Isolation of eastern equine encephalitis virus from Aedes albopictus in Florida.

Fourteen strains of eastern equine encephalitis (EEE) virus were isolated from Aedes albopictus mosquitoes collected in Polk County, Florida. These are the first isolations of an arbovirus of proven public health and veterinary importance from naturally infected Ae. albopictus in the United States since established populations of this introduced mosquito were first discovered in 1985. The widespread distribution of Ae. albopictus in Florida and in other areas of the United States where EEE is endemic raises concern that this species may become an epizootic and epidemic vector of EEE virus.

A new arbovirus from Aedes albopictus, an Asian mosquito established in the United States.

Ten strains of a new arbovirus belonging to the Bunyamwera group (Bunyaviridae) were recovered from field-collected Aedes albopictus mosquitoes in Potosi, Missouri. This evidence indicates that this species may serve as an arbovirus vector in the United States. The urban-suburban distribution, aggressive biting behavior, and broad viral susceptibility of Ae. albopictus may lead to the transmission of viruses of known public health importance and perhaps of viruses hitherto not transmitted to humans because of the feeding pattern of their usual vectors.

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes.

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Antigenic relationships of group A arboviruses by plaque reduction neutralization testing.

Antigenic relationships of 20 group A arboviruses were assessed by the plaque reduction neutralization test, using highly specific hyperimmune mouse ascitic fluids and antisera. The existence of three complexes of group A viruses was verified. With rare exceptions, heterologous neutralization reactions were observed only among viruses in the same complex; however there were at least one or two immune reagents in each complex which were broadly cross-reactive within that complex.

Serological reactions of fractionated hamster immunoglobulins with California group viruses.

Immunoglobulin M and immunoglobulin G (early and late) were separated by sucrose density gradient centrifugation from single inoculation hamster immune sera prepared against La Crosse, California encephalitis and snowshoe hare viruses. Fractionated antibodies were tested for differences in serological specificity by hemagglutination-inhibition, neutralization, and complement-fixation reactions. None of the immunoglobulins demonstrated a consistent pattern of superior specificity in serological reactions with closely related California group viruses. Results were most uniform in the neutralization test in that immunoglobulin M and early and late immunoglobulin G all showed similar cross-reactivities. By hemagglutination-inhibition and complement-fixation, a given immunoglobulin type from one serum would show good specificity while the same type from another serum would be quite cross-reactive. Immunoglobulin M neutralizing antibody fractions fixed complement as effectively as late immunoglobulin G neutralizing antibody fractions. Unlike results obtained by others with flavivirus immune sera, the immunoglobulin M fractions from California group immune sera were not more specific than immunoglobulin G fractions by neutralization, hemagglutination-inhibition, or complement-fixation tests. Single inoculation hamster antisera, prepared to California group viruses and collected late (28-30 days), were as specific in the neutralization test as antisera collected early (7-10 days).

Antigenic uniformity of topotype strains of Thogoto virus from Africa, Europe, and Asia.

Prototype Thogoto virus from Kenya and Thogoto virus isolates from Portugal, Italy, Nigeria, Central African Republic, Cameroon, and Iran were cross-tested by hemagglutination inhibition and serum dilution plaque reduction neutralization. Considering the broad geographic distribution of these strains, the antigenic differences seen appear to be essentially minor. This uniformity suggests that the distribution of Thogoto virus is due to repeated introduction of virus from common sources and that such introduction may depend on the movement of tick-infested domestic animals.

California serogroup viruses from mosquitoes collected in the USSR.

Four California serogroup viruses isolated from mosquitoes in the USSR were tested for antigenic analogy with prototype viruses of the California serogroup. The topotype isolates are biologically similar to, but antigenically different from each other. One is a subtype of snowshoe hare virus, two are different subtypes of Tahyna, Lumbo, and snowshoe hare viruses, and one is identical to Inkoo virus, previously isolated only in Finland. The results indicate that molecular studies of these viruses are necessary to comprehend their evolution.

Isolation of a newly recognized alphavirus from mosquitoes in Vietnam and evidence for human infection and disease.

During studies of arboviral epidemiology in Vietnam, five virus isolates were recovered from Culex tritaeniorhynchus mosquitoes. Three of the five isolates were identified as strains of Japanese encephalitis virus, but the others, collected at Me Tri village, Hanoi, were shown to represent an alphavirus, for which we propose the name Me Tri virus. This newly recognized virus is most closely related to Semliki Forest virus. The two isolates appear to be antigenic subtypes of a single virus, and each was associated with central nervous system illnesses in children. Serologic surveys indicate widespread distribution of these viruses in both humans and livestock in Vietnam. We suggest that Me Tri virus is an etiologic agent of human disease in southeast Asia.

Genomic sequencing of deer tick virus and phylogeny of powassan-related viruses of North America.

