Adenoviruses. Laboratory diagnosis of diseases.
Herpesviruses. Laboratory diagnosis of herpes infections
Adenovirus Family
Adenoviruses consist of 2 genera, one that infects birds and another that infects mammals. Human adenoviruses are divided into 5 groups (A-E) based on their physical, chemical, and biologic properties. There are at least 37 antigenic types of human adenoviruses that may produce subclinical infection, respiratory tract or eye diseases, and occasionally other disorders. A few types serve as models for cancer induction in animals.
Properties of the Virus. Structure: Infective virions, 70-90 nm in diameter, are icosahedrons with capsids composed of 252 capsomeres. Three structural proteins, produced in large excess, constitute “soluble antigens” A, B, and C (Table). There is no envelope. The DNA is linear and double-stranded (MW 20-30 x 106). The guanine-cytosine (G + C) content of the DNA is lowest (48-49%) in group A (types 12, 18, and 31), which are the most strongly oncogenic types. The DNA can be isolated in an infectious form capable of transforming cells in culture.
Table.
Comparative data on adenovirus type 2 morphologic and antigenic subunits and protein components
Dodecon: Hemagglutinin made up of 12 pentons
with their fibers.
Animal Susceptibility and Transformation of Cells. Most laboratory animals are not readily infected with adenoviruses. Newborn hamsters sustain a fatal infection with type 5 and develop malignant tumours when inoculated with any of 8 or more types, including types 12, 18, and 31. Adenovirus cannot be recovered from these tumours, but in the tumour a new antigen can be detected by complement fixation or immunofluorescence. This tumour, or T, antigen also develops in hamster cells that are infected or transformed by oncogenic adenovirus types. Transformed cells produce tumours when inoculated into adult hamsters but do not contain infectious virus. Only a small part (< 10%) of the adenovirus genome is present in many transformed cells. This explains the inability to recover infectious virus from such cells.
Adenovirus messenger RNA (mRNA) can be detected in transformed or tumour cells. Different types of adenovirus result in different mRNA in transformed cells.
In human tumours, adenovirus DNA or mRNA has never been found.
Antigenic Properties. All adenoviruses contain a common complement-fixing antigen mat persists in suspensions of virus treated with heat or formalin to inactivate infectivity. At least 47 antigenic types have been isolated from humans and many additional ones from various animals. They are typed by cross-neutralization tests or hemagglutination-inhibition.
The major antigens, their size, and their structural position in the virion are shown in Table 1. Group-reactive complement-fixing antigens are hexons that form a majority of capsomeres and are 8 nm in diameter. Pentons have a similar size, occur at the 12 vertices of the capsid, and have a fiber protruding from them. The penton base carries a toxinlike activity that results in detachment of cells from the surface on which they are growing. Pentons and fibers are associated with hemagglutinating activity.
Group B adenoviruses (types 3,7,11, 14, 16,21, 34, 35) clump rhesus but not rat erythrocytes: group D (types 8, 9, 10, 13, 15, 17, 19. 22, 23, 24, 26, 27, 29, 30, 32, 33, 36, 37) clump rat but not rhesus erythrocytes; groups C (types 1, 2, 5, 6) and D (type 4) only partly clump rat cells. Types 20, 25, and 28 are atypical in that they have the physical and chemical properties of group D but agglutinate only rhesus cells. Group A (oncogenic types 12, 18, and 31) adenoviruses usually fail to hemagglutinate. Inhibition of hemagglutination by type-specific sera can be used for typing isolates. Some cross-reactions, however, do occur.
Virus Growth in Cell Culture. Adenoviruses are cytopathic for human cell cultures, particularly primary kidney and continuous epithelial cells. Growth of virus in tissue culture is associated with a stimulation of acid production (increased glycolysis) in the early stages of infection. The cytopathic effect usually consists of marked rounding and aggregation of affected cells into grapelike clusters. The infected cells do not lyse even though they round up and leave the glass surface on which they have been grown.
In HeLa cells infected with adenovirus types 3,4, and 7, rounded intranuclear inclusions containing DNA are seen. The virus particles develop in the nucleus and frequently exhibit crystalline arrangement. Many cells infected with type 5 virus also contain crystals, but these crystals are composed of a protein that has not been clearly identified.
During adenovirus replication in cultures of human cells, at least 12 virus-specific polypeptides are synthesized. These peptides are cataloged and their relationship to the virus structure is shown in Table 1.
Adenovirus DNA replication occurs in the nucleus and requires host cell DNA polymerase. Adenovirus mRNA is also made in the nucleus in a complex sequence that requires first the synthesis of larger molecules which are broken up and some sections of which are respliced by special enzymes. The spliced mRNA is translated into virus proteins.
Adenovirus-specific proteins are synthesized in the cytoplasm of infected cells and then move rapidly into the nucleus, where viral maturation occurs. In the adenovirus growth cycle in human epithelial cells, new virus particles can be detected about 16-20 hours after inoculation and continue to be formed at a uniform rate for the next 24 hours. About 7000 virus particles are produced per infected cell, and most of them remain intracellular. Particles having a density of 1.34 are infectious (one particle in 5 is infectious), whereas those having densities of less than 1.30 are noninfectious, since they lack the DNA core. Crude infected cell lysates show huge quantities of capsomeres, sometimes partially assembled into viral components.
When infecting cells derived from species other than humans, the human adenoviruses undergo an abortive replication cycle. Adenovirus tumour antigen, mRNA, and DNA are all synthesized, but no capsid proteins or infectious progeny are produced.
Adenovirus-SV40 “Hybrids”: Certain adenoviruses grown in monkey kidney cell cultures have become “contaminated” with the monkey virus SV40. While some of it was free in the mixture, other SV40 genomes became covalently linked to the adenovirus, so that stable “hybrids” were formed. Two types of hybrids have been identified. One is a defective adenovirus-SV40 genome encased in an adenovirus capsid. The other consists of nondefective (i.e., self-replicating) adenovirus type 2 that carries 5-40% of the SV40 genome. These hybrids have been used in genetic analysis but have no manifest medical relevance.
Adenoassociated Virus (AAV): In some adenovirus preparations, small 20-nm particles were found. These proved to be small viruses that could not replicate unless adenovirus (or sometimes herpesvirus) was present as a helper. AAV contains single-stranded DNA (MW 1.6 x 106) and is serologically unrelated to adenovirus. Four antigenic types of AAV are known, 3 of which infect humans but do not seem to produce disease. AAV can infect cells in the absence of an adenovirus helper and induce a latent infection. AAV enters the cell nucleus and is uncoated there, but no mRNA synthesis occurs. Upon addition of an adenovirus, AAV is “rescued” and replication occurs
Pathogenesis. Adenoviruses infect epithelial cells of mucous membranes, the cornea, and other organ systems. They can be isolated from such structures during acute illness and may persist for long periods. Types 1,2,5 and 6 can be isolated from surgically removed adenoids or tonsils of most children by growing the epithelium in vitro. Gradual removal of antibody during long culture in vitro permits the viruses to grow, as they cannot be isolated directly from suspensions of such tissues.
Most human adenoviruses grow in intestinal epithelium after ingestion but usually do not produce symptoms or lesions.
Clinical Findings. Adenovirus diseases include syndromes designated as undifferentiated acute respiratory disease, pharyngoconjunctival fever, nonstreptococcal exudative pharyngitis, and primary atypical pneumonia not associated with the development of cold agglutinins.
Pharyngoconjunctival fever may be caused by several adenovirus types. It is characterized by fever, conjunctivitis, pharyngitis, malaise, and cervical lymphadenopathy. The conjunctivitis is readily reproduced when any adenovirus is swabbed onto the eyes of volunteers, However, under natural conditions, only types 3 and 7 regularly cause outbreaks in which conjunctivitis is a predominant symptom. Types 1, 2, 5, 6, 37, and many others have produced sporadic cases of conjunctivitis.
Types 8 and 19 cause epidemic keratoconjunctivitis (shipyard eye). The disease is characterized by an acute conjunctivitis, with enlarged, tender preauricular nodes, followed by keratitis that leaves round, subepithelial opacities in the cornea for up to 2 years. Type 8 infections have been characterized by their lack of associated systemic symptoms except in infants. Intussusception of infancy has been ascribed to adenoviruses 1, 2, 3, and 5.
Types 11 and 21 may be a cause of acute hemorrhagic cystitis in children. Virus commonly occurs in the urine of such patients- Type 37 occurs in cervical lesions and in male urethritis and may be sexually transmitted.
A newly discovered serotype has been associated with infantile gastroenteritis. The virus is abundantly present in stools but has not been grown in cell culture.
Laboratory Diagnosis. Recovery of Virus. The viruses are isolated by inoculation of tissue cultures of human cells in which characteristic cytopathic changes are produced.
The viruses have been recovered from throat swabs, conjunctival swabs, rectal swabs, stools of patients with acute pharyngitis and conjunctivitis, and urine of patients with acute hemorrhagic cystitis. Virus isolations from the eye are obtained mainly from patients with conjunctivitis.
A new serotype that has not been isolated in cell cultures can be detected by direct examination of fecal extracts by electron microscopy or by enzyme-linked immunosorbent assay.
Serology. In most cases, the neutralizing antibody titer of infected persons shows a 4-fold or greater rise against the type recovered from the patient and a lesser response to other types. Neutralizing antibodies are measured in human cell cultures using the cytopathic end point in tube cultures or the colour test in panel cups. The latter test depends upon the phenomenon that adenovirus growing in HeLa cell cultures produces an excess of acid over that of uninfected control cultures. This viral lowering of pH can be prevented by immune serum. The pH is measured by incorporating phenol red into the medium and observing the colour changes after 3 days of incubation. Serum and cell control cultures reach a pH of 7.4; virus activity is indicated by a pH of 7.0; and neutralization is presumed to have occurred when the pH is 0.2 unit above that of the virus control.
Infection of humans with any adenovirus type stimulates a rise in complement-fixing antibodies to adenovirus antigens of all types. The CF test, using the common antigen, is an easily applied method for detecting infection by any member of the group.
A sensitive radioimmunoassay can measure serum antibody to type 5 fiber antigen. In response to vaccination with the fiber subunit, volunteers exhibited a 54-fold increase in antifiber antibody.
Immunity. Studies in volunteers revealed that type-specific neutralizing antibodies protect against the disease but not always against reinfection. Infections with the viruses were frequently induced without the production of overt illness.
Neutralizing antibodies against one or more types may be present in over 50% of infants 6-11 months old. Normal healthy adults generally have antibodies to several types. Neutralizing antibodies to types 1 and 2 occur in 55-70% of individuals age 6-15, but antibodies to types 3 and 4 are less prevalent. Neutralizing antibodies probably persist for life.
Infants are usually born without complement-fixing antibodies but develop these by age 6 months. Older individuals with neutralizing antibodies to 4 or more strains frequently give completely negative complement fixation reactions. For military recruits, the incidence of infection (especially due to types 3 and 4) was not influenced by the presence of group complement-fixing antibodies.
Epidemiology. Adenoviruses can readily spread from person to person. Type 1, 2, 5, and 6 infections occur chiefly during the first years of life and are associated with fever and pharyngitis or asymptomatic infection. These are the types most frequently obtained from the adenoids and tonsils.
In children and young adults, types 3 and 7 commonly cause upper respiratory illness, pharyngitis, and conjunctivitis. While the illness is usually mild, occasionally there is high fever, cervical lymphadenitis, and even pneumonitis. Sometimes enteric infection produces gastroenteritis, but more commonly it is asymptomatic. Types 11 and 21 can produce acute hemorrhagic cystitis in children.
In adolescents and young adults, eg, college populations, only 2-5% of respiratory illness is caused by adenoviruses. In sharp contrast, respiratory disease due to types 3, 4, 7, 14, and 21 is common among military recruits. Adenovirus disease causes great morbidity when large numbers of persons are being inducted into the armed forces; consequently, its greatest impact is during periods of mobilization. During a 1-year study, 10% of recruits in basic training were hospitalized for a respiratory illness caused by an adenovirus. During the winter, adenovirus accounted for 72% of all the respiratory illness. However, adenovirus disease is not a problem in seasoned troops.
