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Among arthropod-borne and related viruses, the Flaviviridae are medically the most important group and biologically one of the most intriguing. Elucidation of perplexingly complex virus- and host-specified factors that underlie virulence and pathogenesis has lagged behind other areas of virology, and the available information is largely descriptive. Ultimately, flavivirus biology and pathogenesis will be understood in terms of viral gene expression, virus receptor—host cell membrane interactions, biochemical alterations in host cells, physiological responses, and immune and nonimmune mechanisms that control virus replication and virus spread, subjects to which other chapters in this book are devoted. There will remain, however, a need to understand and synthesize this information with observations relating to infection at the level of the intact organism (virus, vector, and host) and of populations of organisms in nature. It is at these levels that the phenomena that require explanation first present themselves.

We studied the capacity of 44 strains of St. Louis encephalitis (SLE) virus to induce viremia in an epidemiologically important wild avian host, the House Sparrow (Passer domesticus). Selected virus strains were also inoculated at varying doses into 3-week-old chicks. Viremic responses were analyzed in terms of the proportion of inoculated nestling and adult birds which became viremic, the mean duration and the mean peak titer of viremia. Infectivity of avian sera was determined by plaque assay in primary duck embryo cell cultures. The susceptibility of the House Sparrow to viremic infection with different SLE virus strains varied markedly. Nestling sparrows 6-10 days of age were generally more susceptible than adult birds. All virus strains isolated during Culex pipiens-borne epidemics in the eastern United States were highly viremogenic [viremia in 80% of birds with a mean duration of greater than or equal to 1.6 days in adults or greater than or equal to 2.7 days in nestlings and a mean peak titer of greater than or equal to 10(3.0) plaque-forming units (PFU)/ml in adults and 10(4.0) PFU/ml in nestlings]. All virus strains isolated from Culex tarsalis in the western United States, strains isolated from rodents in South America. and six of 16 strains isolated from various sources elsewhere in tropical America were partially or fully attenuated. A high degree of concordance was demonstrated between experimental viremia in sparrows, viremia in 3-week-old chicks, and neurovirulence for weanling mice. The epidemilogic significance of these findings is discussed.

Rhesus monkeys inoculated with yellow fever (YF) (DakH 1279) virus had a predictable course. Death occurred 108–135 hours after infection and was preceded by a 14- to 25-hour period during which clinical signs and physiological changes were present. Viremia was first detected 48 hours after inoculation and peaked at 96 hours. Abnormal liver function (elevated serum bilirubin and transaminases and retention of bromsulfalein) was not detected until approximately 24 hours before, nor hypoglycemia until 8 hours or less before death. Chemical hepatic dysfunction and histopathologic alterations developed precipitously; needle liver biopsies up to 23 hours before death revealed only Kupffer cell changes without hepatocellular degeneration, whereas at necropsy there was coagulative necrosis of the entire liver. Renal function was also not impaired until the terminal 24-hour period, during which urine output diminished, proteinuria, and cylindruria appeared, and serum creatinine rose. A marked fall in 24-hour urinary Na+ excretion was a feature of the early oliguric phase, and needle biopsies of the kidney were normal. At autopsy, there was necrosis of the tubular epithelium and accumulation of cell debris and proteinaceous material in the tubular lumina. These findings suggest that the acute tubular necrosis may be the end result of prerenal hemodynamic alterations and azotemia, and thus be preventable. Acid-base and electrolyte disturbances were late findings. Between 2 and 9 hours before death, arterial pCO2 fell; there followed a modest decline in pH, and systolic and pulse pressures gradually decreased. In the final 1–2 hours, pCO2 rose acutely and severe acidemia, hyperkalemia, and shock developed. These pathophysiologic changes suggest the need to investigate therapeutic countermeasures. No definitive evidence for disseminated intravascular coagulation was obtained in our experiments. An interesting finding was the marked acute necrosis of B cell areas of lymphoid tissues, and further studies are indicated to define possible immunosuppressive effects of YF viral lymphoid injury. The lymphoid necrosis may be diagnostically helpful; examination of spleen and nodes has been overlooked in the histopathologic diagnosis of human YF. Speculations on the possible role of bacterial endotoxemia in the pathogenesis of fatal YF are discussed.

