tions over a period of 1-2 years are recommended in the primary series. A booster immunization is neces­sary every 2-3 years to maintain immunity. Killed vaccine induces humoral antibodies, but, upon expo­sure, virus is still able to multiply in the gut.

Oral vaccines contain live attenuated virus now grown in human diploid cell cultures. The vaccine is stabilized by molar MgCl₂ so that it can be kept with­out losing potency for a year at 4 °C and for a month at room temperature. Nonstabilized vaccine must be kept frozen until used.

The live poliovaccine multiplies, infects, and thus immunizes. In the process, infectious progeny of the vaccine virus are disseminated in the community. Although the viruses, particularly type 3 and type 2, mutate in the course of their multiplication in vacci­nated children, only extremely rare cases of paralytic poliomyelitis have occurred in recipients of oral poliovaccine or their close contacts. Repeat vaccina­tions seem to be important to establish permanent immunity. The vaccine produces not only IgM and IgG antibodies in the blood but also secretory IgA an­tibodies in the intestine, which then becomes resistant to reinfection.

A potential limiting factor for oral vaccine is that of interference. The alimentary tract of the child may be infected with another enterovirus at the time the vaccine is fed. This interferes with the establishment of infection and immunity and is an important problem in areas (particularly in tropical regions) where en­terovirus infections are common.

Trivalent oral poliovaccine is used in the USA. The American Academy of Pediatrics recommends that primary immunization of infants begin at 2 months of age simultaneously with the first DTP inoculation. The second and third doses should be given at 2-month intervals thereafter, and a fourth dose at 1¹/₂ years of age. A trivalent vaccine booster is recommended for all children entering elementary school. No further boosters are presently recom­mended.

Adults residing in the continental USA have only a small risk of exposure. However, adults who are at increased risk because of contact with a patient or who are anticipating travel to an endemic or epidemic area should be immunized. Pregnancy is neither an indica­tion for nor a contraindication to required immuniza­tion.

Both killed and live virus vaccines induce an­tibodies and protect the central nervous system from subsequent invasion by wild virus. Low levels of anti­body resulting from killed vaccine have little effect on intestinal carriage of virus. The gut develops a far greater degree of resistance after live virus vaccine, which seems to be dependent on the extent of initial vaccine virus multiplication in the alimentary tract rather than on serum antibody level.

Live vaccine should not be administered to immunodeficient or immunosuppressed individuals. Only killed (Salk) vaccine is to be used.

On very rare occasions, a live vaccine strain can induce neurologic or paralytic disease in persons who are not evidently immunodeficient. Such cases are carefully studied by public health agencies, and it is estimated that there has been one vaccine-associated case for every 10 million persons vaccinated.

Immune human serum globulin (gamma globu­lin), 0.3 mL/kg, can provide protection for a few weeks against the paralytic disease but does not pre­vent subclinical infection. Gamma globulin is effec­tive only if given shortly before infection; it is of no value after clinical symptoms develop.

The prevention of poliomyelitis depends on vac­cination. Quarantine of patients or intimate contacts is ineffective in controlling the spread of the disease. This is understandable in view of the large number of inapparent infections that occur.

During epidemic periods (defined now as 2 or more local cases caused by the same type in any 4-week period), children with fever should be placed at bed rest. Undue exercise or fatigue, elective nose and throat operations, or dental extractions should be avoided. Food and human excrement should be pro­tected from flies. Once the poliovirus type responsible for the epidemic is determined, oral poliovaccine should be administered to susceptible persons in the population.

Patients with poliomyelitis can be admitted to general hospitals provided appropriate isolation pre­cautions are employed. All pharyngeal and bowel discharges are considered infectious and should be disposed of quickly and safely.

 

COXSACKIEVIRUSES

The coxsackieviruses comprise a large subgroup of the enteroviruses. They produce a variety of ill­nesses in human beings, including aseptic meningitis, herpangina, pleurodynia, hand, foot, and mouth disease, myo- and pericarditis, common colds, and possibly diabetes. Coxsackieviruses have been divided into 2 groups, A and B, having different pathogenic potentials for mice. Coxsackie B viruses are the most commonly identified causative agents of viral heart disease in humans.

 

Properties of the Viruses

General Properties: Coxsackieviruses are typical enteroviruses, with a diameter of 28 nm.

Animal Susceptibility and Growth of Virus. Coxsackieviruses are highly infective for newborn mice. Certain strains (Bl-6, A7, 9, 16) also grow in monkey kidney cell culture. Some group A strains grow in human amnion and human embryonic lung fibroblast cells. Chimpanzees and cynomolgus mon­keys can be infected subclinically; virus appears in the blood and throat for short periods and is excreted in the feces for 2-5 weeks. Type A14 produces poliomyelitislike lesions in adult mice and in monkeys, but in suckling mice this type produces only myositis. Type A7 strains produce paralysis and severe central ner­vous system lesions in monkeys.

Group A viruses produce widespread myositis in the skeletal muscles of newborn mice, resulting in flaccid paralysis without other observable lesions. Group B viruses may produce focal myositis, encepha­litis, and, most typically, necrotizing steatitis involv­ing mainly fetal fat lobules. The genetic makeup of inbred strains determines their susceptibility to coxsackie B viruses. Some B strains also produce pancreatitis, myocarditis, endocarditis, and hepatitis in both suckling and adult mice. Corticosteroids may enhance the susceptibility of older mice to infection of the pancreas. Normal adult mice tolerate infections with group B coxsackieviruses. However, severely malnourished or immunodeficient mice have greatly enhanced susceptibility.

Antigenic Properties: At least 32 different immunologic types of coxsackieviruses are now rec­ognized; 26 are listed as group A and 6 as group B types.

Resistance. Coxsackie and ECHO viruses possess relatively high resistance. They survive for a long period of time in a frozen state at -70° C. In glycerin and horse serum at room temperature they persist for 70 days. In a refrigerator they survive for more than a year.

The Coxsackie viruses resemble the poliomyelitis viruses in that they are resistant to various concentrations of hydrogen ions. They survive at pH 2.3-9.4 for 24 hours and at pH 4.0-8.0 for 7 days. They are resis­tant to antibiotics, 70° ethyl alcohol, and 5 per cent lysol solutions but are extremely sensitive to solutions of hydrochloric acid and formalde­hyde. A temperature of 50-55° C kills the viruses in 30 minutes.

Pathogenesis & Pathology. Virus has been recovered from the blood in the early stages of natural infection in humans and of experimental infection in chimpanzees. Virus is also found in the throat for a few days early in the infection and in the stools for up to 5-6 weeks. The distribution of virus is similar to that found with the other en­teroviruses.

Group B coxsackieviruses may cause acute fatal encephalomyocarditis in infants. This appears to be a generalized systemic disease with virus replication and lesions in the central nervous system, heart muscle, and other organs.

Clinical Findings. The incubation period of coxsackievirus infection ranges from 2 to 9 days. The clinical manifestations of infection with various coxsackie viruses are diverse and may present as distinct disease entities.

A. Herpangina: This disease is caused by certain group A viruses (2, 4, 5, 6, 8, 10), There is an abrupt onset of fever, sore throat, anorexia, dysphagia, vom­iting, or abdominal pain. The pharynx is usually hyperaemic, and characteristic discrete vesicles occur on the anterior pillars of the fauces, the palate, uvula, tonsils, or tongue. The illness is self-limited and most frequent in small children.

B. Summer Minor Illnesses: Coxsackieviruses are often isolated from patients with acute febrile ill­nesses of short duration that occur during the summer or fall and are without distinctive features.

C. Pleurodynia (Epidemic Myalgia, Bornholm Disease): This disease is caused by group B viruses. Fever and chest pain are usually abrupt in onset but are sometimes preceded by malaise, head­ache, and anorexia. The chest pain may be located on either side or substernally, is intensified by movement, and may last from 2 days to 2 weeks. Abdominal pain occurs in approximately half of cases, and in children this may be the chief complaint. The illness is self-limited, and recovery is complete, although relapses are common.

D. Aseptic Meningitis and Mild Paresis: This syndrome is caused by all types of group B cox­sackieviruses and by coxsackie viruses A7, A9, and A24. Fever, malaise, headache, nausea, and abdomi­nal pain are common early symptoms. Signs of meningeal irritation, stiff neck or back, and vomiting may appear 1-2 days later. The disease sometimes progresses to mild muscle weakness suggestive of paralytic poliomyelitis. Patients almost always recover completely from nonpoliovirus paresis. Early in asep­tic meningitis, the cerebrospinal fluid shows pleocytosis (up to 500 cells/mcL) with up to 50% polymorphonuclear neutrophils.

E. Neonatal Disease: Neonatal disease may be caused by group B coxsackieviruses. with lethargy, feeding difficulty, and vomiting, with or without fever. In severe cases, myocarditis or pericarditis can occur within the first 8 days of life; it may be preceded by a brief episode of diarrhea and anorexia. Cardiac and respiratory embarrassment are indicated by tachycardia, dyspnea, cyanosis, and changes in the electrocardiogram. The clinical course may be rapidly fatal, or the patient may recover completely. The disease may sometimes he acquired transplacentally. Myocarditis has also been caused by some group A coxsackieviruses.

F. Colds: A number of the enteroviruses have been associated with common colds; among these are coxsackieviruses A10, A21, A24, and B3.

G. Hand, Foot, and Mouth Disease: This disease has been associated particularly with coxsack-ievirus A16, but A4, A5, A7, A9, and A10 have also been implicated. Virus may be recovered riot only from the stool and pharyngeal secretions but also from vesicular fluid-

The syndrome is characterized by oral and pharyngeal ulcerations and a vesicular rash of the palms and soles that may spread to the arms and legs. Vesicles heal without crusting, which clinically differ­entiates them from the vesicles of herpes- and pox-viruses. The rare deaths are caused by pneumonia.

H. Myocardiopathy: Coxsackie virus B infec­tions are increasingly recognized as a cause of primary myocardial disease in adults as well as children. Cox­sackieviruses of group A and echoviruses have been implicated to a lesser degree.

I. Acute Hemorrhagic Conjunctivitis: Coxsackievirus A24 is one of the agents that can cause this disease.

J. Diabetes Mellitus: Serologic studies suggest an association of diabetes of abrupt onset with past infection by Coxsackievirus B4 and perhaps other members of the B group. Experimental studies support the findings in humans. Another picornavirus, encephalomyocarditis virus, induces lesions in mice in the pancreatic islets of Langerhans as well as an ac­companying diabetes.

K. Swine Vesicular Disease: The agent of this disease is an enterovirus that antigenically is related to Coxsackievirus B5. Furthermore, the swine virus can also infect humans.

 

At autopsy, virus has been demonstrated in the myocardium, endocardium, and pericardial fluid by immunofluorescence, peroxidase-labeled antibody, or ferritin-labeled antibody. About 5% of all symptoma­tic Coxsackie virus infections induce heart disease. The virus may affect the endocardium, pericardium, myocardium, or all three. Acute myocardiopathies have been shown to be caused by coxsackieviruses A4, A14, Bl-5, and others, and also by echovirus types 9 and 22 and others.

Monkeys infected with Coxsackie virus B4 de­velop pancarditis, with a pathologic picture strikingly similar to that of rheumatic heart disease,

In experimental animals, the severity of acute viral myocardiopathy is greatly increased by vigorous exercise, hydrocortisone, alcohol consumption, preg­nancy, and undernutrition and is greater in males than in females. In human illnesses, these factors may simi­larly increase the severity of the disease.