The follicular conjunctivitis caused by many adenovirus types resembles chlamydial conjunctivitis and is self-limited.
Epidemic keratoconjunctivitis caused by type 8 spread in 1941 from Australia via the Hawaiian Islands to the Pacific Coast. There it spread rapidly through the shipyards and other industries, thence to the East Coast, and finally to the Midwest. A large outbreak caused by type 8 occurred in
Canine hepatitis virus is an adenovirus. Therefore, humans infected with adenoviruses develop group complement-fixing antibodies that also react with canine hepatitis virus.
In prospective family studies, adenovirus infections have been found to be predominantly enteric; they may be abortive or invasive and followed by persistent intermittent excretion of virus. Such excretion is most characteristic of types 1,2,3, and 5, which are usually endemic. Infection rates are highest among infants, but siblings who introduce the infection into a household are more effective in spreading the disease than are infants; similarly, duration of excretion is more important than the mode. In the families studied, neutralizing antibodies provided immunity (85% protective) against homotypic but not heterotypic infection. The contribution of adenoviruses to all infectious illness in the families, based on virus-positive infections only, was 5% in infants and 3% in the 2- to 4-year-old age group.
Prevention & Control. A trivalent vaccine was prepared by growing type 3,4, and 7 viruses in monkey kidney cultures and then inactivating the viruses with formalin. However, when it was found that the vaccine strains were contaminated genetically with SV40 tumour virus determinants, this vaccine was withdrawn from use. Subsequently, it was found that most adenovirus strains do not replicate in monkey cells unless SV40 is present as a helper virus. Thus, a vaccine had to be made from noncontaminated live virus that could be grown in human diploid cells, The vaccine is given orally in a coated capsule to liberate the virus into the intestine. By this route, the live vaccine produces a subclinical infection that confers a high degree of immunity against wild strains. It does not spread from a vaccinated person to contacts. Such live virus vaccines against type 4 and type 7 are licensed and recommended for immunization of military populations. When both are administered simultaneously, vaccines respond with neutralizing antibodies against both virus types.
Rigid asepsis during eye examination is essential in the control of epidemic keratoconjunctivitis.
Herpesvirus Family
All herpesviruses have a core of double-stranded DNA surrounded by a protein coat that exhibits icosahedral symmetry and has 162 capsomeres. The nucleocapsid is surrounded by an envelope. The enveloped form measures 150-200 nm; the “naked” virion, 100 nm. The double-stranded DNA (MW 85-150 x lO6) has a wide range of guanine + cytosine content in different herpesviruses, There is little DNA homology among different herpesviruses, except herpes simplex types 1 and 2.
Various classifications for herpesviruses have been proposed, but individual virus names are generally used. Common and important herpesviruses of humans include herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr (EB) virus, and cytomegalovirus. They have a propensity for subclinical infection, latency following the primary infection, and reactivation thereafter.
Herpesviruses that infect lower animals are B virus of Old World monkeys; herpesviruses saimiri, aotus, and ateles; marmoset herpesvirus of New World monkeys, pseudorabies virus of pigs; virus III of rabbits; infectious bovine rhinotracheitis virus; and many others. Herpesviruses are also known for birds, fish, fungi, and oysters, although the only link between some of these viruses is their appearance in the electron microscope.
Herpesviruses have been linked with malignant diseases in humans and lower animals: herpes simplex virus type 2 with cervical and vulvar carcinoma; EB virus with Burkilt ‘s lymphoma of African children and with nasopharyngeal carcinoma; Lucke virus with renal adenocarcinomas of the frog; Marek’s disease virus with a lymphoma of chickens; Hinze virus with a lymphoma of rabbits; and a number of New World primate herpesviruses with reticulum cell sarcomas and lymphomas in these animals.
HERPES SIMPLEX (Human Herpesvirus 1 & 2) (Herpes Labialis, Herpes Genitalis, & Many Other Syndromes).
Infection with herpes simplex virus (herpesvirus hominis) may take several clinical forms. The infection is most often inapparent. The usual clinical manifestation is a vesicular eruption of the skin or mucous membranes. Infection is sometimes seen as severe keratitis, meningoencephalitis, and a disseminated illness of the newborn.
Properties of the Virus. Morphologically and chemically, herpes simplex virus has been studied in great detail (see Fig. 1). The envelope is derived from the nuclear membrane of the infected cell. It contains lipids, carbohydrate, and protein and is removed by ether treatment. The double-stranded DNA genome is linear (MW 85-106 x 106). Types 1 and 2 show 50% sequence homology. Treatment with restriction endonucleases yields characteristically different cleavage patterns for type 1 and 2 viruses and even for different strains of each type. This “fingerprinting” of strains allows epidemiologic tracing of a given strain, whereas in the past, the ubiquitousness of herpes simplex virus made such investigations impossible.
Animal Susceptibility and Growth of Virus. The virus has a wide host range and can infect rabbits, guinea pigs, mice, hamsters, rats, and the chorioallantois of the embryonated egg.
In rabbits, herpesvirus produces a vesicular eruption in the skin of the inoculated area, sometimes progressing to fatal encephalitis. Corneal inoculation results in dendritic keratitis, which may progress to encephalitis, The virus may remain latent in the brains of survivors, and anaphylactic shock can precipitate an acute relapse of encephalomyelitis. Herpetic keratitis heals, but infective herpesvirus may be recovered from the eye intermittently with or without clinical activity. The virus remains latent in the trigeminal ganglion.
In the chorioallantoic membrane of embryonated eggs the lesions are raised white plaques, each induced by one infectious virus particle. The plaques produced by herpesvirus type 2 are larger than the tiny plaques produced by type 1 virus. The virus grows readily and produces plaques in almost any cell culture. Infected cells develop inclusion bodies and then undergo necrosis (cytopathic effect).
In Chinese hamster cells, which contain 22 chromosomes, the virus causes breaks in region 7 of chromosome No. 1 and in region 3 of the X chromosome. The Y chromosome is unaffected.
Virus Replication. The virus enters the cell either by fusion with the cell membrane or by pinocytosis. It is then uncoated, and the DNA becomes associated with the nucleus. Normal cellular DNA and protein synthesis virtually stop as virus replication begins. The virus induces a number of enzymes, at least 2 of which — thymidine kinase and DNA polymerase — are virus-coded. Thymidine kinases produced by different herpesviruses are serologically different from each other and different from the enzyme in uninfected cells. Phosphonoacetic acid specifically inhibits herpesvirus replication by inhibiting viral DNA polymerase.
Viral proteins are made in a controlled sequence that must proceed stepwise. They are made in the cytoplasm and most are transported to the nucleus, where they take part in virus DNA synthesis and the assembly of nucleocapsids, Maturation occurs by budding of nucleocapsids through the altered inner nuclear membrane. Enveloped virus particles are then released from the cell through tubular structures that are continuous with the outside of the cell or from vacuoles that release their contents at the cell surface.
Defective Interfering Herpesvirions: Serial passage of undiluted herpes simplex virus results in cyclic production of infectious and defective virions. The DNA in defective virions is made up of reiterated sequences of small fragments of the virus DNA. Defective virions interfere with the replication of standard virus and stimulate overproduction of a large polypeptide, which may have a regulatory function. The biologic role of the defective virions is not known.
Antigenic Properties. There are 7-12 precipitating antigens that represent structural and non-structural viral proteins. Some of these antigens are common to both types 1 and 2 and some are specific for one type. A number of tests, eg, fluorescent antibody, complement fixation, virus neutralization, and radioimmunoassay, have been used to detect herpesvirus antigens.
Differentiation of Types 1 and 2. Herpes simplex virus types 1 and 2 cross-react serologically but may be distinguished by a number of tests: (1) The use of type-specific antiserum prepared by adsorption of the viral antiserum with heterotypically infected cells or by inoculation of rabbits with individual type-specific proteins. (2) The greater temperature sensitivity of type 2 infectivity. (3) Preferential growth in different cell species. (4) Restriction enzyme patterns of virus DNA molecules. (5) Differences in the polypeptides produced by type 1 and type 2.
Oncogenic Properties: After inactivation of their lytic capabilities by ultraviolet irradiation or other means, herpesvirus types 1 and 2 can cause transformation of cultured hamster cells, which may induce tumours when inoculated into newborn hamsters. Viral genetic information can be demonstrated in the tumour cells.
Pathogenesis & Pathology. The lesion in the skin involves proliferation, ballooning degeneration, and intranuclear acidophilic inclusions. In fatal cases of herpes encephalitis, there are meningitis, perivascular infiltration, and nerve cell destruction, especially in the cortex. Neonatal generalized herpes infection causes areas of focal necrosis with a mononuclear reaction and formation of intranuclear inclusion bodies in all organs. Survivors may sustain permanent damage.
The fully formed early inclusion (Cowdry type A inclusion body) is rich in DNA and virtually fills the nucleus, compressing the chromatin to the nuclear margin. Later, the inclusion loses its DNA and is separated by a halo from the chromatin at the nuclear margin.
Clinical Findings. Herpesvirus may cause many clinical entities, and the infections may be primary or recurrent. Primary infections occur in persons without antibodies and often result in the virus assuming a latent state in sensory ganglia of the host. Latent infections persist in persons with antibodies, and recurrent lesions are common (eg, recurrent herpes labialis). The primary infection in most individuals is clinically inapparent but is invariably accompanied by antibody production.
The recurrent attacks, in the presence of viral neutralizing antibody, follow non-specific stimuli such as exposure to excess sunlight, fever, menstruation, or emotional stresses.
Herpesvirus Type 1. The clinical entities attributable to herpesvirus type 1 include the following:
1. Acute herpetic gingivostomatitis (aphthous stomatitis, Vincent’s stomatitis). This is the most common clinical entity caused by primary infections with type 1 herpesvirus. It occurs most frequently in small children (1-3 years of age) and includes extensive vesiculoulcerative lesions of the mucous membranes of the mouth, fever, irritability, and local lymphadenopathy. The incubation period is short (about 3-5 days), and the lesions heal in 2-3 weeks.
2. Eczema herpeticum (Kaposi’s varicelliform eruption). This is a primary infection, usually with herpesvirus type
3. Keratoconjunctivitis. The initial infection with herpesvirus may be in the eye, producing severe keratoconjunctivitis. Recurrent lesions of the eye appear as dendritic keratitis or corneal ulcers or as vesicles on the eyelids. With recurrent keratitis, there may be progressive involvement of the corneal stroma, with permanent opacification and blindness.
4. Encephalitis. A severe form of encephalitis may be produced by herpesvirus. In adults, the neurologic manifestations suggest a lesion in the temporal lobe. Pleocytosis (chiefly of lymphocytes) is present in the cerebrospinal fluid; however, definite diagnosis during the illness can usually be made only by isolation of the virus (or by demonstrating viral antigens by immunofluorescence) from brain tissue obtained by biopsy or at post-mortem. The disease carries a high mortality rate, and those who survive often have residual neurologic defects.
5. Herpes labialis (cold sores, herpes febrilis). This is the most common recurrent disease produced by type 1. Clusters of localized vesicles occur, usually at the mucocutaneous junction of the lips. The vesicle ruptures, leaving a painful ulcer that heals without scarring. The lesions may recur, repeatedly and at various intervals of time, in the same location. The permanent site of latent herpes simplex virus is the trigeminal ganglion.
Herpesvirus Type 2. The clinical entities associated with herpesvirus type 2 include the following:
1. Genital herpes (herpes progenitalis). Genital herpes is characterized by vesiculoulcerative lesions of the penis of the male or the cervix, vulva, vagina, and perineum of the female. The lesions are more severe during primary infection and may be associated with fever, malaise, and inguinal lymphadenopathy. In women with herpesvirus antibodies, only the cervix or vagina may be involved, and the disease may therefore be asymptomatic. Recurrence of the lesions is common. Type 2 virus remains latent in lumbar and sacral ganglia. Changing patterns of sexual behaviour are reflected by an increasing number of type 1 virus isolations from genital lesions and of type 2 from facial lesions, presumably as a result of oral-genital sexual activity.