Powassan (POW) virus is responsible for central nervous system infection in humans in North America and the eastern parts of Russia. Recently, a new flavivirus, deer tick (DT) virus, related to POW virus was isolated in the United States, but neither its pathogenic potential in human nor the taxonomic relationship with POW virus has been elucidated. In this study, we obtained the near-full-length genomic sequence of the DT virus and complete sequences of 3 genomic regions of 15 strains of POW-related virus strains. The phylogeny revealed 2 lineages, one of which had the prototype POW virus and the other DT virus. Both lineages can cause central nervous system infection in humans. By use of the combination of molecular definition of virus species within the genus Flavivirus and serological distinction in a 2-way cross-neutralization test, the lineage of DT virus is classified as a distinct genotype of POW virus.

Ovine fetal malformations induced by in utero inoculation with Main Drain, San Angelo, and LaCrosse viruses.

The teratogenic potential of three bunyaviruses, two California serogroup bunyaviruses, LaCrosse virus and San Angelo virus, and a Bunyamwera serogroup member, Main Drain virus, in sheep was studied following in utero inoculation of ewes in early gestation. Although Main Drain virus appeared to be most teratogenic, all three viruses induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios. The teratogenic effects of these viruses are identical to those described in ovine infections by Cache Valley and Akabane viruses. Demonstration of a common bunyaviral tropism for fetal tissue infection that results in congenital brain and musculoskeletal malformations provides evidence that human in utero infection by bunyaviruses could result in similar malformations in human infants.

Seroepidemiology of La Crosse virus infection in humans in western North Carolina.

On the Cherokee Indian Reservation and surrounding area of western North Carolina, an area-wide serosurvey was conducted to determine the prevalence of neutralizing antibody to La Crosse (LAC) virus. A questionnaire was used to identify risk factors important in exposure to virus-infected mosquitoes in populations near the reservation. Of 1,008 serum samples tested, 9.6% were positive for LAC virus antibody. For samples solely collected from on (n = 311) or off (n = 697) the reservation, the prevalence of seropositive samples was 20.6% on the reservation and only 4.7% off the reservation. Seropositivity increased directly with age, indicating that transmission of LAC virus was highly endemic. Age and location residence (on versus off the reservation) were significant risk factors for exposure to LAC virus. Persons on the reservation were 5.5 times more likely to have been exposed to LAC virus than were people who reside off the reservation. An additive increase in risk of 1.5 times over each age group was found, so that the oldest age group ( > or = 75 years) was 7.5 times more likely to have been exposed to LAC virus than was the youngest age group ( < 1-14 years).

Reevaluation of the western equine encephalitis antigenic complex of alphaviruses (family Togaviridae) as determined by neutralization tests.

Fourteen viruses closely related to the Fleming strain of western equine encephalitis (WEE) virus were cross-tested by serum dilution-plaque reduction neutralization. The results demonstrate that strains McMillan, R-43738, AG80-646, BeAr 102091, and Y62-33 are subtypes or varieties of western equine encephalitis virus strain Fleming. Ockelbo, Kyzylagach, and Babanki are subtypes of the prototype strain (EgAr 339) of Sindbis virus. Fort Morgan and Buggy Creek viruses are closely related to each other, whereas Highlands J and Aura viruses are distinct from other members of this antigenic complex. There appear to be parallels between geographic distribution and antigenic relatedness. We hypothesize that birds, the principal vertebrate hosts for these viruses, spread the progenitor viruses north and south and from continent to continent. Viruses of the WEE complex with lesser antigenic differences may develop in discrete ecologic conditions.

Identification of Highlands J virus from a Florida horse.

A virus, strain 64A-1519, isolated from the brain of a horse dying of encephalitis in Florida in 1964, was identified as western equine encephalomyelitis (WEE) virus. Recently, we used polyclonal and monoclonal immune reagents to identify this isolate by comparing it to 2 strains of WEE virus and to Highlands J (HJ) virus in hemagglutination-inhibition, immunofluorescent antibody, and plaque-reduction neutralization tests. These tests demonstrate that strain 64A-1519 is a strain of HJ virus distinct from WEE virus.

Kairi virus identified from a febrile horse in Argentina.

A virus isolated from the blood of a febrile horse in Argentina was identified as a strain of Kairi virus. This is the fifth Bunyamwera serogroup virus isolated from livestock and wild animals in the Americas. Bunyamwera serogroup viruses have been isolated from febrile humans in the Americas and Africa.

Distribution of Bunyamwera serogroup viruses in North America, 1956-1984.