 

Laboratory Diagnosis

Recovery of Virus. The virus is isolated readily from throat washings during the first few days of illness and in the stools during the first few weeks. In Coxsackievirus A21 infections, the largest amount of virus is found iasal secretions. In cases of aseptic meningitis, strains have been recovered from the cerebrospinat fluid as well as from the alimentary tract. In hemorrhagic conjunctivitis cases, A24 virus is isolated from eye washings.

Specimens are inoculated into tissue cultures and also into suckling mice. In tissue culture, a cytopathic effect appears within 5-14 days. In suckling mice, signs of illness appear usually within 3-8 days with group A strains and 5-14 days with group B strains.

The virus is identified by the pathologic lesions it produces and by immunologic means.

Serology. Neutralizing antibodies, which are detected, appear early during the course of infection. Nt antibodies tend to be specific for the infecting virus and persist for years. CF an­tibodies exhibit cross-reactions and disappear in 6 months. Serologic tests are difficult to evaluate (be­cause of the multiplicity of types) unless the antigen used in the test has been isolated from a specific patient or during an epidemic outbreak.

Serum antibodies can also be detected and titrated by the immunofluorescence technique, using infected cell cultures on coverslips as antigens. These can be preserved frozen for years.

Immunity. In humans, Nt and CF antibodies are transferred passively from mother to fetus. Adults have antibodies against more types of coxsackieviruses than do chil­dren, which indicates that multiple experience with these viruses is common and increases with age.

 

Epidemiology

Viruses of the coxsackie group have been encoun­tered around the globe. Isolations have been made mainly from human feces, pharyngeal swabbings, sewage, and flies. Antibodies to various coxsackievi­ruses are found in serum collected from persons all over the world and in pooled gamma globulin.

Coxsackieviruses are recovered much more fre­quently during the summer and early fall. Also, chil­dren develop neutralizing and complement-fixing an­tibodies during the summer, indicating infection by these agents during this period. Such children have much higher incidence rates for acute, febrile minor illnesses during the summer than children who fail to develop Coxsackievirus antibodies.

Familial exposure is important in the acquisition of infections with coxsackieviruses. Once the virus is introduced into a household, all susceptible persons usually become infected, although all do not develop clinically apparent disease.

In herpangina, only about 30% of infected per­sons within households develop faucial lesions. Others may present a mild febrile illness without throat le­sions. Virus has been found in 85% of patients with herpangina, in 65% of their neighbours, in 40% of family contacts, and in 4% of all persons in the com­munity.

The coxsackieviruses share many properties with the echo-and polioviruses. Because of their epidemiclogic similarities, enteroviruses may occur together in nature, even in the same human host or the same specimens of sewage or flies.

 

ECHOVIRUSES

The echoviruses (enteric cytopathogenic human orphan viruses) are grouped together because they infect the human enteric tract and because they can be recovered from humans only by inoculation of certain tissue cultures. Over 30 serotypes are known, but not all cause human illness. Aseptic meningitis, febrile illnesses with or without rash, common colds, and acute hemorrhagic conjunctivitis are among the diseases caused by echoviruses.

Properties of the Viruses

General Properties. Echoviruses are typical enteroviruses measuring 24-30 nm.

Growth of Virus. Monkey kidney cell culture is the method of choice for the isolation of these agents. Some also multiply in human amnion cells and cell lines such as HeLa.

Certain echoviruses agglutinate human group 0 erythrocytes. The hemagglutinins are associated with the infectious virus particle but are not affected by neuraminidase.

Initially, echoviruses were distinguished from coxsackieviruses by their failure to produce pathologic changes iew-born mice, but echovirus-9 can produce paralysis iew-born mice. Conversely, strains of some coxsackievirus types (especially A9) lack mouse pathogenicity and thus resemble echoviruses. This var­iability in biologic properties is the chief reason why new enteroviruses are no longer being subclassified as echo- or coxsackieviruses,

Antigenic Properties. Over 30 different antigenic types have been identified. The different types may be separated on the basis of cross-Nt or cross-CF tests. Variants exist that do not behave exactly like the prototypes. After human infections, Nt antibodies per­sist longer than CF antibodies.

Animal Susceptibility. To be included in the echo group, prototype strains must not produce disease in suckling mice, rabbits, or monkeys. In the chimpan­zee, no apparent illness is produced, but infection can be demonstrated by the presence and persistence of virus in the throat and in the feces and by the type-specific antibody responses.

Pathogenesis & Pathology. The pathogenesis of the alimentary infection is similar to that of the other enteroviruses. Virus may be recovered from the throat and stools: in certain types (4, 5. 6, 9, 14. and IS) associated with aseptic menin­gitis, the virus has been recovered from the cerebrospinal fluid.

Clinical Findings. To establish etiologic association of echovirus with disease, the following criteria are used; (1) There is a much higher rate of recovery of virus from patients with the disease than from healthy individuals of the same age and socioeconomic level living in the same area at the same time. (2) Antibodies against the virus develop during the course of the disease. If the clinical syndrome can be caused by other known agents, then virologic or serologic evidence must be negative for concurrent infection with such agents. (3) The virus is isolated from body fluids or tissues manifesting le­sions, e.g., from the cerebrospinal fluid in cases of aseptic meningitis.

Echoviruses 4, 6, 9, 11, 14, 16, 18, and others have been associated with aseptic meningitis. Rashes are common in types 9, 16 (“Boston exanthem disease”), 18, and 4. Rashes are commonest in young children. Occasionally, there is conjunctivitis, muscle weakness, and spasm (types 6,9, and others). Infantile diarrhea may be associated with some types (e.g., 18, 20). Echovirus type 28 isolated from upper respiratory illness causes “colds” in volunteers and has been reclassified as rhinovirus type 1. For many echoviruses (and some coxsackieviruses), no disease entities have been defined.

With the virtual elimination of polio in developed countries, the central nervous system syndromes as­sociated with echo- and coxsackieviruses have as­sumed greater prominence. The latter in children under age 1 may lead to neurologic sequelae and mental impairment. This does not appear to happen in older children.

Laboratory Diagnosis

It is impossible in an individual case to diagnose an echovirus infection on clinical grounds. However, in the following epidemic situations, echoviruses must be considered: (1) summer outbreaks of aseptic meningitis; (2) summer epidemics, especially in young children, of a febrile illness with rash; and (3) out­breaks of diarrheal disease in young infants from whom no pathogenic enterobacteria can be recovered.

The diagnosis is dependent upon laboratory tests. The procedure of choice is isolation of virus from throat swabs, stools, rectal swabs, and, in aseptic meningitis, cerebrospinal fluid. Serologic tests are impractical — because of the many different virus types — unless a virus has been isolated from a patient or during an outbreak, of typical clinical illness. Nt and HI antibodies are type-specific and may persist for years. CF antibodies give many heterotypic responses.

If an agent is isolated in tissue culture, it is tested against different pools of antisera against en­teroviruses. Determination of the type of virus present depends upoeutralization by a single serum. Infec­tion with 2 or more enteroviruses may occur simulta­neously.

Epidemiology. The epidemiology of echoviruses is similar to that of other enteroviruses. They occur in all parts of the globe. Unlike the enterobacteria, which are constantly present in the intestinal tract, the enteroviruses pro­duce only transitory infections. They are more apt to be found in the young than in the old. In the temperate zone, infections occur chiefly in summer and autumn and are about 5 times more prevalent in children of lower income families than in those living in more favourable circumstances.

Control. Avoidance of contact with patients exhibiting acute febrile illness, especially those with a rash, is advisable for very young children. Members of institu­tional staffs responsible for caring for infants should be tested to determine whether they are carriers of en­teroviruses. This is particularly important during out­breaks of diarrheal disease among infants.

 

OTHER ENTEROVIRUS TYPES

Four enteroviruses (types 68-71) grow in mon­key kidney cultures, and 3 of them cause human disease.

Enterovirus 68 was isolated from the respiratory tracts of children with bronchiolitis or pneumonia.

Enterovirus 70 is the chief cause of acute hemorrhagic conjunctivitis. Acute hemorrhagic conjunctivitis has a sudden onset of subconjunctival hemorrhage ranging from small petechiae to large blotches covering the bulbar conjunctiva. There may also be epithelial keratitis and occasionally lumbar radiculomyelopathy. The disease is com­monest in adults, with an incubation period of 1 day and a duration of 8-10 days. Complete recovery is the rule. The virus is highly communicable and spreads rapidly under crowded or unhygienic conditions. There is no effective treatment.

Enterovirus 71 was isolated from patients with meningitis, encephalitis, and paralysis resembling po­liomyelitis. It continues to be one of the main causes of central nervous system disease, sometimes fatal, around the world. In some areas, particularly in Japan and Sweden, the virus has caused outbreaks of hand, foot, and mouth disease.

 

 

Aphthovirus

The genus Aphthovirus consists of three species, Foot-and-mouth disease virus, Equine rhinitis A virus. and Bovine rhinitis B virus. Recent nucleotide sequence data has shown that bovine rhinoviruses (three serotypes previously classified as tentative members of the now defunct genus Rhinovirus) should be re-classified in the genus Aphthovirus.

Current taxonomy

Genus: Aphthovirus

Species:
  Foot-and-mouth disease virus (7 serotypes)
  Equine rhinitis A virus (1 serotype)
  Bovine rhinitis B virus (formerly bovine rhinovirus 2) (1 serotype)

Unassigned members of the genus:
  Bovine rhinovirus 1
  Bovine rhinovirus 3

 

Aphthovirus (from the Greek aphtha-, vesicles in the mouth) is a viral genus of the family Picornaviridae. Aphthoviruses infect vertebrates, and include the causative agent of foot-and-mouth disease. Foot-and-mouth disease virus (FMDV) is the prototypic member of the Aphthovirus genus.^[1] There are seven FMDV serotypes: A, O, C, SAT 1, SAT 2, SAT 3 and Asia 1 belonging to the species FMDV, and one non-FMDV serotype, equine rhinitis A virus (ERAV) belonging to the species Equine rhinitis A virus.

 

Morphology and Genome Structure

Aphthoviruses are non-enveloped and have an icosahedral nucleocapsid with a diameter of around 27 to 30 nm. The aphthoviruses are differentiated from other picornaviruses as they have a larger genome (ca. 8.2 kilobases.) The genome is linear and non-segmented. It consists of a single molecule of (+) sense RNA, with a 5′ genome linked protein (VPg), which is associated to the genome via a phosphodiester bond linked to a tyrosine residue. The 5′ endof the genome has a ‘poly C’ region, while the 3′ end is polyadenylated.

The virions consist of a non-enveloped virus capsid, which is round and displays icosahedral symmetry.

Aphthovirus

Replication, Ecology and Pathology

Aphthoviruses replicate in a similar fashion to all picornaviruses. Replication is cytoplasmic and initially involves attachment of the exogenous virus to the cell membrane. Attachment to the membrane and subsequent entry into the cell is mediated by a membrane receptor. After genome replication within the cytoplasm, virion assembly occurs and new virus particles aggregate within the cell. Release of virus particles is mediated by cell lysis.

REPLICATION

CYTOPLASMIC

1.     Virus attaches to host receptors, formation of a coated vesicle.

2.     Uncoating, and release of the viral genomic RNA into the cytoplasm possibly through the formation of a pore in the host cell membrane.

3.     VPg is removed from the viral RNA, which is then translated into a processed polyprotein.

4.     Shutoff of cellular cap-dependent translation through the cleavage of the translation initiation factor eIF4G by the leader protease.