A study was undertaken to investigate an increase in reported cases of clinical encephalitis due to eastern equine encephalitis (EEE) virus in horses and to determine the natural vertebrate hosts of that virus. Horses, birds, and small mammals were sampled at sites in a contiguous area in St. Joseph and Kalamazoo counties, Michigan, from 25 August to 5 September 1980. Serum samples from four horses acutely ill with encephalitis and 16 of 39 pasture mates of ill horses had neutralizing (N) antibody against EEE virus (46.5%); no viruses were isolated from these 43 sera. None of 24 draft horses from a site in St. Joseph County 12 km southeast of the affected sites had EEE antibody. A strain of Cache Valley virus was isolated from the blood of one of the 24 draft horses. No viruses were isolated, and no antibodies to EEE virus were detected in 28 blood samples from small mammals captured at sites where equine cases of encephalitis were occurring. Six strains of EEE virus, five of Highlands J virus, and one of Flanders virus were isolated from the blood of 401 wild birds belonging to 42 species captured at eight sites in both counties. A total of 29.9% of the wild birds had EEE antibody. Five species of domestic birds, mostly chickens and ring-necked pheasants, were sampled in both counties. Six strains of EEE virus were isolated from 152 ring-necked pheasants; these included three isolates from the brains of dead birds. About 13% of 51 pheasants tested from two small flocks in backyard pens in Kalamazoo County and 9% of 103 pheasants tested from a large game farm in St. Joseph County had antibody to EEE virus. The source of the EEE virus and the factors responsible for this epizootic are unknown; however, the epizootic probably represented an explosive expansion of an enzootic level of virus transmission.

Sera from patients with St. Louis encephalitis were tested with an immunoglobulin M (IgM) antibody capture enzyme immunoassay (MAC ELISA). The assay used five reagents: antihuman IgM, test serum, sucrose-acetone-extracted mouse brain antigen, broadly cross-reactive flavivirus monoclonal antibody conjugated to alkaline phosphatase, and substrate (p-nitrophenyl phosphate). MAC ELISA endpoint titers correlated (r = 0.893) with the absorbance value of a 1:100 dilution of patient serum. Significant (fourfold or greater) changes in the endpoint titers between paired sera corresponded to a critical ratio (ratio of absorbance values at the 1:100 dilution) of greater than or equal to 1.3. IgM antibodies were detected in 71% of patients bled at 0 to 3 days after the onset of illness, in 99% bled at 4 to 21 days, and in 91% bled at 22 to 67 days. Thereafter, the IgM seropositivity rate declined; however, 29% of sera were still positive at 115 to 251 days after the onset of illness. MAC ELISA titers were significantly correlated with hemagglutination inhibition (r = 0.258) and neutralization (r = 0.711) titers. Because IgM antibodies appeared early and waned rapidly, a diagnosis was made on the basis of a decrease in titer more often by MAC ELISA than by hemagglutination inhibition, complement fixation, or neutralization tests. IgM antibodies generally showed a high degree of specificity; heterologous cross-reactions were, however, present in 4 of 14 sera examined. The MAC ELISA is useful for the rapid, early diagnosis of St. Louis encephalitis.

Monoclonal antibodies directed against the envelope glycoprotein and the NV3 non-structural viral protein of yellow fever (YF) were tested by the indirect fluorescent antibody technique against a variety of YF virus strains and heterologous flaviviruses. Monoclonal antibodies directed against the envelope glycoprotein exhibited YF strain-specificity, YF type-specificity, broad group cross-reactivity, or limited subgroup reactivity (YF + Banzi or YF + Koutango + Zika + Usutu + Uganda S). Monoclonal antibodies directed against NV3 reacted either with YF + Koutango or with YF + Banzi. These findings generally correlated with the results of biological tests reported previously. Monoclonal antibodies that were type-specific to YF will be useful for the rapid specific identification of YF virus isolates and are available from the Centers for Disease Control on request.

An epizoodemic of Venezuelan equine encephalitis (VEE), subtype I variant B, occurred in Ecuador during the rainy and hot season of 1969. In this paper, a general description of the epidemic is given and virus isolations from humans are reported. A serologic survey was performed in order to determine the extension of the epidemic along the coastal zone of the country. It is not clear whether the higher antibody rate in older people was because they were at greater risk of infection or was the result of an accumulated immunity with time. The latter could be an indication of endemic virus activity, not yet proven for the VEE IB virus variant. Mosquito surveillance and attempted control by aerial spraying were carried out.

LeDantec virus, originally recovered in 1965 from a patient with a febrile illness in Senegal, was observed by thin section electron microscopy to be bullet-shaped, representative of members of the rhabdovirus group, with mean dimensions of 164 X 78 nm. Particles were observed budding from the plasma membrane and moderate numbers accumulated in the intercellular spaces. In three mammalian cell lines, LeDantec virus was rapidly cytopathic and replicated to moderately high titres. In complement fixation tests with other known rhabdoviruses, LeDantec was found to be related to Keuraliba virus, a previously ungrouped agent isolated from rodents in Senegal in 1968. We propose the formation of a new LeDantec serogroup comprising these two viruses.