2. Neonatal herpes. Herpesvirus type 2 may be transmitted to the newborn during birth by contact with herpetic lesions in the birth canal. The spectrum of illness produced in the newborn appears to vary from subclinical or local to severe generalized disease with a fatal outcome. Severely affected infants who survive may have permanent brain damage. To avoid infection, delivery by cesarean section has been used in pregnant women with genital herpes lesions. To be effective, cesarean section must be performed before rupture of the membranes.
Severe generalized disease of the newborn can be acquired postnatally by exposure to either type 1 or 2. Efforts should be made to prevent exposure to active lesions among family and especially among hospital personnel.
Transplacental infection of the fetus with types 1 and 2 herpes simplex virus may cause congenital malformations, but this phenomenon is rare.
Miscellaneous. Localized lesions of the skin caused by type 1 or 2 may occur in abrasions that become contaminated with the virus (traumatic herpes), These lesions are seen on the fingers of dentists, hospital personnel (herpetic whitlow), or persons with genital lesions and on the bodies of wrestlers.
Primary and recurrent herpes can occur in the nose (acute herpetic rhinitis).
Mild aseptic meningitis has been attributed to the virus, and recurrent episodes of meningeal irritation have been observed.
Epidemiologic evidence has demonstrated that in most geographic areas, patients with cervical and vulvar cancer have a high frequency of type 2 antibodies. In addition, herpesvirus type 2 non-structural antigens have been detected by immunofluorescence in biopsies of cervical and vulvar carcinomas.
Laboratory Diagnosis
Recovery of Virus. The virus may be isolated from herpetic lesions (skin, cornea, or brain). It may also be found in the throat, saliva, and stools, both during primary infection and during asymptomatic periods. Therefore, the isolation of herpesvirus is not in itself sufficient evidence to indicate that this virus is the causative agent of a disease under investigation.
inoculation of tissue cultures is used for virus isolation. The appearance of typical cytopathic effects in cell culture suggests the presence of herpesvirus in 18-36 hours. The agent is then identified by neutralization test or immunofluorescence staining with specific antiserum.
Scrapings or swabs from the base of early herpetic lesions contain multinucleated giant cells.
Serology. Antibodies may be measured quantitatively by neutralization tests in cell cultures. In the early stage of the primary immune response, neutralizing antibody appears that is detectable only in the presence of fresh complement. This antibody soon is replaced by neutralizing antibody that can function without complement.
Since the only hope for treatment of herpes simplex virus encephalitis lies in early diagnosis, a rapid means of diagnosis is needed. The fluorescent antibody test using brain biopsy material is the method of choice. Passive hemagglutinating antibodies in the cerebrospinal fluid are a better indicator of the presence of infectious virus than are antibody titters in serum.
A soluble complement-fixing antigen of much smaller size than the virus can be prepared from infected chorioallantoic membranes or from tissue culture. This soluble antigen of herpesvirus can detect dermal hypersensitivity in previously infected persons. There is a good correlation between dermal hypersensitivity and the presence of serum antibodies.
Antibodies appear in 4-7 days; can be measured by neutralization, complement fixation, radioimmunoassay, or immunofluorescence; and reach a peak in 2-4 weeks. They persist with minor fluctuations for the life of the host. The majority of adults have antibodies in their blood at all times.
After a primary type I infection, the IgM neutralizing antibody response is type-specific, but after a primary type 2 infection the IgM that develops neutralizes both type 1 and type 2 virus. Subsequently, IgG antibodies react with both type 1 and type 2 antigens, albeit in varying ratios.
There is also some cross-stimulation between herpes simplex and varicella-zoster antigens in patients with pre-existing antibody to the other virus.
Immunity. Many newborns have passively transferred maternal antibodies. This antibody is lost during the first 6 months of life, and the period of greatest susceptibility to primary herpes infection occurs between ages 6 months and 2 years. Type 1 antibodies begin to appear in the population in early childhood; by adolescence they are present in most persons. Antibodies to type 2 (genital herpesvirus) rise during the age of adolescence and sexual activity.
After recovery from a primary infection (inapparent, mild, or severe), the virus is usually carried in a latent state, in the presence of antibodies.
Treatment. Topically applied idoxuridine (5-iodo-2′-deoxy-uridine, IUDR), trifluorothymidine, vidarabine (ade-nine arabinoside, ara-A), acyclovir, and other inhibitors of viral DNA synthesis are effective in herpetic keratitis. These drugs inhibit herpesvirus replication and may suppress clinical manifestations. However, the virus remains latent in the sensory ganglia, and the rate of relapse is similar in drug-treated and untreated individuals. Some drug-resistant virus strains have emerged. Most strains of type 2 herpesvirus are suppressed less effectively than type 1.
For systemic administration, vidarabine (15 mg/kg/d intravenously) is accepted in herpes encephalitis diagnosed by biopsy. Best results are obtained if treatment is begun early in the disease, before coma sets in. Vidarabine also has some effect in disseminated herpes simplex.
Other drugs, especially acyclovir, are undergoing clinical trial, and many new antiherpes compounds are being developed. Acyclovir has low toxicity and has been administered systemically to suppress the activation of a latent herpes infection in immunosuppressed patients.
Epidemiology. The epidemiology of type 1 and type 2 herpesvirus differs. Herpesvirus type 1 is probably more constantly present in humans than any other virus. Primary infection occurs early in life and is often asymptomatic or produces acute gingivostomatitis. Antibodies develop, but the virus is not eliminated from the body; a carrier state is established that lasts throughout life and is punctuated by transient attacks of herpes. If primary infection is avoided in childhood, it may not occur in later life, perhaps because the thicker adult epithelium is less susceptible or because the opportunity for contact with the virus is diminished (less contact with saliva of infected persons).
The highest incidence of type I virus carriage in the oropharynx of healthy persons occurs among children 6 months to 3 years of age. By adulthood, 70-90% of persons have type 1 antibodies.
Type 1 virus is transmitted more readily in families of lower socioeconomic groups; the most obvious explanation is their more crowded living conditions and lower hygienic standards. The virus is spread by direct contact (saliva) or through utensils contaminated with the saliva of a virus shedder. The source of infection for children is usually a parent with an active herpetic lesion.
Type 2 is usually acquired as a sexually transmitted disease, and the age distribution of primary infection is a function of sexual activity. The neonate may acquire type 2 infection from an active lesion in the mother’s birth canal.
Control. Neonates and persons with eczema should be protected from evident active herpetic lesions. Although certain drugs are effective in treatment of herpesvirus infections, once a latent infection is established there has beeo known treatment that would prevent recurrences until the recent successful results with acyclovir in immunosuppressed patients.
Little Is known about vaccines. Herpes recurs in the presence of circulating antibody, so a vaccine would be of little use in a person who already had a primary infection. A vaccine currently made in Europe has not been adequately tested.
Adenovirus, a DNA virus, was first isolated in the 1950s in adenoid tissue–derived cell cultures, hence the name. These primary cell cultures were ofteoted to spontaneously degenerate over time, and adenoviruses are now known to be a common cause of asymptomatic respiratory tract infection that produces in vitro cytolysis in these tissues.
About Adenovirus Infections
Adenoviruses are a group of viruses that can infect the membranes (tissue linings) of the respiratory tract, eyes, intestines, and urinary tract. They account for about 10% of acute respiratory infections in kids and are a frequent cause of diarrhea.
Adenoviral infections affect babies and young children much more often than adults. Childcare centers and schools sometimes have multiple cases of respiratory infections and diarrhea caused by adenovirus.
Adenoviral infections can occur at any time of the year, but:
respiratory tract problems caused by adenovirus are more common in late winter, spring, and early summer
conjunctivitis (pinkeye) and pharyngoconjunctival fever caused by adenovirus tend to affect older kids, mostly in the summer
Adenoviral infections can affect children of any age, but most occur in the first years of life — and most kids have had at least one before age 10. There are many different types of adenoviruses, so some kids can have repeated adenoviral infections.
Signs and Symptoms
Depending on which part of the body is affected, the signs and symptoms of adenoviral infections vary:
Febrile respiratory disease, an infection with fever of the respiratory tract, is the most common result of adenoviral infection in kids. The illness often appears flu-like and can include symptoms of pharyngitis (inflammation of the pharynx, or sore throat), rhinitis (inflammation of nasal membranes, or a congested, runny nose), cough, and swollen lymph nodes (glands). Sometimes the respiratory infection leads to acute otitis media, an infection of the middle ear.
Adenovirus often affects the lower respiratory tract as well, causing bronchiolitis, croup, or viral pneumonia, which is less common but can cause serious illness in infants. Adenovirus can also produce a dry, harsh cough that can resemble whooping cough (pertussis).
Gastroenteritis is an inflammation of the stomach and the small and large intestines. Symptoms include watery diarrhea, vomiting, headache, fever, and abdominal cramps.
Genitourinary infections: Urinary tract infections can cause frequent urination, burning, pain, and blood in the urine. Adenoviruses are also known to cause a condition called hemorrhagic cystitis, which is characterized by blood in the urine. Hemorrhagic cystitis usually resolves on its own.
Eye infections:
Pinkeye (conjunctivitis) is a mild inflammation of the conjunctiva (membranes that cover the eye and inner surfaces of the eyelids). Symptoms include red eyes, discharge, tearing, and the feeling that there’s something in the eye.
Pharyngoconjunctival fever, often seen in small outbreaks among school-age kids, occurs when adenovirus affects both the lining of the eye and the respiratory tract. Symptoms include very red eyes and a severe sore throat, sometimes accompanied by low-grade fever, rhinitis, and swollen lymph nodes.
Keratoconjunctivitis is a more severe infection that involves both the conjunctiva and cornea (the transparent front part of the eye) in both eyes. This type of adenoviral infection is extremely contagious and occurs most often in older kids and young adults, causing red eyes, photophobia (discomfort of the eyes upon exposure to light), blurry vision, tearing, and pain.
Contagiousness
Adenovirus is highly contagious, so multiple cases are common in close-contact settings like childcare centers, schools, hospitals, and summer camps.
The types of adenovirus that cause respiratory and intestinal infections spread from person to person through respiratory secretions (coughs or sneezes) or fecal contamination. Fecal material can spread via contaminated water, eating food contaminated by houseflies, and poor hand washing (such as after using the bathroom, before eating or preparing food, or after handling dirty diapers).
A child might also pick up the virus by holding hands or sharing a toy with an infected person. Adenovirus can survive on surfaces for long periods, so indirect transmission can occur through exposure to the contaminated surfaces of furniture and other objects.
The types of adenovirus causing pinkeye may be transmitted by water (in lakes and swimming pools), by sharing contaminated objects (such as towels or toys), or by touch.
Once a child is exposed to adenovirus, symptoms usually develop from 2 days to 2 weeks later.
Back Treatment
Adenoviral illnesses often resemble certain bacterial infections, which can be treated with antibiotics. But antibiotics don’t work against viruses. To diagnose the true cause of the symptoms so that proper treatment can be prescribed, your doctor may want to test respiratory or conjunctival secretions, a stool specimen, or a blood or urine sample.
The doctor will decide on a course of action based on your child’s condition. Adenoviral infections usually don’t require hospitalization. However, babies and young children may not be able to drink enough fluids to replace what they lose during vomiting or diarrhea and so might need to be hospitalized to treat or prevent dehydration. Also, young (especially premature) infants with pneumonia usually need to be hospitalized.
In most cases, a child’s body, with the help of the immune system, will get rid of the virus over time. Antibiotics cannot treat a viral infection, so it’s best to just make your child more comfortable.
If your child has a respiratory infection or fever, getting plenty of rest and taking in extra fluids are essential. A cool-mist humidifier (vaporizer) may help loosen congestion and make your child more comfortable. Be sure to clean and dry the humidifier thoroughly each day to prevent bacterial or mold contamination. If your child is under 6 months old, you may need to clear his or her nose with nasal saline drops and a bulb syringe.