5.     Replication of viral RNA takes place on membrane vesicles derived from the ER. A negative-sense complementary ssRNA is synthesized using the genomic RNA as a template.

6.     New genomic RNA synthesized using the negative-sense RNA as a template is believed to be packaged into preformed procapsids.

7.     Cell lysis and virus release.

8.     Maturation of provirions by an unknown host protease.

 

Aphthoviruses include the causative agents of foot-and-mouth disease (FMD), which primarily affects livestock such as cattle, swine, sheep and goats. FMD was first discovered in Italy in the 16th century. Since then, the prevalence of the disease has remained, despite many countries being declared FMD-free. Endemic regions of the disease include areas of Europe, Asia and South America. The virus commonly persists in animal feed and is able to survive environmentally for up to one month. Eradication of FMD in endemic areas has been difficult, despite the availability of a vaccine

Equine rhinitis A virus (ERAV) was first isolated from horses in the 1960s and 1970s showing acute febrile respiratory disease signs, including fever, cough, clear nasal discharge and lethargy. Given its similarity to the common cold in humans (caused by another picornavirus, rhinovirus), ERAV was initially named “equine rhinovirus 1”. Modern molecular biology techniques such as nucleotide sequencing demonstrated that ERAV was in fact more closely related to FMDV, and was reclassified to the aphthovirus genus.

Foot and Mouth Disease

Foot-and-mouth disease is one of the most contagious animal diseases and causes death mainly to young animals through inflammation of the heart muscle (i.e. myocarditis). It infects animals in the families Bovidae (cattle, sheep, goats, antelope) and Suidae (pigs, warthogs).  Symptoms include the formation of blisters in the mouth and between the claws, hence the name. People can be infected through skin wounds or the mucous membranes in the mouth by handling diseased stock, handling the virus in the laboratory, or by drinking infected milk, but not by eating meat from infected animals. However, infection of humans is temporary and mild and foot-and-mouth disease is not considered a public health problem. 

Transmission. Infection can spread all too easily through direct or indirect contact. This can be through movement of humans, animals, vehicles and implements, and through the air, especially in temperate zones (up to 60 km overland and 300 km by sea). Cattle are mainly infected by inhalation, often from pigs, which excrete large amounts of virus through their breathing and are considered highly important in disease spread. Aphthovirus, which causes this disease, is in the same family of viruses as the Common Cold viruses (Rhinovirus) and you know just how easily they can spread! Newly infected animals start spreading the virus before symptoms appear so it is often difficult to control this disease before it has spread too far. African Buffalo are important carriers of foot-and-mouth disease. Recovered cattle may be carriers for 18 to 24 months; sheep for 1 to 2 months, while pigs are not carriers. 

Distribution. Foot-and-mouth disease is endemic in parts of Asia, Africa, the Middle East and South America with sporadic outbreaks in free areas.  Foot-and-mouth disease was last reported in 1929 in the U.S.A., 1952 in Canada, and 1954 in Mexico. There have been a notably high number of outbreaks recently, the most publisised being that in the UK.  Foot-and-mouth disease was reported from the following countries from 1999 to March 2001: Argentina, Bhutan, Brazil, Colombia, Egypt, France, Georgia, Greece, Iran, Israel, Japan, Kazakhstan, Korea, Kuwait, Malawi, Malaysia, Mauritania, Mongolia, Namibia, Peru, Philippines, Russia, Saudi Arabia, South Africa, Swaziland, Taipei China, Tajikistan, Turkey, United Kingdom / Great Britain, Northern Ireland (isolated case stemming from outbreak in England), Uruguay, Zambia and Zimbabwe (information from OIE). The outbreak in KwaZulu-Natal in South Africa was first detected on 12 September 2000.

Symptoms. Fever (pyrexia), lack of appetite (anorexia), reduction in milk production for 2-3 days.  The blisters that form in the mouth and nasal cavities make the animal smack its lips, grind its teeth and drool. The blisters that form between the claws cause lameness, stamping and kicking of feet. In sheep and goats the blistering is less pronounced. 

Prevention and Control. 

·      Animal movement control through construction of border fences and surveillance of trucks, etc where roads and railways pass through these borders.

·      Slaughter of infected, recovered, and susceptible contact animals.

·      Disinfection of premises and infected materials (implements, cars, clothes, etc.).

·      Destruction of cadavers and susceptible animals products in the infected areas.

·      Quarantine measures. The incubation period is 2-21 days (average 3-8 days) so animals passing out of infected areas would presumably need to be held in quarantine for at least 14 days before the disease could be reliably tested.  

·      Vaccination. Tests for foot-and-mouth disease involve testing for antibodies to this disease and as both the real disease and the vaccination lead to the build up of antibodies, vaccinated animals are excluded because of the risk of bringing in animals that actually are infected. However, new tests are currently being developed that will enable differentiation of vaccinated and infected livestock.  The rationale behind this method is as follows: (1) the vaccination involves injecting the animal with inactivated viral particles made up of structural proteins; (2) the animal develops antibodies against these structural proteins; (3) the live viral particles include structural proteins as well as non-structural proteins which are produced during viral replication; (4) the animal develops antibodies against both these forms of proteins when infected by the live virus; (5) the test therefore distinguishes each of these main types of antibodies – if only the antibodies against structural proteins are detected, then there can be certainty that it is vaccinated and not infected. 

 

 

RHINOVIRUS GROUP

Rhinoviruses are isolated commonly from the nose and throat but very rarely from feces. These viruses, as well as coronaviruses and some reo-,   adeno-, entero-, parainfluenza, and influenza viruses, cause upper respiratory tract infections, including the “common cold.”

General Properties: Rhinoviruses are picornaviruses similar to enteroviruses but differing from them in having a CsCI buoyant density of 1.40 g/mL and in being acid-labile.

Animal Susceptibility and Growth of Virus. These viruses are infectious only for humans and chimpanzees. They have been grown in cultures of human embryonic lung fibroblasts (WI-38) and in organ cultures of ferret and human trachea! epithelium. They are grown best at 33 °C in rolled cultures.

Antigenic Properties: Over 100 serotypes are known. Some cross-react (e.g., types 9 and 32).

Pathogenesis & Pathology. The virus enters via the upper respiratory tract. High titers of virus iasal secretions — which can be found as early as 2-4 days after exposure – are as­sociated with maximal illness. Thereafter, viral titers fall, although illness persists.

Histopathologic changes are limited to the submucosa and surface epithelium. These include engorgement of blood vessels, oedema, mild cellular infil­tration, and desquamation of surface epithelium, which is complete by the third day. Nasal secretion increases in quantity and in protein concentration.

Experiments under controlled conditions have shown that chilling, including the wearing of wet clothes, does not produce a cold or increase suscepti­bility to the virus. Chilliness is an early symptom of the common cold.

Clinical Findings. The incubation period is brief, from 2 to 4 days, and the acute illness usually lasts for 7 days although a non-productive cough may persist for 2-3 weeks. The average adult has 1-2 attacks each year. Usual symp­toms in adults include irritation in the upper respiratory tract, nasal discharge, headache, mild cough, malaise, and a chilly sensation. There is little or no fever. The nasal and nasopharyngeal mucosa become red and swollen, and the sense of smell becomes less keen. Mild hoarseness may be present. Prominent cervical adenopathy does not occur. Secondary bacterial infec­tion may produce acute otitis media, sinusitis, bron­chitis, or pneumonitis, especially in children. Type-specific antibodies appear or rise with each infection.

Epidemiology. The disease occurs throughout the world. In the temperate zones, the attack rates are highest in early fall and winter, declining in the late spring. Members of isolated communities form highly susceptible groups. The virus is believed to be transmitted through close contact, by large droplets. Under some circum­stances, transmission of the virus by self-inoculation through hand contamination may be a more important mode of spread than that by airborne particles.

Colds in children spread more easily to others than do colds in adults. Adults in households with a child in school have twice as many colds as adults in households without school children.

In a single community, many rhinovirus serotypes cause outbreaks of disease in a single season, and different serotypes predominate during different respi­ratory disease seasons.

Treatment & Control. No specific treatment is available. The develop­ment of a potent rhinovirus vaccine is unlikely because of the difficulty in growing rhinoviruses to high titer in culture, the fleeting immunity, and the many serotypes causing colds. In addition, many rhinovirus serotypes are present during single respiratory disease outbreaks and may recur only rarely in the same area. Injection of purified vaccines has shown that the high levels of serum antibody are frequently not associated with similar elevation of local secretory antibody, which may be the most significant factor in disease preven­tion.

 

Diagnosis of ENTEROVIRAL INFECTION

Human intestinal viruses (enteroviruses) belong to the genus Enterovirus of the family Picornaviridae. They include viruses of poliomyelitis (3 serovars), Coxsackie A (24 serovars), Coxsackie B (6 serovars) [Coxsackie A 23 is identical to ECHO 9 virus],  ECHO (34 serovars) [ECHO 28 is referred to rhinoviruses, ECHO 34 is a variant of Coxsa­ckie A 24], and human enteroviruses of the 69th-72nd serovars (serotypes). Enterovirus 70 induces acute haemorrhagic conjunctivitis, enterovirus 72, hepatitis A. The genus Enterovirus also  includes intestinal viruses of animals (cattle, monkeys, swine, mice) and insects. Enteroviruses of animals are characterized by  species specificity.

Having infected the man, enteroviruses cause damage to the  central nervous system (encephalitis, meningoencephalitis, polio­myelitis, meningitis), stomach, and kidneys (diarrhoea, hepatitis, pancreatitis), as well as to the respiratory tract (rhinitis, pharyngitis, pneumonia of new–borns, etc.) and the cardiovascular system (myo­carditis, pericarditis). The development of herpetic angina, exan­thema, vesicular stomatitis, myalgia, and conjunctivitis is also possible. Various enteroviruses may be responsible for identical clinical manifestations. Yet, on the other hand, one and the same enterovirus may cause various syndromes.

Material for examination includes faeces, washings off the nasal portion of the throat, cerebrospinal fluid (CSF), blood, serum, the contents of vesicles, urine, and ascitic fluid. Specimens are collected within the first hours of the disease. At autopsy, the following materi­al is collected: blood, cerebrospinal fluid, tissue of the spinal cord and medulla oblongata, pons varolii, pieces of the intestines and their contents, pieces of the liver, spleen, lung, pancreas, myocardium, and lymph nodes. Post-mortem specimens are taken within 3-4 hrs after death.

Blood (some 10 ml) for virological analysis and preparation of the serum is withdrawn on a fasting stomach at the beginning of the disease and then 3-4 weeks later.

Faeces (4-6 g) are collected at an interval of 1-2 clays. A 10 percent suspension is prepared in Hanks’ solution, shaken, and bleached by centrifugation for 30 min at 3000 X g (if possible, the material is centrifuged at 10 000 X g). The supernatant is treated with ether (to a bleached suspension of faeces add 50 per cent (by the volume) of diethyl ether and let the mixture stand in the refrigerator under a cotton-gauze plug for 12-16 hrs) and antibiotics.

Rectal tampons are put into a test tube with 1-2 ml of Hanks’ or Earle’s solution, rinsed, and squeezed dry. The material is centri­fuged and treated with ether and antibiotics.

The throat washing in a volume of 20 ml is obtained by gargling with sterile saline or distilled water (twice at a 3-min interval). Brush the posterior wall of the throat, tonsils, and palate arches with swabs and place them in a test tube with 1-2 ml of Hanks’ solution. The material is centrifuged and treated with ether and antibiotics. If the cerebrospinal fluid (about 3 ml) is sterile and transparent, it is used for virus isolation without preliminary treatment. Turbid cerebrospinal fluid, as well as that containing erythrocytes, is centri­fuged. A mid-stream urine sample (10 ml) is collected into a sterile vessel and treated with antibiotics.