We studied the transmission of Fort Morgan (FM) virus within colonies of nesting Cliff Swallows and House Sparrows under three bridges in Morgan County, Colorado during 1976. Nests were examined, and blood or brain specimens were collected from nestlings once or twice a week. Flying birds and small mammals were also studied. We analyzed nesting activity, virus isolations from nestlings of both species, fledging success, multiple infections within a brood of nestlings, infection frequency by age of nestlings, nestling mortality, and infection frequencies by avian species and bridge site. Fort Morgan virus was isolated from 7% (80/1, 156) of the blood and brain samples collected from nestlings. The duration of viremia for nestling House Sparrows was at least 3-4 days based on virus isolation from sequential blood samples. Viremia of nestling Cliff Swallows and House Sparrows did not reduce fledging success, nor were young nestling sparrows viremic more frequently than older nestling sparrows. Nest destruction (by falling down) was a more important cause of nestling mortality than FM virus infection. All age groups of nestling sparrows were viremic at equal rates, but younger nestlings (less than or equal to 7 days old) were more likely than older nestlings (greater than 7 days old) to develop an encephalitic infection. Among nestling House Sparrows, FM virus infections were clustered in time and space. Nestling House Sparrows with FM virus-infected nest-mates were infected more often than conspecifics whose nest-mates were not infected. We concluded that nestling Cliff Swallows and symbiotic House Sparrows that reside in swallow nesting colonies are the principal vertebrate hosts for the maintenance and amplification of FM virus.

A series of 57 Saint Louis encephalitis (SLE) virus isolates from humans, birds, rodents, and mosquitoes showed extensive variability in their RNase T 1 oligonucleotide fingerprints. The fingerprints of virion RNA did not contain an obvious poly(A) tract and were identical when the virus was grown in either mosquito or mammalian cells. Analysis and comparison of long oligonucleotides, representing approximately 10% of the genome of SLE isolates from the Central and Atlantic states, indicated the viruses share at least 80% of their long oligonucleotides. Analysis of the large T 1 -resistant oligonucleotides by RNase A digestion revealed chemical similarities in the composition of common oligonucleotides derived from the genomes of four isolates. Analysis of 57 SLE strains from North America indicated that on the basis of the similarity of the oligonucleotide fingerprints, SLE isolates could be divided into genotypic sets representing different geographic regions in North America. These geographic varieties, designated “topotypes,” represent isolates from: (1) the Central/Atlantic states, (2) Florida, (3) and the Western United States. Variants of each “topotype” have been characterized whose oligonucleotide fingerprints are similar to that of the “topotype” but are sufficiently distinct to permit separation of the highly and less virulent SLE strains.

Only 6 isolations of Saint Louis encephalitis (SLE) virus have been made from Argentina. These include 2 from patients with undifferentiated fever, 2 from rodents (CorAn-9124 and CorAn-9275), and 2 from mosquitoes (78V-6507 and 79V-2533). The infectivity of the last 4 strains for colonized Culex pipiens quinquefasciatus from Argentina was determined following intrathoracic inoculation of virus or by feeding mosquitoes on virus-laden suspensions. Comparison of the replication and dissemination of viral strains CorAn-9275 and 79V-2533 was made by serially dissecting orally infected mosquitoes and by testing heads, alimentary tracts, and body remnants separately. Each of the 4 viral strains replicated to high titer in Cx. p. quinquefasciatus following intrathoracic inoculation; however, day-20 infection rates never exceeded 5.1% in groups of mosquitoes fed on suspensions of CorAn-9124 and CorAn-9275. In contrast, infection rates were 90.9 and 87.5% in mosquitoes fed on suspensions of 78V-6507 and 79V-2533, and oral ID50's were 103.5 and 102.9 Vero cell PFU, respectively. Further, CorAn-9275 did not replicate well in Cx. p. quinquefasciatus alimentary tracts (avg. titers 102.1 and 102.5 PFU/ml on days 10 and 13) and rarely passed the mesenteronal barrier. In contrast, 79V-2533 readily infected mosquitoes by the oral route, replicated to high titer (avg. 104.8 PFU/ml) in the alimentary tract, and began disseminating by day 5. Widespread dissemination of virus occurred by day 10 but virus spread and multiplication continued at least through day 13, by which time viral antigen was detectable in head squashes of practically all mosquitoes tested. The infectivity of the 4 viral strains for Cx. p. quinquefasciatus correlates well with published reports on the virulence characteristics of these viral strains in vertebrates.