Don’t give any over-the-counter (OTC) cold remedies or cough medicines without checking with your doctor. You can use acetaminophen to treat a fever (your doctor will tell you the proper dose); however, do not give aspirin because of the risk of Reye syndrome, a life-threatening illness.
If your child has diarrhea or is vomiting, increase fluid intake and check with the doctor about giving an oral rehydration solution to prevent dehydration.
To relieve the symptoms of pinkeye, use warm compresses and, if your doctor recommends them, a topical eye ointment or drops
Duration
Most adenoviral infections last from a few days to a week. However:
severe respiratory infections may last longer and cause lingering symptoms, such as a cough
pneumonia can last anywhere from 2-4 weeks
pinkeye can persist for another several days to a week
more severe keratoconjunctivitis can last for several weeks
adenovirus can cause diarrhea that lasts up to 2 weeks (longer than other viral diarrhea episodes)
Prevention
There’s no way to completely prevent adenoviral infections in kids. To reduce their spread, parents and other caregivers should encourage frequent hand washing, keep shared surfaces (such as countertops and toys) clean, and remove kids with infections from group settings until symptoms pass.
When to Call the Doctor
Most of these adenoviral conditions and their symptoms are also associated with other causes. Call your doctor if:
a fever continues more than a few days
symptoms seem to get worse after a week
your child has breathing problems
your child is under 3 months old
any swelling and redness around the eye becomes more severe or painful
your child shows signs of dehydration, such as appearing tired or lacking energy, producing less urine or tears, or having a dry mouth or sunken eyes
Remember, you know your child best. If he or she appears to be severely ill, don’t hesitate to call your doctor right away
VARICELLA-ZOSTER VIRUS (Human Herpesvirus 3)
VARICELLA (Chickenpox) ZOSTER (Herpes Zoster, Shingles, Zona)
Varicella (chickenpox) is a mild, highly infectious disease, chiefly of children, characterized clinically by a vesicular eruption of the skin and mucous membranes. However, in immunocompromised children the disease may be severe. The causative agent is indistinguishable from the virus of zoster.
Zoster (shingles) is a sporadic, incapacitating disease of adults (rare in children) that is characterized by an inflammatory reaction of the posterior nerve roots and ganglia, accompanied by crops of vesicles (like those of varicella) over the skin supplied by the affected sensory nerves.
Both diseases are caused by the same virus. Varicella is the acute disease that follows primary contact with the virus, whereas zoster is the response of the partially immune host to a reactivation of varicella virus present in latent form in sensory ganglia.
Properties of the Virus. Varicella-zoster virus is morphologically identical with herpes simplex virus. The virus propagates in cultures of human embryonic tissue and produces typical intranuclear inclusion bodies. Supernatant fluids from such infected cultures contain a complement-fixing antigen but no infective virus. Infectious virus is easily transmitted by infected cells. The virus has not been propagated in laboratory animals. Virus can be isolated from the vesicles of chickenpox or zoster patients or from the cerebrospinal fluid in cases of zoster aseptic meningitis.
Inoculation of vesicle fluid of zoster into children produces vesicles at the site of inoculation in about 10 days. This may be followed by generalized skin lesions of varicella. Generalized varicella may occur in such inoculated children without local vesicle formation. Contacts of such children develop typical varicella after a 2-week incubation period. Children who have recovered from zoster virus-induced infection are resistant to varicella, and those who have had varicella are no longer susceptible to primary zoster virus.
Antibody to varicella-zoster virus can be measured by CF, gel precipitation, neutralization, or indirect immunofluorescence to virus-induced membrane antigens.
The virus has a colchicinelike effect on human cells. Arrest in metaphase, overcontracted chromosomes, chromosome breaks, and formation of micronuclei are often seen.
Pathogenesis & Pathology. Varicella: The route of infection is probably the mucosa of the upper respiratory tract. The virus probably circulates in the blood and localizes in the skin. Swelling of epithelial cells, ballooning degeneration, and the accumulation of tissue fluids result in vesicle formation. Iuclei of infected cells, particularly in the early stages, eosinophilic inclusion bodies are found.
Zoster: In addition to skin lesions — histopathologically identical with those of varicella — there is an inflammatory reaction of the dorsal nerve roots and sensory ganglia. Often only a single ganglion may be involved. As a rule, the distribution of lesions in the skin corresponds closely to the areas of innervation from an individual dorsal root ganglion. There is cellular infiltration, necrosis of nerve cells, and inflammation of the ganglion sheath.
Varicella virus seems able to enter and remain within dorsal root ganglia for long periods. Years later, various insults (eg, pressure on a nerve) may cause a flare-up of the virus along posterior root fibers, whereupon zoster vesicles appear. Thus, varicella-zoster and herpes simplex viruses are similar in their ability to induce latent infections with clinical recurrence of disease in humans. However, zoster rarely occurs more than once.
Clinical Findings. Varicella: The incubation period is usually 14-21 days. Malaise and fever are the earliest symptoms, soon followed by the rash, first on the trunk and then on the face, the limbs, and the buccal and pharyngeal mucosa. Successive fresh vesicles appear in crops during the next 3-4 days, so that all stages of papules, vesicles, and crusts may be seen at one time. The eruption is found together with the fever and is proportionate to its severity. Complications are rare, although encephalitis does at times occur about 5-10 days after the rash. The mortality rate is much less than 1% in uncomplicated cases. Ieonatal varicella (contracted from the mother just before or just after birth), the mortality rate may be 20%. In varicella encephalitis, the mortality rate is about 10%, and another 10% are left with permanent injury to the central nervous system. Primary varicella pneumonia is rare in children but may occur in about 20-30% of adult cases, may produce severe hypoxia, and may be fatal.
Children with immune deficiency disease or those receiving immunosuppressant or cytotoxic drugs are at high risk of development of very severe and sometimes fatal varicella or disseminated zoster.
Zoster: The incubation period is unknown. The disease starts with malaise and fever that are soon followed by severe pain in the area of skin or mucosa supplied by one or more groups of sensory nerves and ganglia. Within a few days after onset, a crop of vesicles appears over the skin supplied by the affected nerves. The eruption is usually unilateral; the trunk, head, and neck are most commonly involved. Lymphocytic pleocytosis in the cerebrospinal fluid may be present.
In patients with localized zoster and no underlying disease, vesicle interferon levels peak early during infection (by the sixth day), whereas those in patients with disseminated infection peak later. Peak interferon levels are followed by clinical improvement within 48 hours. Vesicles pustulate and crust, and dissemination is halted.
Zoster tends to disseminate when there is an underlying disease, especially if the patient is taking immunosuppressive drugs or has lymphoma treated by irradiation.
Laboratory Diagnosis. In stained smears of scrapings or swabs of the base of vesicles, multinucleated giant cells are seen. In similar smears, intracellular viral antigens can be demonstrated by immunofluorescence staining.
Virus can be isolated in cultures of human or other fibroblastic cells in 3-5 days. It does not grow in epithelial cells, in contrast to herpes simplex, and does not infect laboratory animals or eggs. An isolate in fibroblasts is identified by immunofluorescence or neutralization tests with specific antisera.
Herpesviruses can be differentiated from pox-viruses by (1) the morphologic appearance of particles in vesicular fluids examined by electron microscopy, and by (2) the presence of antigen in vesicle fluid or in an extract of crusts as determined by gel diffusion tests with specific antisera to herpes, varicella, or vaccinia viruses, which give visible precipitation lines in 24-48 hours.
A rise in specific antibody titer can be detected in the patient’s serum by CF, Nt (in cell culture), indirect immunofluorescence tests, or enzyme immunoassay, Zoster can occur in the presence of relatively high neutralizing antibody in the blood just prior to onset. The role of cell-mediated immunity is unknown.
Immunity. Varicella and zoster viruses are identical, the 2 diseases being the result of differing host responses. Previous infection with varicella leaves the patient with enduring immunity to varicella. However, zoster may occur in persons who have contracted varicella earlier. This is a reactivation of a varicella virus infection that has been latent for years.
Prophylaxis & Treatment. Gamma globulin of high specific antibody titer prepared from pooled plasma of patients convalescing from herpes zoster (zoster immune globulin) can be used to prevent the development of the illness in immunocompromised children who have been exposed to varicella. Standard immune serum globulin is without value because of the low titer of varicella antibodies.
Zoster immune globulin is available from the American Red Cross Blood Services (through 13 regional blood centres) for prophylaxis of varicella in exposed high-risk immunodeficient or immunosuppressed children. It has no therapeutic value once varicella has started.
Idoxuridine and cytarabine inhibit replication of the viruses in vitro but are not an effective treatment for patients.
Adenine arabinoside (vidarabine, ara-A) has been beneficial in adults with severe varicella pneumonia, immunocompromised children with varicella, and adults with disseminated zoster. Human leukocyte interferon in large doses appears to be similarly beneficial.
Epidemiology. Zoster occurs sporadically, chiefly in adults and without seasonal prevalence. In contrast, varicella is a common epidemic disease of childhood (peak incidence is in children age 2-6 years, although adult cases do occur). It is much more common in winter and spring than in summer. Almost 200,000 cases are reported annually in the USA.
Varicella readily spreads, presumably by droplets as well as by contact with skin. Contact infection is rare in zoster, perhaps because the virus is absent in the upper respiratory tract.
Zoster, whether in children or adults, can be the source of varicella in children and can initiate large outbreaks.
Control. None is available for the general population. Varicella may spread rapidly among patients, especially among children with immunologic dysfunctions or leukemia or in those receiving corticosteroids or cytotoxic drugs. Varicella in such children poses the threat of pneumonia, encephalitis, or death. Efforts should be made to prevent their exposure to varicella. Zoster immune globulin may be used to modify the disease in such children who have been exposed to varicella.
A live attenuated varicella vaccine has been developed in Japan and tested in hospitalized immune-suppressed children who were exposed to varicella. It appeared to prevent spread of chickenpox. The vaccine is being used experimentally for similar high-risk children in the USA.
A number of problems are envisioned for the use of such a vaccine for the general population as opposed to high-risk patients. The vaccine would need to confer immunity comparable to that of natural infections. A short-lasting immunity might result in an increased number of susceptible adults, in whom the disease is more severe. Furthermore, any such vaccine would need to be evaluated for later morbidity due to zoster as compared to that following natural childhood infections with varicella virus.
CYTOMECALOVIRUS (Human Herpesvirus 5) (Cytomegalic Inclusion Disease)
Cytomegalic inclusion disease is a generalized infection of infants caused by intrauterine or early postnatal infection with the cytomegaloviruses. The disease causes severe congenital anomalies in about 10,000 infants in the USA per year. Cytomegalovirus can be found in the cervix of up to 10% of healthy women. Cytomegalic inclusion disease is characterized by large intranuclear inclusions that occur in the salivary glands, lungs, liver, pancreas, kidneys, endocrine glands, and. occasionally, the brain. Most fatalities occur in children under 2 years of age. Inapparent infection is common during childhood and adolescence. Severe cytomegalovirus infections are frequently found in adults receiving immunosuppressive therapy.
Properties of the Virus. Morphologically, cytomegalovirus is indistinguishable from herpes simplex or varicella-zoster virus.
In infected human fibroblasts, virus particles are assembled in the nucleus. The envelope of the virus is derived from the inner nuclear membrane. The growth cycle of the virus is slower, and infectious virus is more cell-associated than herpes simplex virus.
Animal Susceptibility. All attempts to infect animals with human cytomegalovirus have failed. A number of animal cytomegaloviruses exist, all of them species-specific in rats, hamsters, moles, rabbits, and monkeys. The virus isolated from monkeys propagates in cultures of monkey as well as human cells.
Human cytomegalovirus replicates in vitro only in human fibroblasts. although the virus is often isolated from epithelial cells of the host. The virus can transform human and hamster cells in culture, but whether it is oncogenic in vivo is unknown.