Specimens of section material are ground in a sterile mortar, a 20 per cent suspension is prepared in Hanks’ solution and centrifuged for 5-10 min at 2000 X g.

The material is treated with antibiotics (penicillin, 1000 U/ml and streptomycin, 500 mg/ml) for 2 hrs at room temperature and is then used for virus isolation. A portion of the material is frozen and kept at — 70 °C. Multiple freezing and thawing is unacceptable since it leads to virus inactivation.

Methods of rapid diagnosis have failed to find wide-scale use in enteroviral infections due to the peculiarities of their pathogenesis. Immunofluorescence may be used for investigation of the cerebrospinal fluid cells. EM and IEM are usually employed in rotaviral infections

Isolation of the virus is conducted in cell cultures and one-day-old suckled white mice (Table).                 

      Table

Methods of the Recovery and Identification of Enteroviruses

 

Polioviruses (the first to the third one), most Coxsackie B viruses, ECHO, some types of Coxsackie A viruses (the 7th, 9th, 14th, 16th, 21st) exert the cytopathic effect upon their propagation in monkey kidney cell culture. In a culture of cells from the human embryo kidneys, human amnion, and of diploid cells WI-38 one observes the reproduction of Coxsackie A viruses (the 11th, 13th, 15th, 18th, 20th, 21st, 24th) and ECHO viruses (the 21st and 34th). Most serovars of Coxsackie A viruses propagate and induce the cytopathic effect in cell culture RD (culture from human rhabdomyosarcoma). To isolate Coxsackie viruses, both cell cultures and newborn mice are infected.

Coxsackie A viruses from patients’ material can be most successful­ly isolated only on one-day-old suckling mice owing to their failure to multiply in the majority of monkey and human cell cultures.

Suckling mice are infected intracerebrally (0.01 ml), subcutaneously (0.03 ml), intraperitoneally (0.05 ml), or by a combined method. The infected animals are observed for 14 days. Upon the development of clinical symptoms a 20 per cent suspension is pre­pared from the brain for further passage of the virus. Pieces of tissue for histological examination are also taken.

The demonstration of enteroviruses is based on the cytopathic effect and plaque formation under agar or bentonite coats and in cell cultures, the development of paralysis in suckling mice and their death, as well as on their physicochemical proper­ties: small size, resistance to lipodissolving agents and low pH values (3.0), and thermal stability at 50 °C in the presence of 1 M magnesium chloride.

To identify enteroviruses, the N, HAI, CF, PG, and IF tests with type-specific immune sera are employed.

The neutralization reaction is run in a cell culture or in suckling mice, according to the conventional technique.

Identification of strains of Coxsackie A and B viruses and ECHO viruses possessing haemagglutinating properties is based on the HAI test, using antigens from infected cell cultures and 1 per cent suspension of human erythrocytes, group 0 (1).

In making the CF test, instead of antisera, one uses the immune ascitic fluid of mice, which displays lower anticomplement activity.

The precipitation reaction in gel yields good results with an antigen concentrated 200-400-fold.

To concentrate enteroviruses, the bentonite method is utilized. To 500 ml of the cultural virus-containing fluid add 0.05-0.1 per cent of bentonite gel in an amount equal to a dry weight of the sorbent and bring the pH of the mixture to 3.5-4.0 by adding 0.1 N of HC1 solution. Shake the mixture for 3-5 min and centrifuge at 2000-3000 X g for 10-15 min. Wash off the sediment (virusbentonite) with 20-40 ml of distilled water. Elution of the virus is achieved by adding 0.05 M solution of tris-buffer (pH 9.0) and intensive shaking for 4-5 min. After this, the mixture is centrifuged and the virus-containing supernatant is studied.

The methods used in serological diagnosis include the N, CF, and HAI tests. A four-fold or higher increase in the antibody litre is of diagnostic significance. The results of serological examination should be compared with virological, epidemiological. and clinical findings.

To perform the neutralization test, 100 doses of the virus are mixed with two-fold diluted sera of the patient. When this reaction is made in cell cultures, the results are read on the 3rd-4th day and 7th-8th day. When suckling mice are used, the results are assessed on the 10th-14th day.

The CF test is utilized for the diagnosis of poliomyelitis. The test is diagnostically important when the titre of antibodies is 1:32 and higher, and when the antibody titre has increased by a four­fold or greater magnitude. The results are better when one uses unheated (poorly reactive) antigens obtained by single freezing and thawing of infected cells after the complete specific destruction has taken place.

The HAI test is rarely utilized for the serological diagnosis of enteroviral infections. The haemagglutination antigen is obtained when the virus titre is high (10⁶-10⁷ CPE₅₀/ml). Patients’ sera are treated to remove spontaneous haemagglutinins and non-specific inhibitors of haemagglutination.

 

 

Orthomyxovirus (Influenza) Family

The name myxovirus was originally applied to influenza viruses. It meant virus with an affinity for mucins. Now there are 2 main groups –the orthomyxoviruses and the paramyxoviruses. All orthomyxoviruses are influenza viruses. Iso­lated strains are named after the vims type (A, B, C), the host and location of initial isolation, the year of isolation, and the aniigenic designation of the hemagglutinin and neuraminidase. Eleven hemagglutinin antigenic subtypes and 8 neuraminidase antigenic subtypes are designated. Both of these are glycopro-teins under separate genetic control, and they vary independently. Examples of influenza designations follow: A/swine/New Jersey/8/76 (H1N1), pre­viously (HswINI), A/Brazil/78 (H1N1), B/Singapore/79,A/Bangkok/79 (H3N2)

 

INFLUENZA

Influenza is an acute respiratory tract infection that usually occurs in epidemics. Three immunologic types of influenza virus are known: A, B, and C.

 

Antigenic changes continually take place within the A group of influenza viruses and to a lesser degree in the B group, whereas influenza C appears to be antigeni-cally stable. Influenza A strains are also known for pigs, horses, ducks, and chickens (fowl plague). Some animal isolates are antigenically similar to the strains circulating in the human population.

Influenza virus type C differs from the type A and type B viruses; its receptor-destroying enzyme does not appear to be a neuraminidase, and its virion struc­ture is not fully understood. The following descrip­tions are based on influenza virus type A.

Properties of the Virus

A. Structure: Influenza virus consists of pleomorphic, approximately spherical particles having an external diameter of about 110 nm and an inner electron-dense core of 70 nm.

The surface of the virus particles is covered with 2 types of projections, or spikes, approximately 10 nm long possessing either the hemagglutinin or the neuraminidase activity of the virus. A model of the influenza virion is shown in Fig.

 

 

Figure 1. A model of the influenza virion The inner most component is the helical ribonucleoprotein (RNP). which is 9 nm in diameter It is 35 yet unknown whether the RNP is one long molecule or is divided into pieces like the virus RNA The nucleocapsid is further organized by the coiling of the whole RNP strand into a double helix 50-60 nm in diameter The protein component of this structure has a molecular weight of 60,000 and is associated with the group-specific CF antigen A protein (M) shell surrounds the nucleoprotein and forms the inner part of the virus envelope It is composed of a small protein (MW 26,000) and constitutes about 40% of the virus protein. About 20% of the virus particle is composed of lipid, apparently derived from the host cell The lipid is formed into a bilayer structure. The hemagglutinin spike is responsible for the agglutination of erythrocytes by this virus.  It is composed of 2 molecules of a glycoprotein (MW 75,000) that may or may not be cleaved to form 2 disulfide-linked glycopeptides of molecular weight 27,000 and 53,000. The smaller of these is present at the end of the molecule which is attached to the lipid. The neuraminidase spike is responsible for the receptor destroying activity of the virus, this activity results in elution of the virus from host cells or erythrocytes. The role of its activity in virus replication is unknown. It is composed of 4 polypeptide molecules with a molecular weight of about 60,000. The arrangement of these molecules is still a matter of debate. Both the hemagglutinin and the neuraminidase spikes have been purified, and a study of the purified protein has helped to explain antigenic changes of the virus.

The RNA genome consists of 8 distinct pieces with an aggregate molecular weight of 2-4 x 10⁶.

Because of a divided genome, viruses of this group exhibit several biologic phenomena such as high recombination frequency, multiplicity reactivation, and ability to synthesize hemagglutinin and neuraminidase after chemical mactivation of viral in­fectivity.

Although viral RNA has not proved to be infec­tious, viral ribonucleoprotein appears to he so. This structure contains the virion-associated RNA-dependent RNA polymerase as well as the genome. Evidently, all messenger RNA is complementary to the virion RNA.

The results of hybridization studies on RNA have supported the immunologic grouping of the hemagglutinins of the influenza A viruses. Similar studies of the neuraminidase genes have been in agreement with N antigen subtype designations based on the results of serologic tests.

B. Reactions to Physical and Chemical Agents: Influenza viruses are relatively stable and may be stored at 0-4 °C for weeks. The virus is less stable at -20 °C than at +4 °C. Ether and protein denaturants destroy infectivity. The hemagglutinin and CF antigens are more stable than the infective virus. Ultraviolet irradiation destroys infectivity, hemagglutinating activity, neuraminidase activity, and CF antigen, in that order. Infectivity and hemagglutmation are more stable at alkaline pH than at acid pH.

C. Animal Susceptibility and Growth of Virus: Human strains of the virus can infect different animals; ferrets are most susceptible Senal passage in mice increases its virulence, producing extensive  pulmonary consolidation and death The developing chick embryo readily supports the growth of virus, but there are no gross lesions.

Wild influenza viruses do not grow well in tissue cultures. In most instances, only an abortive growth cycle occurs, ie, viral subunits are synthesized but little or no new infectious progeny is formed. From most influenza strains mutants can be selected that will grow in cell culture. Because of the poor growth  of many strains in cell culture, initial isolation attempts should employ inoculation both of the amniotic cavity of the embryonated egg and of monkey cell cultures.

The process of infection begins by adsorption of the virus onto its receptor sites (neuramimc acid- containing glycoproteins). The hemagglutinin protein is involved in this reaction. The other spike protein, neuraminidase, can destroy the site. The virus particle is taken into the cell, where it is disrupted, causing a decrease in detectable virus shortly after infection Intracellular synthesis of the viral RNA and protein then occurs. Viral RNA pieces are synthesized individually m the nucleus within 2-3 hours All viral proteins are synthesized in the cytoplasm Structural proteins bind to the cell membrane and are joined by the ribonucleoprotein At 8 hours, new virus particles bud through the membrane. Neuraminidase may be important in release of the completed virion.

In most influenza virus systems, noninfectious particles capable of hem agglutination are produced(von Magnus phenomenon). These particles, called “incomplete,” increase iumber upon serial, high-multiplicity passage of the vims The incomplete panicles are smaller and more pleomorphic than standard virus, and they interfere with replication of standard virus. They are known as defective interfering, or DI, particles The largest virus RNA piece is missing from such particles.

D. Biologic Properties:

1. Hemagglutination. All strains of influenza virus agglutinate erythrocytes from chickens, guinea pigs, and humans and—unlike paramyxo viruses – agglutinate erythrocytes from many other species a.s well. Agglutination of red blood cells occurs when the hemagglutinin interacts with a specific receptor on the red blood ceil membrane This receptor is a glycoprotein (MW 3 x 10⁴) that contains sialic acid. This glycoprotein serves both as the receptor site for the hemagglutinin and as the substrate for the viral neuraminidase. Cleavage of the glycoprotein by the enzyme dissociates the virion from the red cell, resulting
in spontaneous elution. After elution, the cell receptors are destroyed and hence cao longer be agglutinated with fresh virus; however, the eluted virus can reattach and agglutinate additional cells.