Diseases: Yellow fever, dengue, St. Louis encephalitis, Japanese encephalitis, Wes- selsbron, tick-borne encephalitis, louping ill, Kyasanur Forest disease, other tick- borne hemorrhagic fevers, Murray Valley encephalitis, Rocio encephalitis, equine encephalitides (eastern, western, Venezuelan), chikungunya, o’nyong-nyong, Ross River, Mayaro, Sindbis, Ockelbo.

On the basis of previous studies, it has long been stated that 17D yellow fever (YF) vaccine generally does not induce complement-fixing (CF) antibodies, and that the presence of CF antibodies could be used in epidemiological studies to distinguish individuals infected with wild YF virus from vaccinated persons. In January 1979, seroepidemiological investigations were conducted during a YF epidemic in The Gambia, West Africa. Since a mass vaccination campaign was also in progress, it was important to confirm that the CF test could be used for serodiagnosis and determination of the incidence of natural YF infections. The serological responses of 58 individuals who received 17D YF vaccine were studied. The vaccinees fell into three gorups: 1) those with prevaccination YF neutralizing (N) antibodies; 2) immunological virgins without prevaccination YF-N antibody or hemagglutination-inhibiting (HI) antibodies to heterologous flaviviruses (Zika, West Nile, dengue 1, Uganda S, Spondweni, or Ntaya; and 3) those without prevaccination YF-N antibodies but with heterologous flaviviral HI antibodies. Vaccination of persons without prior flaviviral immunological experience resulted in monotypic YF HI and/or N antibody seroconversions, but no CF antibody response. The presence of prevaccination YF N antibodies blocked serological response to the vaccine in a high proportion of the cases; however, 24% of vaccinees in this group had a marked rise in log2 YF CF antibody titer (mean increase of 3.9). Thirteen (46%) of 28 persons without prevaccination YF N, but with heterologous flaviviral HI antibodies demonstrated YF CF antibody seroconversion or increase in titer following vaccination; in this group the mean increase in log2/ YF CF antibody titer was 2.1. The CF antibody response was generally broadly cross-reactive; but in a few individuals, the YF CF antibody response was homotypic. Nine different patterns of HI and CF homologous and heterologous antibody responses were defined and are discussed. The practical significance of these studies is that they demonstrate that in a high percentage of persons with prior flavivirus exposure, anamnestic serological responses to YF vaccine result in CF antibodies similar to those induced by natural YF virus infection. In Africa and tropical America, where the background of flaviviral immunity is high, it is imperative that seroepidemiologic investigations during or after YF outbreaks be conducted prior to vaccination.

Laboratory-acquired infections encountered between 1963 and 1977 among personnel of the Virus Research Laboratory, Ibadan, Nigeria, are reported. Two cases of chikungunya infection occurred and one each with Dugbe, Wesselsbron, and dengue viruses. In each case, virus was isolated or development of antibody demonstrated. Among virus and two each to chikungunya and Rift Valley fever viruses, without experiencing any clinically recognized disease.

Excerpt In august 1985,Aedes albopictusmosquitoes (Figure 1) were found breeding in tire dumps in Harris County (Houston), Texas (1). Although lone examples of this Asian species have been found in...

Immunization of monkeys with yellow fever virus-specified nonstructural protein NS1 resulted in protection against fatal hepatitis as well as marked reduction in the magnitude of viremia after subcutaneous challenge with yellow fever virus. The results may be relevant to the design of possible subunit or recombinant flavivirus vaccines.

The growth of four viruses isolated from lizards in Brazil (Marco, Chaco, and Timbo viruses) and Australia (Almpiwar virus) was studied in a variety of continuous cell lines of mammalian, reptilian, amphibian, and piscine origin. Although replication was found in certain cell lines derived from the coldblooded species, cytopathic effect (CPE) was absent or minimal and growth was less than or equal to that in mammalian cells. Those observations appear to limit the value of poikilothermic cells for primary isolation of viruses from field-collected, cold-blooded vertebrates or arthropods that feed upon them. The four reptilian viruses were found to be naturally occurring temperature sensitive agents, with optima for growth of approximately 30 degrees C. Electron microscope studies showed three of the viruses (Marco, Chaco, and Timbo) to be new members of the family Rhabdoviridae. Marco virus particles were conically shaped and resembled bovine ephemeral fever virus, and two lyssaviruses (Kotonkan and Obodhiang). Chaco and Timbo viruses were cylindrical viruses resembling other rhabdoviruses with particle lengths longer than the prototype VSV. No serologic relationships were found in cross complement fixation tests between these viruses, Marco virus, and 34 other rhabdoviruses.