Pathogenesis & Pathology. In infants, Cytomegalic inclusion disease is con-genitally acquired, probably as a result of primary infection of the mother during pregnancy. The virus can be isolated from the urine of the mother at the time of birth of the infected baby, and typical cytomegalic cells, 25-40 mcm in size, occur in the chorionic villi of the infected placenta.
Foci of cytomegalic cells are found in fatal cases in the epithelial tissues of the liver, lungs, kidneys, gastrointestinal tract, parotid gland, pancreas, thymus, thyroid, adrenals, and other regions. The cells can be found also in the urine or adenoid tissue of healthy children. The route of infection in older infants, children, and adults is not known.
The isolation of the virus from urine and from tissue cultures of adenoids of healthy children suggests subclinical infections at a young age. The virus may persist in various organs for long periods in a latent state or as a chronic infection. Virus is not recovered from the mouths of adults. Disseminated inclusions in adults occur in association with other severe diseases.
Clinical Findings. Congenital infection may result in death of the fetus in utero or may produce the clinical syndrome of cytomegalic inclusion disease, with signs of prematurity, jaundice with hepatosplenomegaly, thrombocytopenic purpura, pneumonitis, and central nervous system damage (microcephaly, periventricular calcification, chorioretinitis, optic atrophy, and mental or motor retardation).
Infants born with congenital cytomegalic inclusion disease may appear well and live for many years. It has been estimated that one in every 1000 babies born in the USA is seriously retarded as a result of this congenital infection.
Inapparent intrauterine infection seems to occur frequently. Elevated IgM antibody to cytomegalovirus or isolation of the virus from the urine occurs in up to 2% of apparently normal newborns. This high rate occurs in spite of the fact that women may already have cytomegalovirus antibody before becoming pregnant. Such intrauterine infections have been implicated as possible causes of mental retardation and hearing loss.
Many women who have been infected naturally with cytomegalovirus at some time prior to pregnancy begin to excrete the virus from the cervix during the last trimester of pregnancy. At the time of delivery, infants pass through the infected birth canal and become infected, although they possess high titters of maternal antibody acquired transplacentally. These infants begin to excrete the virus in their urine at about 8-12 weeks of age. They continue to excrete the virus for several years but remain healthy.
Acquired infection with cytomegalovirus is common and usually inapparent, In children, acquired infection may result in hepatitis, interstitial pneumonitis, or acquired hemolytic anemia. The virus is shed in the saliva and urine of infected individuals for weeks or months.
Cytomegalovirus can cause an infectious mononucleosis-like disease without heterophil antibodies. “Cytomegalovirus mononucleosis” occurs either spontaneously or after transfusions of fresh blood during surgery (“postperfusion syndrome”). The incubation period is about 30-40 days. There is cytomegaloviruria and a rise of cytomegalovirus antibody. Cytomegalovirus has been isolated from the peripheral blood leukocytes of such patients. Perhaps the postperfusion syndrome is caused by cytomegalovirus harboured in the leukocytes of the blood donors.
Patients with malignancies or immunologic defects or those undergoing immunosuppressive therapy for organ transplantation may develop cytomegalo-virus pneumonitis or hepatitis and occasionally generalized disease. In such patients a latent infection may be reactivated when host susceptibility to infection is increased by immunosuppression. In seronegative patients without evidence of previous cytomegalovirus infection, the virus may be transmitted exogenously. Eighty-three percent of seronegative patients who received kidneys from seropositive transplant donors developed infection. Thus, the kidneys seemed to be the source of virus.
Laboratory Diagnosis. Recovery of Virus: The virus can be recovered from mouth swabs, urine, liver, adenoids, kidneys, and peripheral blood leukocytes by inoculation of human fibroblastic cell cultures. In cultures, 1-2 weeks are usually needed for cytotogic changes consisting of small foci of swollen, rounded, translucent cells with large intranuclear inclusions. Cell degeneration progresses slowly, and the virus concentration is much higher within the cell than in the fluid. Prolonged serial propagation is needed before the virus reaches high titters.
Rapid diagnosis of cytomegalovirus infection in infants is possible by detection of inclusion-bearing “owl cells” in the urine. These are desquamated cells from infected kidney tubules.
Serology. Antibodies may be detected by neutralization, complement fixation, or immunofluorescence tests. Such tests may be useful in detecting congenitally infected infants with no clinical manifestations of disease.
Immunity. Complement-fixing and neutralizing antibodies occur in most human sera. In young children possessing CF antibodies, virus may be detected in the mouth and in the urine for many months.
Virus may occur in the urine of children even though serum-neutralizing antibody is present. This suggests that the virus propagates in the urinary tract rather than being filtered from the bloodstream. Virus is not found in young children who lack antibody.
Intrauterine infection may produce a serious disease in the newborn. Infants infected during fetal life may be born with antibody that continues to rise after birth in the presence of persistent virus excretion. (This is similar to the situation in congenital rubella infection.)
Most infants infected with cytomegalovirus in the perinatal period are asymptomatic, and infection continues in the presence of high antibody titters.
Treatment. There is no specific treatment. Neither immune gamma globulior DNA virus-inhibitory drugs have any effect.
Epidemiology. The mechanism of virus transmission in the population remains unknown except in congenital infections and those acquired by organ transplantation, blood transfusion, and reactivation of latent virus. Infection with cytomegaloviruses is widespread. Antibody is found in 80% of individuals over 35 years of age. The prolonged shedding of virus in urine and saliva suggests a urine-hand-oral route of infection. Cytomegalovirus can also be transmitted by sexual contact.
Control. Specific control measures are not available. Isolation of newborns with generalized cytomegalic inclusion disease from other neonates is advisable.
Screening of transplant donors and recipients for cytomegalovirus antibody may prevent some transmissions of primary cytomegalovirus. The cytomegalovirus-seronegative transplant recipient population represents a high-risk group for cytomegalovirus infections as well as other lethal superinfections and would be a target population for a vaccine.
A live cytomegalovirus “vaccine” has been developed and has had some preliminary clinical trials, Since cytomegalovirus, like other herpesviruses, causes latent persistent infection, there is doubt that such a “vaccine” would be useful for the population at large. The possible benefits and dangers of a vaccine program for prevention of cytomegalovirus congenital infections require further study.
EB HERPESVIRUS (Human Herpesvirus 4).
(Infectious Mononucleosis, Burkitt’s Lymphoma, Nasopharyngeal Carcinoma).
EB (Epstein-Barr) virus is the causative agent of infectious mononucleosis and has been associated with Burkitt’s lymphoma and nasopharyngeal carcinoma. The virus is an antigenically distinct herpesvirus.
Properties of the Virus. Morphology: EB virus is indistinguishable in size and structure from other herpesviruses.
Antigenic Properties: EB virus is distinct from all other human herpesviruses. Many different EB virus antigens can be detected by CF, immunodiffusion, or immunofluorescence tests. A lymphocyte-detected membrane antigen (LYDMA) is the earliest-detected virus-determined antigen. EBNA is a complement-fixing nuclear antigen. Early antigen (EA) is formed in the presence of DNA inhibitors and membrane antigen (MA), the neutralizing antigen, is a cell surface antigen. The virus capsid antigen (VCA) is a late antigen representing virions and structural antigen.
C. Virus Growth: Human blood B lymphocytes infected in vitro with EB virus have resulted in the establishment of continuous cell lines, suggesting that these cells have been transformed by the virus.
This transformation by EB virus enables B lymphocytes to multiply continuously, and all cells contain many EB virus genomes and express EBNA. Some EB virus cell lines express certain antigens but produce no virus particles or VCA; others produce virus particles. EB virus is carried in lymphoid cell lines derived from patients with African Burkitt’s lymphoma, nasopharyngeal carcinoma, or infectious mononucleosis. Non-virus-producing B lymphocyte cell lines can be established in vitro from the blood of patients with infectious mononucleosis. Such lines represent a latent state of the virus; the cells contain EB virus genomes but express only the earliest antigen (LYDMA) and possibly EBNA.
Owl monkeys and marmosets inoculated with cell-free EB virus can develop fatal malignant lymphomas. Lymphoblastoid cells from such monkeys cultured as continuous cell lines give positive reactions with EB virus antisera by immunofluorescence.
Immunity. The most widely used and most sensitive serologic procedure for detection of EB virus infection is the indirect immunofluorescence test with acetone-fixed smears of cultured Burkitt’s lymphoma cells. The cells containing the EB virus exhibit fluorescence after treatment with fluorescent antibody. Detectable levels of antibody persist for many years.
Early in acute disease, a transient rise in IgM antibodies to VCA occurs, replaced within 2 weeks by IgG antibodies to VCA, which persist for life. Slightly later, antibodies to MA and to EBNA arise and persist throughout life.
Epidemiology. Seroepidemiologic studies using the immunofluorescence technique and CF reaction indicate that infection with EB virus is common in different parts of the world and that it occurs early in life. In some areas, including urban parts of the USA, about 50% of children 1 year old. 80-90% of children over age 4, and 90% of adults have antibody to EB virus.
In groups at a low socioeconomic level, EB virus infection occurs in early childhood without any recognizable disease. These inapparent infections result in permanent seroconversion and total immunity to infectious mononucleosis. In groups living in comfortable social circumstances, infection is often postponed until adolescence and young adulthood. Again, the majority of these adult infections are asymptomatic, but In almost half of cases the infection is manifested by heterophil-positive infectious mononucleosis.
Antibody to EB virus is also present ionhuman primates.
EB Virus & Human Disease. Most EB virus infections are clinically inapparent. The virus causes infectious mononucleosis and is strongly associated with Burkitt’s lymphoma and nasopharyngeal carcinoma.
Infectious mononucleosis (glandular fever) is a disease of children and young adults characterized by fever and enlarged lymph nodes and spleen. The total white blood count may range from 10,000/mcL to 80,000/mc, with a predominance of lymphocytes. Many of these are-large “atypical” cells with vacuo-lated cytoplasm and nucleus. These atypical lymphocytes, probably T cells, are diagnostically important. During mononucleosis, there often are signs of hepatitis.
During the course of infection, the majority of patients develop heterophil antibodies, detected by sheep cell agglutination or the mononucleosis spot test.
Although the pathogenesis of infectious mononucleosis is still not understood, infectious EB virus can be recovered from throat washings and saliva of patients (“kissing disease”). Infectious virus is produced by B lymphocytes in the oropharynx and perhaps in special epithelial cells of this region. Virus cannot be recovered from blood, but EB virus genome-containing B lymphocytes are present in up to 0.05% of me circulating mononuclear leukocytes as demonstrated by me establishment of cell lines. These EB virus genome-containing cells express the earliest antigen, LYDMA, which is specifically recognized by killer T cells.
These T cells reach large numbers and can lyse EB virus genome-positive but not EB virus genome-negative target cells. Part of the infectious mononucleosis syndrome may reflect a rejection reaction against virally converted lymphocytes.
Patients with infectious mononucleosis develop antibodies against EB virus, as measured by immunofluorescence with virus-bearing cells. Antibodies appear early in the acute disease, rise to peak levels within a few weeks, and remain high during convalescence. Unlike the short-lived heterophil antibodies, those against EB virus persist for years.
The role that EB virus may play in Burkitt’s lymphoma (a tumour of the jaw in African children and young adults) and nasopharyngeal carcinoma (common in males of Chinese origin) is less well established. The association with EB virus is based primarily on the finding that the prevalence of antibody is greater and the antibody titters are higher among patients with Burkitt’s lymphoma and nasopharyngealcarcinoma than in healthy matched controls or individuals with other types of malignancies. The significance of these associations is uncertain at present. All cells from Burkitt’s lymphoma of African origin and from nasopharyngeal carcinoma carry multiple copies of the EB virus genome and express the antigen EBNA.