2. Group antigen. All influenza A virus strains share a common antigen, distinct from those of influenza B and C. This soluble (S) antigen is found in the medium from infected cell cultures and is a component of the nbonucleoprotein of the virus It can be identified by CF. Antibody to this nucleoprotein antigen does not induce resistance to the virus in humans. The other internal proteins and the RNA polymerase also have group-specific antigenic activity.

3. Specific antigens. The infectious vims parti­cles induce in animals the development of virus-neutralizing and other antibodies, and the inoculated animals become resistant to infection. Influenza vims administered in large amounts is toxic- The effect is apparently associated directly with the virus particles and can be prevented by specific antibody.

Human influenza virus

Influenza A/Bangkok/1/79(H3N2)

Influenza A/Singapore/1/57(H2N2)

Influenza B/Ann Arbor/1/86

 

A/Sydney/5/97 (H3N2)

 

Virions contain 2 subtype or strain-specific antigens – the hemagglutinin and the neuraminidase. The hemagglutinin is the principal specific envelope antigen, and differences in this antigen among strains of virus can be shown by HI tests. Antibody to the hemagglutinieutralizes vims and is a protective mechanism.

Neuraminidase is antigenically distinct from the hemagglutinin and is governed by a separate gene (RNA fragment); hence, it can vary independently of the hemagglutinin. The antigens of the hemagglutinin and the neuraminidase of the virus are the basis for classifying new strains. Antibody against the neuraminidase does not neutralize the vims, but it modifies the infection, probably by its effect on the release of vims from the cells. The antibody against the neuraminidase occurs in sera of humans who experi­ence infection. The presence of antineuraminidase antibody results in marked protection against disease.

4. Recombination. The multisegment nature of the influenza vims genome allows recombination to occur with high frequency by reassortment between orthomyxoviruses of the same group. The RNA frag­ments of different influenza A vimses migrate at dif­ferent rates in polyacrylamide gets. Similarly, the polypeptides of different influenza A vimses can be differentiated. Thus, using 2 different parental vimses and obtaining recombinants between them, it is possi­ble to tell which parent donated which RNA fragment to the recombinant. These techniques enable rapid and more complete analysis of recombinants that emerge iature,

Antigenic drift

 

 

 

Pathogenesis and Pathology

The virus enters the respiratory tract in airborne droplets. Viremia is rare. Vims is present in the nasopharynx from 1-2 days before to 1-2 days after onset of symptoms. The neuraminidase lowers the viscosity of the mucous film in the respiratory tract, laying bare the cellular surface receptors and promo­ting the spread of virus-containing fluid to lower por­tions of the tract. Even when neutralizing antibodies are in the blood they may not protect against infection.

Antibodies must be present in sufficient concentration at the superficial cells of the respiratory tract. This can be achieved only if the antibody level in the blood is high or if antibody is secreted locally.

Inflammation of the upper respiratory tract causes necrosis of the ciliated and goblet cells of the tracheal and bronchial mucosa but does not affect the basal layer of epithelium. Interstitial pneumonia may occur with necrosis of bronchiolar epithelium and may be fatal. The pneumonia is often associated with second­ary bacterial invaders: staphylococci, pneumococci, streptococci, and Haemophilus influenzae.

Clinical Findings

The incubation period is 1 or 2 days. Chills, malaise, fever, muscular aches, prostration, and respi­ratory symptoms may occur. The fever persists for about 3 days; complications are not common, but pneumonia, myocarditis, pericarditis, and central ner­vous system complications occur rarely. The latter include encephalomyelitis, polyneuritis, Guillain-Barre syndrome, and Reye’s syndrome (see below).

When influenza appears in epidemic form, the clinical findings are consistent enough so that the disease can be diagnosed in most cases. Sporadic cases cannot be diagnosed on clinical grounds. Mild as well as asymptomatic infections occur. The severity of the pandemic of 1918-1919 has been attributed to the fact that bacterial pneumonia often developed.

> 20 million died of the flu during WW I

A new influenza vaccine must be developed yearly

 

The lethal impact of an influenza epidemic is reflected in the excess deaths due to pneumonia and cardiovascular and renal diseases. Pregnant women and elderly persons with chronic illnesses have a higher risk of complications and death.

Reye’s syndrome occurs mainly in children. It is characterized by encephalopathy and fatty degenera­tion of the liver, and the mortality rate is high. In 1979-1980, more than 400 cases were reported in the USA, with a mortality rate near 30%. Reye’s syn­drome is associated with influenza B, rarely with in­fluenza A, and sometimes with other viral diseases such as chickenpox and zoster.

 

Laboratory Diagnosis

Influenza is readily diagnosed by laboratory pro­cedures. For antibody determinations, the first serum should be taken less than 5 days after onset and the second 10-14 days later.

For rapid detection of influenza virus in clinical specimens, positive smears from nasal swabs may be demonstrated by specific staining with fluorescein-labeled antibody.

A. Recovery of Virus: Throat washings or garglings are obtained within 3 days after onset and should be tested at once or stored frozen. Penicillin and strep­tomycin are added to limit bacterial contamination, and embryonated eggs are inoculated by the amniotic route. Amniotic and allantoic fluids are harvested 2-4 days later and tested for hemagglutinins. If results are negative, passage is made to fresh embryos. If hemagglutinins are not detected after 2 such passages, the result is negative.

If a strain of virus is isolated – as demonstrated by the presence of hemagglutinins—it is titrated in the presence of type-specific influenza sera to determine its type. The new virus belongs to the same type as the semm that inhibits its hemagglutinating power.

Primate cell cultures (human or monkey) are sus­ceptible to certain human strains of influenza virus. Rapid diagnosis can be made by growing the virus from the clinical specimen in cell culture and then staining the cultured cells with fluorescent influenza antibody 24 hours later, when infected celts are rich in antigen even though they may appear normal.

The phenomenon of hemadsorption is utilized for the early detection of virus growth in cell cultures. Guinea pig red cells or human 0 cells are added to the cultures 24-48 hours after the clinical specimens have been inoculated and are viewed under the low power lens. Positive hemadsorption shows red blood cells firmly attached to the cell culture sheets as rosettes or chains. The cytopathogenic effects of the influenza viruses are often negligible. Hemadsorption provides a more sensitive testing procedure.

Membrane iminunofluorescence has also been recommended as a simple, rapid, and accurate method for typing current influenza A isolates. Surface anti­gens of infected, unfixed monkey kidney cells are stained in suspension by the indirect immunofluores-cence method using anti-H3N2 and anti-H1N1 antisera.

C. Serology: Paired sera are used to detect rises in HI, CF, or Nt antibodies. The HI antibody is used most often. Normal sera often contain nonspecific mucoprotein inhibitors that must first be destroyed by treatment with RDE (receptor-destroying enzyme of Vibrio cholerae cultures), trypsin, or periodate. Be­cause normal persons usually have influenza an­tibodies, a 4-fold or greater increase in titer is neces­sary to indicate influenza infection. Peak levels of antibodies are present 2—4 weeks after onset, persist for about 4 weeks, and then gradually fall during the course of a year to preinfection levels.

Complement-fixing antigens are of 2 types. One is soluble (S antigen) and type-specific but not strain-specific. The other is part of the virus particle (V antigen) and is highly strain-specific. It is useful for demonstrating antibody rise when the first serum specimen was not taken early in the disease, because the peak CF titer occurs in the fourth week.

Immunity

Three immunologically unrelated types of influ­enza virus are known and are referred to as influenza A, B, andC. In addition, the swine, equine, and avian influenza viruses are antigenically related to the human influenza A virus. Influenza C virus exists as a single and stable antigenic type.

At least 18 different antigenic components have been determined in type A strains of influenza virus by quantitative adsorption methods. More undoubtedly exist. Strains share their antigenic components, but in varying proportions. A strain generally shares its anti­gens with strains prevalent within a few years of its isolation.

Two possible mechanisms for the antigenic varia­tion of influenza virus have been suggested:

(1) All possible configurations may be present in a pool of antigens that exist throughout the globe; from these, highly infectious strains arise and initiate epidemics. High antibody levels to receni strains in the human population will inhibit strains with major anti­gens that were dominant in recently prevalent strains and will select strains of different antigenic composi­tion.

Serial passage of virus in mice vaccinated with the homologous strain yields a vims with an apparent rearrangement of antigens or the appearance of new antigens. The change in antigenic character evolves slowly on passage (antigenic drift).

(2) Antigenically different strains may be selected by means of genetic recombination induced by selection factors such as passage in a partially immune host. When 2 strains of influenza virus are simultaneously injected into mice or eggs, a new strain sharing the properties of each parent strain may be recovered; this has been attributed to genetic recombi­nation (antigenic shift).

Antibodies are important in immunity against in­fluenza, but they must be present at the site of virus invasion. Resistance to initiation of infection is related to antibody against the hemagglutinin. Decreased ex­tent of viral invasion and decreased ability to transmit virus to contacts are related to antibody directed against the neuraminidase.

Virus-neutralizing antibody occurs earlier iasal secretions and rises to high liters sooner among those already possessing high concentrations ofIgA in their nasal washings prior to the infection. Even though infected with influenza virus, such individuals remain well. In contrast, those with low nasal wash IgA levels prior to infection are highly susceptible not only to infection but also to clinical illness.

Prevention and Treatment by Drugs

Amantadine hydrochloride and its analog rimantadine are antiviral drugs for systemic use in the prevention of influenza A. The drugs block penetration of or uncoat influenza A virus in the host cell and prevent virus replication. The established ef­fect is prophylaxis, and amantadine (200 mg/d) must be given to high-risk persons during epidemics of influenza A if protection is to result. Amantadine is relatively nontoxic but may produce central nervous system stimulation with dizziness and insomnia, par­ticularly in the elderly. It should be considered for persons with chronic obstructive respiratory disease, cardiac insufficiency, or renal disease, particularly if they have not been vaccinated yearly or if a new influen/.a A strain is epidemic. Amantadine may also modify the severity of influenza A if started within 24-48 hours after onset of illness.

Trivalent Influenza virus vaccines

1999-2000

Ø A/Sydney/05/97 (H3N2)

Ø A/Beijing/262/95 (H1N1)

Ø B/Yamanashi/166/98

 

2000-2001

Ø         A/Moscow/10/99(H3N2)-like

Ø A/New Caledonia/20/99 (H1N1)-like

Ø B/Beijing/184/93-like

 

To day

Ø  A/Brisben/59/2007 (H1N1)

Ø  A/Brisben/10/2007 (H3N2)

Ø  B/Florida/4/2006

 

Prevention and Treatment

 

Ø RIMANTADINE (blocks the M2 ion channel) (M2)

Ø type A only, needs to be given early

Ø  AMANTADINE (blocks the M2 ion channel) (M2)

Ø type A only, needs to be given early

Ø  ZANAMIVIR (neuraminidase inhibitors)  (NA)

Ø types A and B, needs to be given early

Ø  OSELTAMIVIR (neuraminidase inhibitors)   (NA)

Ø types A and B, needs to be given early

 

Epidemiology

Influenza occurs in successive waves of infec­tion, with peak incidences during the winter. Influenza A infections may vary from a few isolated cases to extensive outbreaks that within a few weeks involve 10% or more of the population, with rates of 50-75% in children of school age. The period between epidemic waves of influenza A is 2-3 years. All known pandemics were caused by influenza A strains. During the pandemic of 1918-1919 more than 20 million persons died, mainly from complicating bacte­rial pneumonias. Recent pandemics occurred in 1957-1958 owing to A influenza (H2N2) and in 1968 owing to A influenza (H3N2). In 1976 in New Jersey, a new type of influenza arose that resembled swine influenza (HswINi), hut it failed to spread in spite of a lack of immunity in most people under age 50 years. An enormous government-sponsored vaccination campaign was stopped because Guillain-Barre syn­drome appeared in some vaccinated individuals. The predominant influenza A in the USA in 1978-1979 was an H1N1 variant of the strains prevalent in the 1950s.