Western equine encephalitis (WEE) virus was found in myocardial tissue of adult mice during the first five days after inoculation of the virus, with a peak titer (5.0 log plaque-forming units/g) at 24 hr. Light microscopy revealed a multifocal necrotizing myocarditis with a prominent inflammatory response and hyaline and granular degeneration of myofibers. Electron microscopy showed cytoplasmic viral nucleoids and budding and free mature WEE viral particles. Serial electrocardiograms showed the development of disturbances of rate and rhythm, defects in conduction, marked elevation in the ST segment, and low voltage. Myocarditis has not been previously recognized as a complication of alphavirus infection in humans. and we found no evidence for myocardial damage in 11 persons with acute WEE virus infections studied electrocardiographically in 1975. Demonstration of myocarditis in the WEE virus-infected mouse, however, suggests the need to monitor human patients for possible cardiac involvement during future epidemics of WEE virus infection.

Studies were conducted in a restricted area of gallery forest along the middle upper Niger River, Nigeria. Yellow fever neutralizing antibodies were found in 25% of the human population and in 57% of the monkeys tested. Both the abundance of monkeys and the ecological conditions in such forest appear to be favourable for year-round maintenance of yellow fever virus.

Enhancement of neutralization of Sindbis, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, and St. Louis encephalitis viruses by labile serum factor (LSF) in human serum and plasma was demonstrated. Human serum and plasma could be diluted 1:8 and 1:16 and still retain some LSF activity. Satisfactory storage temperatures for retention of LSF activity were −20 or −56 C. Repeated freeze-thaw cycles of serum did not alter LSF activity, but the activity was completely eliminated by heating at 56 C for 5 min. LSF of human serum equally enhanced neutralization by Sindbis immune mouse and rabbit sera; these results suggest a lack of species specificity. Rehydrated lyophilized gunea pig complement did not restore LSF activity to heated human plasma. Serum components responsible for LSF activity were not dialyzable. Discovery of fresh serum without LSF activity established the need to pretest all sera used as LSF sources.

Human sera were treated with kaolin, acetone, and dextran sulfate to determine the best method for removing nonspecific hemagglutination inhibitors. Results indicated that on surveys for group A, group B, and some group C arbovirus HI antibodies, dextran sulfate treatment of sera could be used effectively. This method, however, has limited usefulness for detecting HI antibody for a number of arboviruses, particularly some members of the Bunyamwera supergroup since nonspecific inhibitors for these antigens were not completely removed. HI antibodies in sera drawn early after dengue and Venezuelan equine encephalitis infection were detected more readily after dextran sulfate treatment than after kaolin treatment. Kaolin, but not dextran sulfate, was shown to remove antibody from IgM fractions of sera.

The role of the reticuloendothelial system (RES), and especially of the macrophage, in host defense against viral infection includes the important functions of clearance and synthesis of antibody and interferon [1-4]. Disease of the RES, splenectomy, and RES "blockade" by injection of colloidal substances may alter host responses to viral infection. Thorium dioxide (Thorotrast)1 has been widely used to produce RES "blockade" [5-9], but its effects on the pathogenesis of viral infections have received relatively little attention. In particular, the effect of Thorotrast on response of humoral antibody to viral infection has not been studied.

St. Louis encephalitis (SLE) virus was isolated from adult Dermacentor variabilis ticks removed from a Raccoon (Procyon lotor) captured in Memphis, Tennessee, USA, on 24 May 1979. Neutralizing antibody against SLE virus was detected in mammals from the same area. The epidemiologic significance of the isolate is unknown.

• Using techniques of stereology, we measured the severity of lesions in ten cases of acute St Louis encephalitis (SLE) from the 1975 epidemic in northern Illinois. Percentage of fractional volume and numerical profile density on area (N/A) of cellular nodules and N/A of blood vessels with perivascular inflammatory cellular infiltration were significantly correlated in 17 anatomic regions of the CNS. Ranking of the severity of lesions in these regions agreed with subjective estimates of other cases of SLE. Immunofluorescent tests established the presence of SLE viral antigen in cell bodies of neurons. Our findings contribute to better understanding of the pathology of SLE in man.