Students Practical activities
1. To inoculate the pig embryo kidneys cell culture by blood of the patient with suspicion on a tick-borne encephalitis.
There is pig embryo kidneys cell culture the sterile bottle. It is on the side of bottle opposite to vertical line. In sterile conditions it is necessary to pour out the medium and to fill in the bottle 1,5 ml of the defibrinated patient’s blood. To close the bottle and to put it on a horizontal surface by the line upwards for 1 h at 37 °C for adsorption of the viruses on the cells surface. After that sterilely to add in the bottle 10,0 ml of medium 199.
In 72-96 hours material is inoculating into the brain of newborn white mice for the identification of viruses.
2. To carry out neutralization test with type specific sera in the pig embryo kidneys cell culture.
The scheme of the neutralization test for viruses identification
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Ingredients |
Tubes |
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1 |
2 |
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Patient’s defibrinated blood or serum (virus-containing specimen) |
0,5 ml |
0,5 ml |
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Tick-borne encephalitis viruses antiserum |
0,5 ml |
– |
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Japanese encephalitis viruses antiserum |
– |
0,5 ml |
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Incubation 1 h, temperature 18-20 °C |
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Pig embryo kidneys cell culture |
5,0 ml |
5,0 ml |
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Incubation 4-7 days, temperature 37 °C |
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Results |
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3. To carry out Complement fixation test with paired sera for serological diagnosis tick-borne encephalitis.
The scheme of the Complement fixation test
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Ingredient, ml |
Number of the test tubes |
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1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
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Isotonic sodium chloride solution |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
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Patient’s serum diluted 1:5 |
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0,2 |
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– |
0,2 |
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II |
0,2 |
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® |
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– |
0,2 |
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Serum dilution |
1:10 |
1:20 |
1:40 |
1:80 |
1:160 |
1:320 |
– |
– |
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Viral diagnosticum (tick-borne encephalitis viruses) |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
– |
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Complement |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
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Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature |
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Hemolytic system (Hemolytic serum and 3 % sheep erythrocytes suspension ) |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
0,4 |
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Incubation for 30-60 min, temperature 37 °C |
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Results |
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Patient’s serum |
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In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).
The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.
4. To carry out put Complement fixation test with specific serum against Crimean-Congo hemorrhagic fever viruses.
The scheme of Complement fixation test for laboratory diagnosis of
Crimean-Congo hemorrhagic fever
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Ingredient, ml |
Number of the test tubes |
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1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
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Dilution of antigen |
1:4 |
1:8 |
1:16 |
1:32 |
1:4 |
1:4 |
1:8 |
1:8 |
|
Investigated antigen |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
|
Specific serum |
0,1 |
0,1 |
0,1 |
0,1 |
– |
– |
– |
– |
|
Non-specific serum |
– |
– |
– |
– |
0,1 |
– |
0,1 |
– |
|
Isotonic sodium chloride solution |
– |
– |
– |
– |
– |
0,1 |
– |
0,1 |
|
Complement (2 U) |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
|
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature |
||||||||
|
Hemolytic system |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
|
Incubation for 30-60 min, temperature 37 °C |
||||||||
|
Results |
|
|
|
|
|
|
|
|
In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).
5. To carry out Hemagglutination inhibition test with paired sera for diagnosis of rubella.
The scheme of Hemagglutination inhibition test for serological diagnosis of rubella
|
Ingredient, ml |
Number of the test tubes |
|||||||
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
Dextrose-gelatin-veronal buffer with 0,4 % of bovine albumin |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
|
Patient’s serum diluted 1:5 |
|
|
|
|
|
|
|
|
|
I |
0,1 |
® |
® |
® |
® |
¯ |
0,1 |
– |
|
II |
0,1 |
® |
® |
® |
® |
¯ |
0,1 |
– |
|
Dilution |
1:10 |
1:20 |
1:40 |
1:80 |
1:160 |
1:320 |
– |
– |
|
Viral diagnosticum (Rubella virus, 4 HAU/ml) |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
– |
0,1 |
|
Incubation for 30 min, temperature 18-20 °C |
||||||||
|
1 % suspension of chicken erythrocytes |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
|
Incubation for 45 min, temperature 18-20 °C |
||||||||
|
Result: Serum I |
|
|
|
|
|
|
|
|
|
II |
|
|
|
|
|
|
|
|
Test results are assessed after complete erythrocyte sedimentation in control (7 well). In the experimental well a markedly localized erythrocytes sediment (“rouleaus”), and in the control well (8) the rapid erythrocytes agglutination with star-like, marginally festooned sediment (“umbrella”) on the bottom are observed. The titer of serum is its biggest dilution, which inhibits hemagglutination. The growth of patient’s antiviral antibodies titers at least in 4 times testifies about disease.
6. To do Complement fixation test for serological diagnosis of rotavirus infection.
The scheme of the Complement fixation test
|
Ingredient, ml |
Number of the test tubes |
|||||||
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
Isotonic sodium chloride solution |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
|
Patient’s serum diluted 1:5 I |
0,1 |
® |
® |
® |
® |
¯ |
– |
0,1 |
|
II |
0,1 |
® |
® |
® |
® |
¯ |
– |
0,1 |
|
Serum dilution |
1:10 |
1:20 |
1:40 |
1:80 |
1:160 |
1:320 |
– |
– |
|
Viral diagnosticum (rotavirus) |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
– |
|
Complement |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
0,1 |
|
Incubation for 18-20 h, temperature 4 °C and then 15 min at room temperature |
||||||||
|
Hemolytic system |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
0,2 |
|
Incubation for 30-60 min, temperature 37 °C |
||||||||
|
Result : Serum I |
|
|
|
|
|
|
|
|
|
II |
|
|
|
|
|
|
|
|
In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (“+++” , “++”), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood). The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.
7. To carry out complement fixation test with patient’s paired sera for serological diagnosis of Herpes simplex.
The scheme of Complement fixation test
|
Ingredient, ml |
Number of the test tubes |
|||||||
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
Isotonic sodium chloride solution |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
|
Patient’s serum diluted 1:5 |
|
|
|
|
|
|
|
|
|
I |
0,5 |
® |
® |
® |
® |
¯ |
– |
0,5 |
|
II |
0,5 |
® |
® |
® |
® |
¯ |
– |
0,5 |
|
Serum dilution |
1:10 |
1:20 |
1:40 |
1:80 |
1:160 |
1:320 |
– |
– |
|
Viral diagnosticum |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
– |
|
Incubation for 45 min, temperature 37 °C |
||||||||
|
Hemolytic system (Hemolytic serum and 3 % sheep erythrocytes suspension ) |
1,0 |
1,0 |
1,0 |
1,0 |
1,0 |
1,0 |
1,0 |
1,0 |
|
Incubation for 30-60 min, temperature 37 °C |
||||||||
|
Results |
|
|
|
|
|
|
|
|
|
Patient’s serum |
|
|
|
|
|
|
|
|
|
I |
|
|
|
|
|
|
|
|
|
II |
|
|
|
|
|
|
|
|
In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (+++ , ++), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).
The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.
The titer of antibody in the second serum must increase in 4 times as compared with the first one.
Arthropod-Borne (Arbo) Viral Diseases
The arthropod-borne viruses, or arboviruses, are a group of infectious agents that are transmitted by blood-sucking arthropods from one vertebrate host to another. They can multiply in the tissues of the arthropod without evidence of disease or damage. The vector acquires a lifelong infection through the inges-tion of blood from a viremic vertebrate. All arboviruses have an RNA genome, and most have a lipid-containing envelope and consequently are inacti-vated by ether or sodium deoxycholate.
Individual viruses were sometimes named after a disease (dengue, yellow fever) or after the geographic area where the virus was first isolated (St. Louis encephalitis. West Nile fever). Although arboviruses are found in all temperate and tropical zones, they are most prevalent in the tropical rain forest with its abundance of animals and arthropods.
There are more than 350 arboviruses, grouped according to their antigenic relationships. An effort is being made to classify them according to their chemical and physical properties, as arranged in Table 1. Many arboviruses are placed among toga-, bunya-, reo-, arena-, picoma-, and rhabdovirus groups.
Togaviruses and Flaviviruses: Spherical particles contain a single-stranded RNA genome (MW 4 x 106) and are surrounded by a lipid-containing envelope. Group A arboviruses are larger (40-80 nm) and are inactivated by proteases, and all multiply in arthropods. Group B arboviruses are smaller (20-50 nm) and are not inactivated fcy proteases, and not all multiply in arthropods, but all are serologically related. All togaviruses multiply in the cytoplasm and mature by budding. Names of representative group A and group B viruses are given in Table 1. Some of the most important ones are discussed below.
Bunyaviruses: Spherical particles contain a single negative strand RNA genome that is segmented. They have a lip id-containing envelope and measure 90-l00 nm. The nucleocapsids have helical symmetry and contain a major viral protein. The envelope has 2 glycoproteins in the lipid bilayer and surface projections (10 nm) of glycopeptides clustered to form hollow cylinders. All bunyaviruses multiply in arthropods. Several produce mosquito-borne en-cephalitides of humans and animals, others hemorrhagic fevers. Some are transmitted by sandflies (Phlebotomus).
Reoviruses: A few arboviruses are placed into this group (subgroup Orbivirus), including African horse sickness and Colorado tick fever. Some infect birds, small mammals, and ticks.
Current taxonomic status of some arboviruses
|
Current Taxonomic Classification |
Arbovirus Members |
|
Togaviridae Genus Alphavirus |
Aura, Chikungunya, eastern equine encephalitis, Getah, Maygro, Middelburg. Mucambo, Ndumu, O’Nyong-nyong, Pixuna, Ross River, Semliki Forest, Sindbis, Una, Venezuelan and Western equine encephalitis, and Whataroa viruses |
|
Flaviviridae Genus Flavivirus |
Brazilian encephalitis (Rocio virus). Bussuquara, dengue, llheus, Israel turkey meningoencephalitis, Japanese B encephalitis, Kunjin, Kyasanur Forest disease, Langat, louping ill, Modoc, Murray Valley encephalitis, Ntaya. Omsk hemorrhagic fever. Powassan, St. Louis encephalitis, Spondweni, tick-borne encephalitis, Uganda S, US bat salivary gland, Wesselsbron, West Nile fever, yellow fever, and Zika viruses |
|
Bunyaviridae Genus Bunyavirus |
Bunyamwera, Bwamba, C, California, Capim, Guama, Koongol, Patois,Simbu, and Tete; 7 unassigned viruses |
|
Genus Uukuniemi |
Uukuniemi, Anopheles A, Anopheles B, Bakau, Crimean-Congo hemorrhagic fever, Kaisodi, Mapputta, Nairobi sheep disease, Phlebotomus fever, and Turlock; 8 unassigned viruses |
|
Reoviridae Genus Orbivirus |
African horse sickness, bluetongue, and Colorado tick fever viruses |
|
Rhabdoviridae Genus Vesiculovirus |
Cocal, Hart Park, Kern Canyon, and vesicular stomatitis viruses |
|
Arenaviridae Genus Arenavirus |
Junin, Lassa, Machupo, and Pichinde viruses |
|
Nodaviridae |
Nodamura virus |
Arenaviruses: Pleomorphic particles contain a segmented single negative strand RNA genome (MW 3-5 x 106), are surrounded by an envelope, and measure 50-300 nm. They contain granules believed to be ribosomes. Several hemorrhagic fever viruses that are antigenically related fall into this group. Most have a rodent host in their natural cycle.
Rhabdoviruses: Several bullet-shaped arboviruses fall into this group.
Nodavirus: Nodamura virus is an insect pathogen that can infect mammals.
Human Infection
About 75 arboviruses can infect humans, but not all cause overt disease. Those infecting humans are all believed to be zoonotic, with humans the accidental hosts who play no important role in the maintenance or transmission cycle of the virus. Exceptions are urban yellow fever and dengue. Some of the natural cycles are simple and involve a nonhuman vertebrate host (mammal or bird) with a species of mosquito or tick (eg, jungle yellow fever, Colorado tick fever). Others, however, are quite complex. For example, many cases of Central European diphasic meningoencephalitis occur following ingestion of raw milk from goats and cows infected by grazing in tick-infested pastures where a tick-rodent cycle is occurring.