Influenza B tends not to spread through com­munities as quickly as influenza A. Its interepidemic period is from 3 to 6 years. Small outbreaks of influ­enza B were frequent in the USA in 1979-1980.

The main reason for the periodic occurrence of epidemic influenza is the accumulation of a sufficient number of susceptibles in a population that harbors the virus in a few subclinical or minor infections through­out the year. Epidemics may be started when the virus mutates to a new antigenic type that has survival ad­vantages and when antibodies in the population are low to this new type. A much more drastic change in the segmented RNA genome occurs when antigenic shift occurs. This involves the recombination of different segments of the RNA, each of which functions as an individual gene.

Surveillance for influenza outbreaks is more ex­tensive than for any other disease in order to identify the early appearance of new strains, with the aim of preparing vaccines against them before an epidemic occurs.

Surveillance also extends into animal popula­tions, especially birds, pigs, and horses. Some believe that pandemic strains arise from recombinants of human and animal strains.

Since the virus causing fowl plague was identified as human influenza A type in 1955, many influenza viruses have been isolated from a wide variety of domestic and wild bird species. Some of these include the major H and N antigens related to human strains.

Avian influenza ranges from highly lethal infec­tions in chickens and turkeys to inapparent infections in these and other avian species that harbor the same strains. Domestic ducks and quail often manifest influ­enza infection by coughing, sneezing, and swelling around the beak, with variable mortality rates. Wildlife species and most domestic fowl show little or no signs of disease.

The possibility that influenza vimses are transmit­ted between birds and mammals, including humans, may seem unlikely, particularly if the transfer were to be only by the respiratory route. However, influenza vimses of ducks multiply in the cells lining the intesti­nal tract and are shed in high concentrations into water. These viruses remain viable for days or weeks in wa­ter. It is possible that influenza among birds is a wa-terbome infection, moving from wild to domestic birds and even to humans.

Current research approaches to better in­fluenza vaccines.

1. A neuraminidase-specific vaccine, which in­duces antibodies only to the neuraminidase antigen of the prevailing influenza virus. Antibody to neuramini­dase reduces the amount of virus replicating in the respiratory tract and the ability to transmit virus to contacts. It reduces clinical symptoms in the infected person but permits subclinical infection that may give rise to more lasting immunity.

2. A live vaccine using temperature-sensitive (ts) mutants. Such ts mutants grow well at the cooler (33 °C) temperature of the upper respiratory tract but fait to grow at the higher (37 “C) temperature of the lung. Mutants selected for this ts property appear to be at­tenuated or avirulent. Thus, they might be given as a live vaccine into the respiratory tract, stimulating local as well as systemic immunity. By recombination of the ts gene with the gene for the current major antigen, potent live vaccines could theoretically be produced and rapidly administered to cope with an influenza epidemic.

Attenuated live influenza virus vaccine has been used in the USSR with reported success. The at­tenuated virus was selected by serial transfer through embryonated eggs rather than by genetic manipula­tion.

3. Combined yearly vaccination of persons at high risk, using the best mix of important antigens, and administration of amantadine or other anti-influenza dmgs at times of particular stress, eg, surgery, hospitalization.

 

Additional materials about laboratory diagnosis of flue

The laboratory diagnosis of influenza includes rapid methods, iso­lation of the virus, and serological examination.

The material for examination consists of washings on the nasal portion of the throat and secretion of the mucosal membrane of the nostrils, which is taken with dry or moist tampons. The tampons are placed in test tubes containing 2-5 ml of buffer (pH 7.0-7.2); or impression smears may be made on glass. Moreover, the virus can be isolated from the blood, cerebrospinal fluid, and autopsy material (pieces of the damaged tissues of the upper respiratory tract, brain, etc.).

The rapid diagnosis of influenza relies on demonstration of the specific viral antigen in the material tested by direct and indirect immunofluorescence. The presence of the influenza virus in the epithe­lial cells is recognized by greenish-yellow luminescence.

The RIHA and ELISA tests may also be employed for isolating the specific influenza antigen. ELISA aimed at identifying the M-protein of the influenza virus appears to be a particularly promis­ing method with regard to the rapid diagnosis of influenza.

An attempt to isolate the virus should be made as early as pos­sible since the feasibility of obtaining a positive result sharply de­clines after the third day of the disease.

To suppress the bacterial flora, the material tested is treated with antibiotics (500-1000 U/ml of penicillin and 200 mg/ml of strepto­mycin) and then introduced (in 0.2-ml quantities) into the amniotic and allantoic cavities of 10-11-day-old chicken embryos.

The material from one sample is used to inoculate at least five embryos which are incubated thereafter for 3-4 days at 37 °C.

Following incubation, the embryos are cooled for 2-4 hrs at 4 °C. The allantoic fluid is aspirated with a syringe or Pasteur pipette, the amniotic one, by means of a syringe fitted with a short needle.

Accumulation of viruses in chicken embryos is detected by the HA test. To carry out this test, the fluids (allantoic and amniotic) are titrated, and a 1 % suspension of chicken red blood cells is added.

Infective material may also be inoculated into cultures of cells of monkey kidneys, human embryo, and others, using nutrient media without serum. Yet, these methods are employed not so frequently as inoculation of chicken embryos. The virus is detected by immunoflu­orescence, determination of the cytopathic effect (CPE), and the haemad-sorption inhibition test with 0.4 per cent suspension of guinea pig red blood cells.

To identify viruses, such tests as Hadsl, HAI, CF, and ELISA are used, with the titre of sera used for Hadsl being no less than 1:160.

If the isolated virus has an altered antigenic structure, Hadsl and HAI tests with available specific sera may be negative. In this case, the complement-fixation test is used, which demonstrates type-specific viral proteins that are not exposed to antigenic alterations.

The neutralization test is widely used to identify the virus in chicken embryos and tissue cultures.

Serological examination is considered to be diagnostically posi­tive of influenza when there is a four-fold or greater increase in the antibody titre.

The first sample of the serum is taken from the patient in the acute period of the disease (within 3-5 days), usually simultaneously with a washing off the nasal portion of the throat, the second one, after the tenth day of the disease. To allow for the simultaneous study of both sera, the first specimen is stored     at – 20 °C.

The CF, HAI, ELISA, and RH tests are performed. To remove non-specific inhibitors of the influenza virus present in patients’ sera, they are treated with a receptor-breaking enzyme. To accomplish this, the enzyme, in a concentration of 100 U/ml, is mixed with the serum in a ratio depending on the titre of the enzyme and is allowed to stand at 37 °C for 10-12 hrs. Then, three volumes of 2.5 per cent sodium citrate solution are added to this mixture, it is heated at 50°C for 30 min, and two volumes of phosphate buffer are added to give the final serum dilution of 1:10.

Antibodies in influenza patients may be determined with the help of the neutralization test on chicken embryos or culture of monkey kidney tissues. This test is very economical when it is made on pieces of tissue of the chorio-allantoic membrane in shells.

 

Paramyxoviruses. Laboratory diagnosis of epidemic parotitis (mumps), measles, parainfluenza, Diseases, which are caused by RS–viruses

 

Paramyxovirus Family

Paramyxoviruses include important human (mumps, measles, parainfluenza, respiratory syncytial) and animal viruses.

 

Properties of the Paramyxoviruses

A.      Structure: The particle has a lipid-containing envelope covered with spikes; a helical ribonucleoproteiucleocapsid 18 nm in diameter is enclosed. The RNA is a single molecule (MW 5-8 x 10⁶).

The envelope of paramyxoviruses contains 2 glycoproteins, HN and F, that form spikelike projec­tions from the surface of the viral membrane. These glycoproteins are involved in the early interactions between virus and cell. The larger glycoprotein, HN, has neuraminidase and hemagglutinating activities and is responsible for virus adsorption. The other glycopro­tein, F, is involved in virus-induced cell fusion and hemolysis and in virus penetration through fusion of viral and cell membranes. The membrane-fusing activ­ity of Ihe F protein is activated by proteolytic cleavage of a precursor (Fo) by a host enzyme to yield 2 disulfide-linked polypeptides (F₁ and F₂). Only then can viral replication begin.

 

B. Biologic Properties:

1. Cell fusion. In the course of infection, para­myxoviruses cause cell fusion, long  recognized as giant cell formation. This ability to fuse cells is now used for the creation of cell hybrids, an important tool in somatic cell genetics.

2. Persistent infection. Most paramyxoviruses can produce a persistent noncytocidal infection of cul­tured cells. The clinical importance of this property may explain subacute sclerosing panencephalitis (SSPE).

3. Antigenic properties. Measles, canine dis­temper, and rinderpest viruses have related antigens. Another antigenically related group includes mumps, parainfluenza, and Newcastle disease viruses.

C. Replication.  The RNA genome of viruses of this group is not infectious and does not function as messenger RNA. Instead, the viral genome is tran­scribed into shorter RNA molecules that serve as mes­senger and are complementary to the genome. The paramyxoviruses possess an RNA-dependent RNA polymerase that is a structural component of the virion and produces the initial messenger RNA.

 

MUMPS (Epidemic Parotitis)

Mumps is an acute contagious disease charac­terized by a nonsuppurative enlargement of one or both of the parotid glands, although other organs may also be involved.

A. Morphology and Biochemical Properties.

The mumps virus particle has the typical paramyxo­virus morphology. Typical also are the biologic properties of hemagglutination, neuraminidase, and hemolysin. Hemagglutination can be inhibited by spe­cific antisera to mumps virus, and this inhibition can be used to measure antibody responses. Similarly, the nucleocapsid of the vims particle forms the major component of the “S” (soluble) complement-fixing antigen.

 

 

B. Reactions to Physical and Chemical Agents. The hemagglutinin, the hemolysin, and the infectivity of the virus are destroyed by heating at 56 °C for 20 minutes. The skin test antigen and the complement-fixing antigen are more heat-stable.

C. Animal Susceptibility and Growth of Vi­rus. In monkeys, mumps can produce a disease that is very much like that in human beings. Parotitis is produced by introducing the virus into Stensen’s duct or directly into the gland by injection. By the use of fluorescent antibody, the virus has been located in the cytoplasm of acinar cells.

Mumps virus CPE

 

The virus grows readily in embryonated eggs and in cell culture. Passage in embryonated eggs reduces pathogenicity for humans, and this method was used to obtain a vaccine strain. Mumps virus growing in cell culture produces multinucieated giant cells (syncytia).

 

Pathogenesis and Pathology

Two theories exist regarding the pathogenesis of mumps. (1) The virus travels from the mouth by way of Stensen ‘s duct to the parotid gland, where it undergoes primary multiplication. This is followed by a gen­eralized viremia and localization in testes. ovaries, pancreas, thyroid, or brain. (2) Primary replication occurs in the superficial epithelium of the respiratory tract. This is followed by a generalized viremia and simultaneous localization in the salivary glands and other organs.