Diseases produced by the arboviruses may be divided into 3 clinical syndromes: (1) fevers of an undifferentiated type with or without a maculopapular rash and usually benign; (2) encephalitis, often with a high case fatality rate; and (3) hemorrhagic fevers, also frequently severe and fatal. These categories are somewhat arbitrary, and some arboviruses may be associated with more than one syndrome, eg, dengue.
The intensity of viral multiplication and its predominant site of localization in tissues determine the clinical syndrome. Thus, individual arboviruses can produce a minor febrile illness in some patients and encephalitis or a hemorrhagic diathesis in others. However, in an epidemic situation, one of the syndromes usually predominates, permitting a tentative diagnosis. A final diagnosis is based on further epide-miologic and serologic data.
After infection with an arbovirus, there is an incubation period during which viral multiplication takes place. This is followed by the abrupt onset of clinical manifestations that are closely related to viral dissemination. Malaise, headache, nausea, vomiting, and myalgia accompany fever, which is an invariable symptom and sometimes the only one. The illness may terminate at this stage, recur with or without a rash, or reveal hemorrhagic manifestations secondary to vascular abnormalities. Frequently, the period of viremia is asymptomatic, with the acute onset of encephalitis following localization of the virus in the central nervous system.
The above clinical categories are utilized in the following sections in discussing some of the most important diseases caused by the arboviruses.
Encephalitis can be produced by many different viruses. Arbovirus encephalitis occurs in distinct geographic distributions and vector patterns (Table 2). Each continent tends to have its own arbovirus pattern, and names are usually suggestive, eg, Venezuelan equine encephalitis (VEE), Japanese B encephalitis (JBE), Murray Valley (Australia) encephalitis (MVE). All of the preceding are togavirus infections spread by mosquitoes with a distinct ecologic distribution. California encephalitis is caused by bunyaviruses, as discussed below. However, on a given continent there may be a shifting distribution depending on virus hosts and vectors in a given year.
Encephalitis or meningoencephalitis can also occur with vimses that involve tissues other than the central nervous system—measles, mumps, hepatitis, chickenpox, zoster, herpes simplex, and others. Some of these viruses replicate actively in the central nervous system, producing inflammation. At other times, the viral infection sets off an immunologic reaction that results in “postinfectious ” encephalo myelitis, with a prominent demyelinating component.
In some parts of the world, epidemics of ar-bovirus infection have involved thousands of individuals with symptomatic infection; many more were asymptomatically infected. In
Characteristics of Togaviruses (genus Alphavirus) and Flaviviruses
A. Properties: Togavimses are small viruses (40 nm for Alphavuruses, 70 nm for Flaviviruses) that are unstable at room temperature, stable at —70 °C, and rapidly mac-tivated by ether or by 1:1000 sodium deoxycholate. This property separates them easily from en-teroviruses.
The viruses infect many cell lines, embryonated eggs, mice, birds, bats, mules, horses, and other animals. In susceptible vertebrate hosts, primary virus multiplication occurs either in myeloid and lymphoid cells or in vascular endothelium. Multiplication in the central nervous system depends on the ability of the virus to pass the blood-brain barrier and to infect nerve cells. Iatural infection of birds and mammals (and in experimental parenteral injection of the virus into animals), an inapparent infection develops in a majority. However, for several days there is viremia, and ar-thropod vectors acquire the virus by sucking blood during this period—the first step in its dissemination to other hosts. The above characteristics apply to the main togavirus infections in the western hemisphere, particularly St. Louis encephalitis (SLE), western equine encephalitis (WEE), eastern equine encephalitis (EEE), and Venezuelan equine encephalitis (VEE) (see Table 2). They also apply to Japanese B encephalitis (JBE), which occurs in the Far East.
B. Replication: All togavimses replicate in the cytoplasm. The genome RNA, released from virus particles, is infectious and, like picomaviruses, can act as a messenger. However, togaviruses have 2 sizes of mRNA: the virion RNA of 42S and a smaller 26S species containing a subset of the 42S sequences. Each mRNA is translated into a large polypeptide that subsequently undergoes cleavage and processing. The 26S mRNA polypeplide is processed to the capsid protein and 2 envelope proteins, whereas that from the 42S mRNA is processed to nonstructural proleins. 2 of which may be part of the rcplicase. Except for the smaller-than-genome mRNA, togavirus replication is similar to that of the picornaviruses. Togaviruses, however, have 2 or more structural potypeptides that undergo glycosylation and cleavage and are incorporated into the cytoplasmic membrane of the cell. The virus particle buds through the altered areas of the membrane to acquire its envelope and the glycosylated polypeptides. Only one polypeptide is found in the nucleocapsid of togaviruses, in contrast to the 4 found in picomavirus nucleocapsids.
C. Measurement of Virus Concentration: Viral multiplication can be measured by cytopathic changes, virus-specific immunofluorescence, or the production of viral hemagglutinin as seen directly in the cell culture by the hemadsorption test. Plaque counts can be done with group A and B viruses in most cultures. Arboviruses exhibit homotypic and heterotypic interference, as well as susceptibility to interferon.
D. Antigenic Properties: Complement-fixing antigens and viral hemagglutinins may be prepared from infected brains of newbom mice (because of their low fat content). The hemagglutinins of these vimses are part of the infectious virus particle and can hemagglutinate goose or newly hatched chick red blood cells. The union between hemagglutinin and red cell is irreversible. The erythrocyte-virus complex is still infective, but it can be neutralized by the addition of antibody, which results in large lattice formations.
Some of these viruses have an overlapping an-tigenicity, most readily demonstrated by cross-reactions in HI tests. The overlapping is due to the presence of one or more cross-reactive antigens in addition to the strain-specific antigen. Thus, immune sera prepared for one strain will contain strain-specific as well as group-specific antibodies.
An immune serum can be made more specific by adsorption with a heterologous virus of the same group. The adsorbed serum tested for hemagglutina-tion-inhibiting (HI) activity reacts only with the homologous and not with the heterologous strain, facilitating the identification of newly isolated strains.
TOGAVIRUS ENCEPHALITIS
Pathogenesis and Pathology
The pathogenesis of the disease in humans has not been well studied, but the disease in experimental animals may afford a model for the human disease. The equine encephalitides in horses are diphasic. In the first phase (minor illness), the virus multiplies ion-neural tissue and is present in the blood 3 days before the first signs of involvement of the central nervous system. In the second phase (major illness), the virus multiplies in the brain, celts are injured and destroyed, and encephalitis becomes clinically apparent. The 2 phases may overlap. It is not known whether in humans there is a period of primary viral multiplication in the viscera with a secondary liberation of virus into the blood before its entry into the central nervous system. The viruses multiply ionneural tissues of experimentally infected monkeys.
High concentrations of virus in brain tissue are necessary before the clinical disease becomes manifest. In mice, the level to which the vims multiplies in the brain is partly influenced by a genetic factor that behaves as a mendelian trait.
The primary encephalitides are characterized by lesions in all parts of the central nervous system, including the basal structures of the brain, the cerebral cortex, and the spinal cord. Small hemorrhages with perivascular cuffing and meningeal infiltration – chiefly with mononuclear cells—are common. Nerve cell degeneration associated with neuronophagia occurs. Purkinje’s cells of the cerebellum may be destroyed. There are also patches of encephalomalacia; acellular plaques of spongy appearance in which medullary fibers, dendrites, and axons are destroyed; and focal microglial proliferation. Thus. not only the neurons but also the cells of the supporting structure of the central nervous system are attacked.
Widespread neuronal degeneration occurs with all arboviruses producing encephalitis, but some localization occurs.
Clinical Findings. Incubation periods of the encephalitides are between 4 and 21 days. There is a sudden onset with severe headache, chills and fever, nausea and vomiting, generalized pains, and malaise. Within 24-48 hours, marked drowsiness develops and the patient may become stuporous. Nuchal rigidity is common. Mental confusion, dysarthria, tremors, convulsions, and coma develop in severe cases. Fever lasts 4-10 days. The mortality rate in encephalitides varies (see Table 30-2). With JBE, the mortality rate in older age groups may be as high as 80%. Sequelae may include mental deterioration, personality changes, paralysis, aphasia, and cerebellar signs-Abortive infections simulate aseptic meningitis or nonparalytic poliomyelitis. Inapparent infections are common.
In California, where both WEE and SLE are prevalent, WEE has a predilection for children and infants. In the same area, SLE rarely occurs in infants, even though both viruses are transmitted by the same arthropod vector (Culex tarsalis}.
Laboratory Diagnosis
A. Recovery of Virus: The virus occurs in the blood only early in the infection, usually before the onset of symptoms. The virus is most often recovered from the brains of fatal cases by intracerebra! inoculation ofnewbom mice, and then it should be identified by serologic tests with known antisera,
B. Serology: Neutralising and hemagglutina-tion-inhibiling antibodies are detectable within a few days after the onset of illness. Complement-fixing antibodies appear later. The neutralizing and the hemagglutination-inhibiting antibodies endure for many years. The complement-fixing antibody may be lost within 2-5 years.
The HI test with newly hatched chick eryth-rocytes is the simplest diagnostic test, but it primarily identifies the group rather than the specific causative virus.
It is necessary to establish a rise in specific antibodies during infection in order to make the diagnosis. The first sample of serum should be taken as soon after the onset as possible and the second sample 2-3 weeks later. The paired specimens must be run in the same serologic test.
The cross-reactivity that takes place within group A or B arboviruses must be considered in making the diagnosis. Thus, following a single infection by one member of the group, antibodies to other members may also appear. These group-specific antibodies are usually of lower titer than the type-specific antibody. Serologic diagnosis becomes difficult when an epidemic caused by one member of the serologic group occurs in an area where another group member is endemic, or when an infected individual has been infected previously by a closely related arbovirus. Under these circumstances, a definite etiologic diagnosis may not be possible. Neutralizing, complement-fixing, and hemagglutination-inhibiting antibodies have a decreasing degree of specificity for the causative viral type (in the order listed).
Immunity
Immunity is believed to be permanent after a single infection. In endemic areas, the population may build up immunity as a result of inapparent infections; the proportion of persons with antibodies to the local arthropod-bome vims increases with age.
Because of antigens common to several members within a group, the response to immunization or to infection with one of the viruses of a group may be modified by prior exposure to another member of the same group. In general, the homologous response is greater than a cross-reacting one. This mechanism may be important in conferring protection on a community against an epidemic of another related agent (eg, no Japanese B encephalitis in areas endemic for West Nile fever).
Treatment
There is no specific treatment. In experimental animals, hyperimmune serum is ineffective if given after the onset of disease. However, if given 1-2 days after the invasion of the virus but before the signs of encephalitis are obvious, specific hyperimmiine serum can prevent a fatal outcome of the infection.
Epidemiology
In severe epidemics caused by the encephalitis viruses, the case rate is about 1:1000. In the large urban epidemic of St. Louis encephalitis that occurred in
SLE is now appearing each year in the USA. In 1976, 372 cases with 17 deaths were reported in the USA.
The epidemiology of the arthropod-bome en-cephalitides must account for the maintenance and dissemination of the vimses iature in the absence of humans. Most infections with the arboviruses occur in mammals or birds, with humans serving as an accidental host. The vims is transmitted from animal to animal through the bite of an arthropod vector. Viruses have been isolated from mosquitoes and ticks, which serve as reservoirs of infection. In ticks, the viruses may pass from generation to generation by the transovarian route, and in such instances the tick acts as a true reservoir of the virus as well as its vector. In tropical climates, where mosquito populations are present throughout the year. arboviruses cycle continuously between mosquitoes and reservoir animals.
It is not known whether in temperate climates the virus is reintroduced each year from the outside (eg, by birds migrating from tropical areas) or whether it somehow survives the winter in the local area. The overwintering mechanism is not known. Three possible mechanisms are (1) that hibernating mosquitoes at the time of their emergence could reinfect birds and thus reestablish a simple bird-mosquito-bird cycle; (2) that the virus could remain latent in winter within birds, mammals, or arthropods; and (3) that coldblooded vertebrates (snakes, turtles, lizards, alligators, frogs) may also act as winter reservoirs—eg, garter snakes experimentally infected with WEE virus can hibernate over the winter and circulate virus in high liters and for long periods the following spring. Normal mosquitoes can be infected by feeding on the emerged snakes and then can transmit the virus. Virus has been found in the blood of wild snakes.
C. Tick-Borne Encephalitis Complex:
1. Russian spring-summer encephalitis. This disease occurs chiefly in the early summer, particularly in humans exposed to the ticks Ixodes persulcatus and Ixodes ricinus in the uncleared forest. Ticks can become infected at any stage in their metamorphosis, and vims can be transmitted transovarially. The virus persists through the winter in hibernating ticks or in vertebrates such as hedgehogs or bats. Vims is se-
creted in the milk of infected goats for long periods, and infection may be transmitted to those who drink unpasteurized milk. Characteristics of the disease are involvement of the bulbar area or the cervical cord and the development of ascending paralysis or hemipa-resis. The mortality rate is about 30%.
2. Louping ill-This disease of sheep in Scotland and northern England is spread by the tick Ixodes ricinus. Humans are occasionally infected.
3. Tick-borne encephalitis (Central European or diphasic meningoencephalitis)-This virus is anti-genically related to Russian spring-summer encephalitis virus and louping ill virus. Typical cases have a diphasic course, the first phase being influenzalike and the second a meningoencephalitis with or without paralysis.
4. Kyasanur Forest disease is an Indian hemor-rhagic disease caused by a virus of the Russian spring-summer encephalitis complex. In addition to humans, langur (Presbylis entellus) and bonnet (Macaco. radiata) monkeys are naturally infected in southern India.
5. Powassan encephalitis-This tick-bome virus is the first member of the Russian spring-summer complex isolated in North America. Human infection is rare. Since 1959, when the original fatal case was reported from Canada, several additional cases have been confirmed in the northeastern portion of the USA.
Control
Biologic control of the natural vertebrate host is generally impractical, especially when the hosts are wild birds. The most effective method is arthropod control. Since the period of viremia in the vertebrate is of short duration (3-6 days for SLE infections of birds), any suppression of the vector for this period should break the transmission cycle. During the 1966 Dallas SLE epidemic, low-volume, high-concentration malathion mist was sprayed aerially over most of Dallas County. A striking decrease in the number and infectivity rate of the mosquito vectors occurred, demonstrating the effectiveness of the treatment.
Killed vaccines have not met with success. Several live attenuated encephalitis vaccines are being investigated. A live attenuated vaccine was successfully used to halt the severe epidemic of VEE in horses in Texas in 1971.
WEST NILE FEVER
West Nile fever is an acute, mild, febrile disease with lymphadenopathy and rash that occurs in the Middle East, tropical or subtropical Africa, and southwest Asia. It is caused by a group B arbovirus, a typical togavirus.
Clinical Findings. The virus is introduced through the bite of a Cuiex mosquito and produces viremia and a generalized systemic infection characterized by lymphadenopathy, sometimes with an accompanying maculopapular rash. Transitory meningeal involvement may occur during the acute stage. The virus may produce fatal encephalitis in older people, who have a delayed (and low) antibody response.
Laboratory Diagnosis. Virus can be recovered from blood taken in the acute stage of the infection. On paired serum specimens, CF, HI, and Nt titer rises may be diagnostic. Nt antibodies persist longer than CF antibodies. During convalescence, heterologous CF and Nt antibodies develop to JBE and SLE. The heterologous response is shorter and of lower titer than the homologous response.
Immunity. Only one antigenic type exists, and immunity is presumably permanent. Maternal antibodies are transferred from mother to offspring and disappear during the first 6 months of life.
Epidemiology and Control. West Nile fever appears to be limited to the Middle East. Antibodies to the virus have been found in Africa, India, and Korea, perhaps because of an antigenically related virus. Ionimmune populations, subclinical or clinical infections are common. In Cairo, 70% of persons over age 4 years have antibodies.
The disease is more common in summer and more prevalent in mral than urban areas. The virus has been isolated from Culex mosquitoes during epidemics, and experimentally infected mosquitoes can transmit the virus. Mosquito abatement appears to be a logical, if unproved, control measure.
YELLOW FEVER
Yellow fever (YF) is an acute, febrile, mos-quito-bome illness. Severe cases are characterized by jaundice, proteinuria, and hemorrhage. YF is a group B arbovirus, a typical togavirus. It multiplies in many different types of animals and in mosquitoes. It grows in embryonated chicks and in cell cultures made from chick embryos.
Strains freshly isolated from humans, monkeys, or mosquitoes are pantropic, ie, the virus invades all 3 embryonal layers. Fresh strains usually produce a severe (often fatal) infection with marked damage to the livers of monkeys after parenteral inoculation. After serial passage in the brains of monkeys or mice, such strains lose much of their viscerotropism; they cause encephalitis after intracerebral injection but only asymptomatic infection after subcutaneous injection. Cross-immunity exists between the pantropic and neurotropic strains of the virus.
During the serial passage of a pantropic strain of YF through tissue cultures, the relatively avirulent 17D strain was recovered. This strain lost its capacity to induce a viscerotropic or neurotropic disease in monkeys and in humans and is now used as a vaccine,
Hemagglutinins and complement-fixing antigens of this group B arbovirus may be prepared from infected tissues. Each antigen has 2 separable components: one is associated with the infectious particle; the other is probably a product of the action of YF virus on tissues it infects.
Pathogenesis and Pathology
Our understanding of the pathogenesis of YF is based on work with the experimental infection in monkeys. The virus enters through the skin and then spreads to the local lymph nodes, where it multiplies. From the lymph nodes, it enters the circulating blood and becomes localized in the liver, spleen, kidney, bone marrow, and lymph glands, where it may persist for days.
The lesions of YF are due to the localization and propagation of the virus in a particular organ. Death may result from the necrotic lesions in the liver and kidney, The most frequent site of hemorrhage is the mucosa at the pyloric end of the stomach.
The distribution of necrosis in the liver may be spotty but is most evident in the midzones of the lobules. The hyaline necrosis may be restricted to the cytoplasm; the hyaline masses are eosinophilic (Councilman bodies), Intranuclear eosinophilic inclusion bodies are also present and are of diagnostic value. During recovery, the parenchymatous cells are replaced, and the liver may be completely restored.
In the kidney, there is fatty degeneration of the tubular epithelium. Degenerative changes also occur in the spleen, lymph nodes, and heart. Intranuclear, acidophilic inclusion bodies may be present in the nerve and glial cells of the brain. Perivascular infiltrations with mononuclear cells also occur in the brain.
Clinical Findings
The incubation period is 3-6 days. At the onset, the patient has fever, chills, headache, and backache, followed by nausea and vomiting. A short period of remission often follows the prodrome. On about the fourth day, the period of intoxication begins with a slow pulse (90-100) relative to a high fever and moderate jaundice. In severe cases, marked proteinuria and hemorrhagic manifestations appear. The vomitus may be black with altered blood. Lymphopenia is present. When the disease progresses to the severe stage (black vomitus and jaundice), the mortality rate is high. On the other hand, the infection may be so mild as to go unrecognized. Regardless of severity, there are no sequelae; patients either die or recover completely.
Laboratory Diagnosis
A. Recovery of Virus: The vims may be recovered from the blood up to the fifth day of the disease by intracerebra! inoculation of mice. The isolated virus is identified by neutralization with specific antiserum.
B. Serology: Neutralizing antibodies develop early (by the fifth day) even in severe and fatal cases. In patients who survive the infection, circufating antibodies endure for life.
Complement-fixing antibodies are rarely found after mild infection or vaccination with the attenuated, live 17D strain. In severe infections, they appear later than the neutralizing antibodies and disappear more rapidly.
The serologic response in YF may be of 2 types. In primary infections of yellow fever, specific hemagglutination-inhibiting (HI) antibodies appear first, followed rapidly by antibodies to other group B viruses. The titers of homologous HI antibodies are usually higher than those of heterologous antibodies. CF and Nt antibodies rise slowly and are usually specific .
In secondary infections where YF occurs in a patient previously infected with another group B ar-bovirus, HI and CF antibodies appear rapidly and to high titers. There is no suggestion of specificity. The highest HI and CF antibodies are usually heterologous. Accurate diagnosis even by Nt test may be impossible.
Histopathologic examination of the liver in fatal cases is useful in those regions where the disease is endemic.
Immunity
Subtle antigenic differences exist between YF strains isolated in different locations and between pan-tropic and vaccine (17D) strains.
An infant born of an immune mother has antibodies at birth that are gradually lost during the first 6 months of life. Reacquisition of similar antibodies is dependent upon the individual’s exposure to the virus under natural conditions or by vaccination.
Epidemiology
Two major epidemiologic cycles of YF are recognized: (1) classic (or urban) epidemic YF and (2) sylvan (or jungle) YF. Urban YF involves person-to-person transmission by domestic ^erfe^ mosquitoes. In the western hemisphere and West Africa, this species is primarily Aedes aegypti, which breeds in the accumulations of water that accompany human settlement. Mosquitoes remain close to houses and become infected by biting a viremic individual. Urban YF is perpetuated in areas where there is a constant influx of susceptible persons, cases of YF, and A aegypti. With the use of intensive measures for mosquito abatement, urban YF has been practically eliminated in South America.
Jungle YF is primarily a disease of monkeys. In South America and Africa, it is transmitted from monkey to monkey by arboreal mosquitoes (ie, Haemagogus, Aedes) that inhabit the moist forest canopy. The infection in animals may be severe or inapparent. Persons such as woodcutters, nut-pickers, or road-builders come in contact with these mosquitoes in the forest and become infected. Jungle YF may also occur when an infected monkey visits a human habitation and is bitten by A aegypll, which then transmits the virus to a human being.
The virus multiplies in mosquitoes, which remain infectious for life. After (he mosquito ingests a virus-containing blood meal, an interval of 12-14 days is required for it to become infectious. This interval is called the extrinsic incubation period.
All age groups are susceptible, but the disease in infants is milder than that in older groups. Large numbers of inapparent infections occur. The disease usually is milder in blacks. Yellow fever has never been reported in India or the Orient, even though the vector, A aegypti, is widely distributed there.
New outbreaks continue to occur. In Bolivia, 145 cases of jungle YF, with over 50% mortality, were reported in 1975. The disease occurred ionimmune persons coming from distant places for the rice harvests . The rice fields, with the jungle adjacent to them, are located near towns. Most cases were in male agricultural workers. The virus had established itself in this area in reservoirs close to the towns. Jungle YF rarely affects the local population, which has developed immunity by having been in contact with the virus through previous minor infections and also by frequent vaccinations. The real number of cases and deaths from jungle YF in such areas is much higher than the reports indicate, as many patients do not go to the hospital but recover or die without any report being made.
Control
Vigorous mosquito abatement programs have virtually eliminated urban YF. The last reported outbreak of YF in the USA occurred in 1905. However, with the speed of modem air travel, wherever A aegypti is present, the threat of a YF outbreak exists. Most countries insist upon proper mosquito control on airplanes and vaccination of all persons at least 10 days before arrival in or from an endemic zone. The yellow fever vaccination requirement for travelers entering the USA was eliminated in 1972.
In
An excellent attenuated, live vaccine is available in the 17D strain. Vaccine is prepared in eggs and dispensed as a dried powder. It is a live virus and must be kept cold. It is rehydrated just before use and injected subcutaneously by skin scarification or by jet injector. A single dose produces a good antibody response in more than 95% of vaccinated persons that persists for at least 10 years. After vaccination, the virus multiplies and may be isolated from the blood before antibodies develop.
References:
1. Ronald M. Atlas. Microbiology in our World, 1995.
2. Handbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 199-204, 210-214.
3. Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P. 221-234, 244-249, 255-256.
4. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P.361-390, 433-440, 441-458, 488-498