Little tissue damage is associated with uncompli­cated mumps. The ducts of the parotid glands show desquamation of the epithelium, and polymorphonu-clear cells are present in the lumens. There are intersti­tial edema and lymphocytic infiltration. With severe orchitis, the testis is congested, and punctate hemor­rhage as well as degeneration of the epithelium of the seminiferous tubules is observed. Central nervous sys­tem pathology may vary from perivascular edema to inflammatory reaction, glial reaction, hemorrhage, or demyelination.

 

Clinical Features

The incubation period is commonly 18-21 days. A prodromal period of malaise and anorexia is fol­lowed by rapid enlargement of parotid glands as well as other salivary glands. Swelling may be confined to one parotid gland, or one gland may enlarge several days before the other. The gland enlargement is as­sociated with pain, especially when tasting acid sub­stances- The salivary adenitis is commonly accom­panied by low-grade fever and lasts for approximately a week.

Epidemic parotitis                Orchitis

 

The testes and ovaries may be affected, especially after puberty. Twenty percent of males over 13 years of age who are infected with mumps virus develop orchitis, which is often unilateral and does not usually lead to sterility. Because of the lack of elasticity of the tunica albuginea, which does not allow the inflamed testis to swell, atrophy of the testis may follow second­ary to pressure necrosis. Secondary sterility does not occur in women because the ovary, which has no such limiting membrane, can swell when inflamed.

Mumps accounts for 10-15% of aseptic menin­gitis observed in the USA and is more common among males than females. Meningoencephalitis usually oc­curs 5-7 days after the inflammation of the salivary glands, but it may occur simultaneously or in the absence of parotitis and is usually self-limiting. The cerebrospinal fluid shows pleocytosis (10-2000//n,L, mostly lymphocytes) that may persist after clinical recovery.

Rare complications of mumps include (1) a self-limiting polyarthritis that resolves without residual de­formity; (2) pancreatitis associated with Transient hy-perglycemia, glycosuria, and steatorrhea (it has been suggested that diabetes mellitus may occasionally fol­low); (3) nephritis; (4) thyroiditis; and (5) unilateral nerve deafness (hearing loss is complete and perma­nent) . Mumps may be a possible causative agent in the production of aqueductal stenosis and hydrocephalus in children. Injection of mumps vims into suckling hamsters has produced similar lesions.

 

Laboratory Diagnosis

Laboratory studies are not usually required to establish the diagnosis of typical cases. However, mumps can sometimes be confused with enlargement of the parotids due to suppuration, foreign bodies in the salivary ducts, tumors, etc. In cases without parotitis, particularly in aseptic meningitis, the laboratory can be helpful in establishing the diagnosis.

A. Recovery of Virus: Virus can be isolated from saliva, cerebrospinal fluid, or urine collected within 4 days after onset of illness. After treatment with antibiotics, the specimens are inoculated into monkey kidney cell cultures. Virus growth can be detected in 5-6 days by adsorption of suitable eryth-rocytes by the infected cells. The isolate can be iden­tified with specific antiserum that can inhibit the hemadsorption. Immunofluore scent serum can also identify a virus isolate in cell culture within 2-3 days.

B. Serology. Antibody rise can be detected in paired sera. The CF test is best for specificity and accuracy, although the HI test may be used. A 4-fold or greater rise in antibody titer is evidence of mumps infection.

A CF test on a single serum sample obtained soon after onset of illness may serve for a presumptive diagnosis. S (soluble) antibodies develop within a few days after onset and sometimes reach a high titer before V (viral) antibodies can be detected. In early convales­cence, both S and V antibodies are present at high levels. Subsequently, S antibodies disappear more rap­idly, leaving V antibodies as a marker of previous infection for several years. The intradermal injection of inactivated virus results in reappearance of V an­tibodies in high titer. Neutralizing antibodies also ap­pear during convalescence and can be determined in cell culture.

C. Skin Test Antigen: Delayed type hypersensitivity may be noted about 3-4 weeks after onset. The skin test is less reliable than serologic tests to establish evidence of past infection.

Immunity. Immunity is permanent after a single infection. Only one antigenic type exists. Passive immunity is transferred from mother to offspring; thus it is rare to see mumps in infants under age 6 months.

Treatment. Gamma globulin is of no value for decreasing the incidence of orchitis, even when given immediately after parotitis is first noted.

Epidemiology. Mumps occurs throughout the world endemically throughout the year. Outbreaks occur where crowding favors dissemination of the virus. The disease reaches its highest incidence in children age 5-15 years, but epidemics occur in army camps. Although morbidity rates are high, the mortality rate is negligible, even when the nervous system is involved.

Humans are the only known reservoir of virus. The virus is transmitted by direct contact, airborne droplets, or fomites contaminated with saliva and, perhaps, urine. The period of communicability is from about 4 days before to about a week after the onset of symptoms. More intimate contact is necessary for the transmission of mumps than for measles or varicella.

About 30-40% of infections with mumps virus are inapparent. Individuals with subclinical mumps acquire immunity. During the course of inapparent infection, they can serve as sources of infection for others.

Antibodies to mumps virus are transferred across the placenta and are gradually lost during the first year of life. In urban areas, antibodies are then acquired gradually, so that the 15-year-old group has about the same prevalence of persons with antibodies as the adult group. Antibodies are acquired at the same rate by persons living under favorable and unfavorable socio-economic conditions.

Control

Mumps is usually a mild childhood disease. A live attenuated vaccine made in chick embryo cell culture is available. It produces a subclinical noncom-municable infection.

The vaccine is recommended for children over age 1 year and for adolescents and adults who have not had mumps parotitis, A single dose of the vaccine given subcutaneously produces detectable antibodies in 95% of vaccinees, and antibody persists for at least 8 years.Combination live virus vaccines (measles-mumps-rubella) produce antibodies to each of the vi­ruses in about 95%.

 

PARAINFLUENZA VIRUS INFECTIONS

The parainfluenza viruses are paramyxoviruses with morphologic and biologic properties typical of the genus. They grow welt in primary monkey or human epithelial ce!! culture bul poorly or not at all in the embryonated egg. They produce a minimal cytopathic effect in cell culture but are recognized by the hemad-sorption method. Laboratory diagnosis may be made by the HI, CF, and Nt tests.

Parainfluenza 1

Included here are Sendai virus, also known as the hemagglutinating virus of Japan (HVJ), and hemadsorption virus type 2 (HA-2). Sendai virus may be a causative agent of pneumonia in pigs and newborn infants. Sendai virus is important in somatic cell genetics, where it is used to produce cell fusion. Clinically, the most important member of this group appears to be the widespread HA-2 virus. It is not cytopathogenic for monkey kidney cell cultures but is detected in such cultures by the hemadsorption test. It is one of the main agents producing croup in children, but it can also cause coryza, pharyngitis, bronchitis, bronchiolitis, or pneumonia. In adults it produces res­piratory symptoms like those of the common cold, with reinfection occurring in persons with antibodies from earlier infections.

Natural infection stimulates antibody appearance iasal secretions and concomitant resistance to rein­fection, An experimental killed vaccine induces serum antibodies but does not protect against infection.

Parainfluenza 2

This group includes the croup-associated (CA) virus of children. The virus grows in human cells (HeLa, lung, amnion) and monkey kidney. Syncytial masses are produced, with loss of cell boundaries. The virus agglutinates chick and human type 0 erythrocytes. Adsorption and hemagglutination occur at    4 °C, and elution of virus takes place rapidly at 37 °C. However, the cells reagglutinate when returned to 4 °C. Mumps patients develop type 2 antibodies.

Parainfluenza virus 2 occurs spontaneously in 30% of lots of monkey kidney cells grown in culture, The monkey virus SV5 is antigenically related.

Parainfluenza 3

The viruses in this group are also known as hemadsorption virus type 1 (HA-1). They are de­tected in monkey kidney cultures by the hemadsorp­tion technique. Serial passage in culture may lead to cytopathic changes. Multinucleated giant cell plaques are produced under agar in certain human cell lines.

The virus has been isolated from children with mild respiratory illnesses, croup, bronchiolitis, or pneumonitis. Strains of type 3 vims have been isolated from nasal secretions of cattle ill with a respiratory syndrome known as “shipping fever. * * At least 70% of market cattle bled at slaughter have parainfluenza 3 antibodies.

Parainfluenza 4 and 5

These viruses are not known to cause any human illness, although antibodies are widespread. Their growth in cell culture can be recognized by the hemad­sorption method.

Clinical Features and Control. Children in the first year of life with primary infections caused by parainfluenza virus type 1, 2, or 3 may have serious illness ranging from laryngo-tracheitis and croup (particularly type 2) to bronchitis, bronchiolitis, and pneumonitis (particularly type 3).

Virtually all infants have maternal antibodies to parainfluenza viruses in serum, yet such antibodies do not prevent infection or disease. Reinfection of older children and adults also occurs in the presence of antibodies arising from an earlier infection. Such rein-fections usually present as nonfebrile upper respiratory infections (“colds”). The incubation for type 1 is 5-6 days; that for type 3 is 2-3 days. Most children have acquired an­tibodies to all 3 types before age 10.

Killed parainfluenza vaccines induce serum anti­bodies but no immunity. Live vaccines arc being inves­tigated.

 

MEASLES (Rubeola)

Measles is an acute, highly infectious disease characterized by a maculopapular rash, fever, and res­piratory symptoms.

Properties of the Virus

A. Morphology and Biologic Properties: Mea­sles virus is a typical paramyxovirus, related to canine distemper and bovine rinderpest. All 3 lack neuraminidase activity. Measles agglutinates monkey erythrocytes at 37 °C but does not elute, and it interacts with a distinct cell receptor. Measles virus also causes hemolysis, and this activity can be separated from that of the hemagglutinin.

 

 

 

B. Animal Susceptibility and Growth of Virus:

The experimental disease has been produced in mon­keys. They develop fever, catarrh, Koplik’s spots, and a discrete papular rash. The virus has been grown in chick embryos; in cell cultures of human, monkey, and dog kidney tissue; and in human continuous cell lines. In cell cultures, multinucleate syncytial giant cells form by fusion of mononucleated ones, and other cells become spindle-shaped in the course of their degenera­tion. Nuclear changes consist of margination of the chromatin and its replacement centrally with an acidophilic inclusion body. Measles virus is relatively unstable after it is released from cells. During the culture of the virus, the intracellular virus liter is 10 or more times the extracellular liter.

 

Pathogenesis and Pathology

The virus enters the respiratory tract, enters cells, and multiplies there. During the prodrome, the virus is present in the blood, throughout the respiratory tract, and in nasopharyngeal, tracheobronchial, and con-junctival secretions. It persists in the blood and nasopharyngeal secretions for 2 days after the appear­ance of the rash, Transplacental transmission of the virus can occur.

Koplik’s spots are vesicles in the mouth formed by focal exudations of serum and endothelial celts, followed by focal necrosis. In the skin the superficial capillaries of the corium are first involved, and it is here that the rash makes its appearance. Generalized lymphoid tissue hyperplasia occurs. Multinucleate giant cells are found in lymph nodes, tonsils, adenoids, spleen, appendix, and skin. In encephalomyelitis, there are petechial hemorrhages, lymphocytic infiltra­tion, and. later, patchy demyelination in the brain and spinal cord.

Measles nucleoprotein antigens have been iden­tified by immunofluorescence within inclusion bodies ierve cells of the brain in subacute sclerosing panencephalitis (SSPE). The virus has been grown by co-cultivating HeLa cells with brain biopsy material or lymph node material from patients. The presence of latent intracellular measles virus in these specimens suggests a tolerant infection with de­fective cell-mediated immunity.

If measles antibody is added to cells infected with measles virus, the viral antigens on the cell surface are altered. By expressing fewer viral antigens on the surface, cells may avoid being killed by antibody- or cell-mediated cytotoxic reactions, yet may retain viral genetic information. This may lead to persistent infec­tion as found in SSPE patients.

 

Clinical Findings

The incubation period is about 10 days to onset of fever and 14 days to appearance of rash. The prodro-mal period is characterized by fever, sneezing, cough­ing, running nose, redness of eyes, Koplik’s spots (enanthems of the buccal mucosa), and lymphopenia. The fever and cough persist until the rash appears and then subside within 1-2 days. The rash spreads over the entire body within 2-4 days, becoming brownish in 5-10 days. Symptoms of the disease are most marked when the rash is at its peak but subside rapidly thereafter.

In measles, the respiratory tract becomes more susceptible to invasion by bacteria, especially hemo-lytic streptococci; bronchitis, pneumonia, and otitis may follow in 15% of cases.

Encephalomyelitis occurs in about 1:1000 cases. There appears to be no correlation between the severity of the measles and the appearance of neurologic com­plications. The cause of measles encephalitis is un­known . It has been suggested that early central nervous system involvement is caused by direct viral invasion of the brain. Later appearance of central nervous sys­tem symptoms is associated with demyelination and may be an immunopathologic reaction. Symptoms re­ferable to the brain usually appear a few days after the appearance of the rash, often after it has faded. There is a second bout of fever, with drowsiness or convulsions and pleocytosis of the cerebrospinal fluid. Survivors may show permanent mental disorders (psychosis or personality change) or physical disabilities, particu­larly seizure disorders. The mortality rate in encepha­litis associated with measles is about 10-30%, and many survivors (40%) show sequelae.

Measles virus appears to be responsible for sub-acute scterosing panencephalitis (SSPE), a fatal de­generative brain disorder. The disease manifests itself in children and young adults by progressive mental deterioration, myoclonic jerks, and an abnormal dec-troencephalogram with periodic high-voltage com­plexes. The disease develops a number of years after the initial measles infection.

 

Laboratory Diagnosis

Measles is usually easily diagnosed on clinical grounds. About 5% of cases lack Koplik’s spots and are difficult to differentiate clinically from infection with rubella virus, certain enteroviruses, and adenoviruses.

A. Recovery of Virus. Measles virus can be iso­lated from the blood and nasopharynx of a patient from 2-3 days before the onset of symptoms to 1 day after the appearance of rash. Human amnion or kidney cell cultures are best suited for isolation of virus.

B. Serology. Specific neutralizing, hemaggluti-nation-inhibiting, and complement-fixing antibodies develop early, with maximal liters near the time of onset of rash. There is only a gradual decline in anti­body liter with age.

Measles and canine distemper share an antigen. Measles patients develop antibodies that cross-react with canine distemper virus. Similarly, dogs, after infection with distemper virus, develop antibodies that fix complement with measles antigen. Rinderpest virus is also related to measles.

Immunity. There appears to be only one antigenic type of measles virus, as one attack generally confers lifelong immunity. Most so-called second attacks represent errors in diagnosis of the initial or the second illness.

Epidemiology. Measles is endemic throughout the world. In gen­eral, epidemics recur regularly every 2-3 years. The state of immunity of the population is the determining factor. The disease flares up when there is an accumu­lation of susceptible children. By age 20 years, over 80% have had an attack of the disease. The severity of an epidemic is a function of the number of susceptible individuals. Only about 1% of susceptible persons fail to contract measles on their first close contact with a patient.

When the disease is introduced into isolated communities where it has not been endemic, all age groups develop clinical measles. A classic example of this was the introduction of measles into the Faroe Islands in 1846; only people over age 60 years, who had been alive during the last epidemic, escaped the disease. In places where the disease strikes rarely, its consequences are often disastrous, and the mortality rate may be as high as 25%.

The highest incidence of measles is in the late winter and spring. Infection is contracted by inhalation of droplets expelled in sneezing or coughing. Measles is spread chiefly by children during the catarrhal pro-dromal period; they are infectious from 1-2 days prior to the onset of symptoms until a few days after the rash has appeared.

Control. Live attenuated measles virus vaccine effectively prevents measles. About 95% of children properly inoculated with live virus vaccine develop antibodies that persist for at least 14 years.

Less attenuated vaccine virus may produce fever and a modified skin rash in a proportion of vaccinees; this reaction can be prevented by the simultaneous administration of gamma globulin (0.02 mL/kg body weight) at a separate site from the vaccine. The more attenuated vaccine vimses do not produce symptoms and do not require the use of gamma globulin. The different vaccine viruses appear to be equally effective in producing immunity.

Measles antibodies cross the placenta and protect the infant during the first 6-10 months of life. Vacci­nation with the live virus fails to take during this period, and measles immunization should be deferred until 15 months of age. This applies both to monova-lent measles vaccine and to combined measles-mumps-rubella vaccine.

When the live vaccine was first introduced, it was often given to infants in the first year of life. This did not produce immunity, and such children must be revaccinated.

Vaccination is not recommended in persons with febrile illnesses or allergies to eggs or other products used in the production of the vaccine, and in persons with immune defects.

Epidemiologic studies have shown that the risk, if any, of SSPE occurring in vaccinated persons is much less than the risk of its occurring in persons who have natural measles.

Killed measles vaccine should not be used, as certain vaccinees become sensitized and develop either local reactions when revaccinated with live attenuated vims or severe atypical measles when infected with wild virus or even with live vaccine vims (see Atypical measles, above).

Measles may be prevented or modified by ad­ministering antibody early in the incubation period. Human gamma globulin contains antibody liters of 200-1000 against 100 TCIDso of virus. With small doses, the disease can be made mild and immunity ensues. With a large dose of gamma globulin, the disease can be prevented; however, the person remains susceptible to infection at a later date. Antibodies given later than 6 days after exposure are not likely to influence the course of the disease.

 

RESPIRATORY SYNCYTIAL (RS) VIRUS

This labile paramyxovirus produces a characteris­tic syncytial effect, the fusion of cells in human cell culture. It is the single most serious cause of bron-chiolitis and pneumonitis in infants.

The particle is slightly smaller (80-120 nm) than other paramyxoviruses, and the nucleocapsid mea­sures 11-15 nm. Although RS is one of the most labile of viruses, it can be stabilized by molar MgSC>4 (like measles and other paramyxoviruses). RS virus does not hemagglutinate. A soluble complement-fixing antigen can be separated from the virus particle. RS virus can be grown in cell culture, but it fails to grow in eggs or in laboratory animals. Immunofluo-rescence can determine the virus antigen in cell cul­ture.

RSV- syncytium formation

 

Immunofluoresent stain

 

RS virus can be isolated from about 40% of in­fants under age 6 months suffering from bronchiolitis and from about 25% with pneumonitis, but it is almost never isolated from healthy infants. RS virus infection in older infants and children results In milder respi­ratory tract infection than in those under 6 months of age. Adult volunteers can be reinfected with RS vims (in spite of the presence of specific antibodies), but the resulting symptoms are those of an upper respiratory infection, a “cold.”

RS viruse spreads extensively in children every year during the winter season. Reinfection commonly occurs in children, but each subsequent infection is milder than the preceding ones. Nosocomial infections occur iurseries and on pediatric hospital wards. Transmission occurs primarily via the hands of staff members. Hand washing after every patient contact, wearing gowns and gloves, and isolation of infected patients reduce nosocomial spread.

RS virus grows slowly in cell cultures (4-8 days). For more rapid results, the direct immunofluorescence test with RS antiserum can be applied to nasopharyn-geal smears containing exfoliated cells.

Maternal RS antibody is transmitted to the fetus, but it does not protect the infant from disease.

The clinical disease in young infants may actually be the result of an antigen-antibody reaction that re­sults when the infecting virus meets maternally trans­mitted antibody. Killed RS vaccines may do more harm than good. Efforts to develop an attenuated vac­cine that infects subclinically and induces nasal anti­body are in progress.

RS virus occurs spontaneously in chimpanzees and has been associated with coryza in these primates.

 

Students’ Practical activities

1. To inoculate cell cultures HeLa with  centrifuged feces from the patient (poliomyelitis is suspected).

There is cell culture HeLa in 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 1,5–2,0 ml the patient’s feces. To close the bottle and to put it on a horizontal surface by the line upwards on 20 minutes at a room temperature for adsorption of the viruses on the cells surface. After that contents of the a bottle sterilely to decant in disinfectant solution and to pour 2,0 ml of medium 199 with phenolrot indicator.

In a few days (4-5) after inoccultation we observe corresponding change in the pH (making the medium orange-coloured, because cell activity in the nutrient medium results in accumulation of acid products).

2. To carry out colorimetric neutralization test with paired sera for serological diagnosis of poliomyelitis.

Schematic Representation of Neutralization test for serological diagnosis of poliomielitis

 

Cell activity in the nutrient medium results in accumulation of acid products, which induces a corresponding change in the pH (making the medium or­ange-coloured). The titer of antibody in the second serum must increase in 4 times as compared with the first one.

3. To carry out of Hemagglutination test with allantoic fluid of chicken embryos, which were previously inoculated by the washings from the nasopharynx of the patient for viruses indication. 

Hemagglutination test is performed in plexiglas plates. In the two wells place: in first – 0,2 ml of Isotonic sodium chloride solution, in second – 0,2 ml allantoic fluid. Then in each wells add 0,2 ml of 1 % suspensions of chicken erythrocytes.

The result of the test are assessed in 30 minutes at a room temperature. In the control well a markedly localized erythrocytes sediment (“rouleaus”), and in the experimental well the rapid erythrocytes agglutination with starlike, marginally festooned sediment (“umbrella”) on the bottom are observed.

4.  To carry out Hemagglutination inhibition test for the determination of influenza viruses subtype .

Table

The scheme Hemagglutination inhibition test for subtype of influenza viruses determination

 

 

Test results are assessed after complete erythrocyte sedimentation in control. In the experimental well a markedly localized erythrocytes sediment (“rouleaus”), and in the control well the rapid erythrocytes agglutination with starlike, marginally festooned sediment (“umbrella”) on the bottom are observed.

5. To carry out Hemagglutination inhibition test for serological diagnosis of influenza (determination of titers of antiviral antibodies increasing).

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 the rapid erythrocytes agglutination with starlike, 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.

The scheme Hemagglutination inhibition test

for serological diagnosis of influenza

 

 

6. To inoculate cell cultures  by saliva of the patient with mumps.

There is cell culture HeLa in 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 1,5–2,0 ml of the patient’s saliva. To close the bottle and to put it on a horizontal surface by the line upwards on 20 minUTes at a room temperature for adsorption of the viruses on the cells surface. After that contents of the a bottle sterilely to decant in disinfectant solution and to pour 2,0 ml of medium 199.

In 48–72 hours after inoccultation by light microscopy we observe, that the mumps viruses cause development of multinuclear gigantic cells with cytoplasmic inclusions. In further the complete destruction of cell monolayer with exfoliation of degenerated cells from a glass has observed.

 7. To carry out Complement fixation test  with paired sera  for mumps diagnosis. 

The scheme of Complement fixation test

 

 

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.