We attempted to tabulate all Bunyamwera serogroup (family Bunyaviridae, genus Bunyavirus) isolates from North America. By summarizing information from the laboratories of the Centers for Disease Control, data generously shared by other laboratories, and the published literature, we were able to accumulate data regarding 1,372 Bunyamwera serogroup viruses. These were: Tensaw (664, including 8 from vertebrates), Cache Valley (396, including 6 from vertebrates), Main Drain (160, including 14 from vertebrates), Lokern (69, including 8 from vertebrates), Northway (13, including 5 from vertebrates), Tlacotalpan (7), Santa Rosa (2), Santa Cruz (1 from a horse), and 60 of undetermined serotype. Virus isolation rates by month of collection were correlated with collection efforts, but associations of viruses and arthropod vectors varied by location, vertebrate host, and arthropod distribution. Tensaw virus was isolated principally from Anopheles crucians mosquitoes (466/656 isolates from arthropods) in the southeastern United States; Cache Valley virus principally from An. quadrimaculatus (94), Coquillettidia perturbans (59), Culiseta inornata (45), Aedes sollicitans (30), Psorophora columbiae (23), An. punctipennis (18), and Ae. vexans and trivittatus (18 each) mosquitoes (total = 305/382 isolates from arthropods from all of the United States and Canada, except the southeastern United States); Main Drain virus from Culicoides variipennis (31), Culicoides (Selfia) sp. (65), and Psorophora (23) and Aedes (21) species mosquitoes in the western United States; Lokern virus from Culicoides species (55/61 isolates from arthropods) in the western United States. Relationships between vector and vertebrate host distributions are discussed briefly in regard to geographic distribution of the Bunyamwera serogroup viruses.

Japanese encephalitis among hospitalized pediatric and adult patients with acute encephalitis syndrome in Hanoi, Vietnam 1995.

The etiologic spectrum of acute encephalitis syndrome (AES) has not been well defined in Vietnam. Cohort and case-control studies were performed on all adult and pediatric AES patients admitted to the Neurology Service of Bach Mai Hospital between June 5 and August 3, 1995. Among pediatric AES patients, 31 (67%) of 46 had acute Japanese encephalitis (JE), compared with only two (6%) of 33 adult AES patients (P < 0.0001). For confirmed JE cases, serum specimens obtained 15-21 days after symptom onset had the highest mean anti-JE IgM signal-to-noise (P/N) ratios (8.08 + 1.09 SE). A serosurvey of adult household members did not reveal any cases of recent subclinical JE infection, although 26% had evidence of past JE infection. The use of bed netting was nearly universal but did not appear to reduce the risk of AES or JE. Given the high incidence of JE, particularly among children, Vietnam seems well suited for the development of a targeted JE vaccination strategy.

Identity of Karelian fever and Ockelbo viruses determined by serum dilution-plaque reduction neutralization tests and oligonucleotide mapping.

The causative agents of Ockelbo disease in Sweden, Pogosta disease in Finland, and Karelian fever in the USSR have been attributed to alphaviruses (family Togaviridae) related to Sindbis virus. We compared prototypes Sindbis, Ockelbo, and Karelian fever viruses by neutralization tests. We also analyzed oligonucleotide fingerprint maps of prototypes Ockelbo and Karelian fever viruses and a strain of Sindbis virus from Czechoslovakia. The results indicate that Ockelbo and Karelian fever viruses are essentially identical and suggest that Ockelbo disease, Pogosta disease, and Karelian fever are synonyms for the same disease.

Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes.

Between July and October 1996, a West Nile (WN) fever epidemic occurred in the southern plain and Danube Valley of Romania and in the capital city of Bucharest, resulting in hundreds of neurologic cases and 17 fatalities. In early October 1996, entomologic and avian investigations of the epidemic were conducted in the city of Bucharest and nearby rural areas. Thirty (41%) of 73 domestic fowl sampled had neutralizing antibody to WN virus, including 5 of 13 ducks (38%), 1 of 1 goose, 19 of 52 chickens (37%), 1 of 1 peahen, and 4 of 6 turkeys (67%). Seroprevalence in domestic fowl (27%, or 7 of 26) from the urban Bucharest site was not significantly different (P = 0.08, by Fisher's exact test) than rates at three rural sites (50%, or 23 of 46). Serum collected from one of 12 Passeriformes, an Erithacus rubecula, was positive for neutralizing antibody to WN virus. A total of 5,577 mosquitoes representing seven taxa were collected. Culex pipiens pipiens accounted for 96% of the mosquitoes collected. A single virus isolate, RO97-50, was obtained from a pool of 30 Cx. p. pipiens females aspirated from the walls and ceiling of a blockhouse located near the center of Bucharest, resulting in a minimum infection rate of 0.19 per 1,000. Antisera prepared against RO97-50 failed to distinguish among RO97-50, WN virus strain Eg101, and Kunjin (KUN) virus strain MRM16. A 2,323-basepair DNA fragment of the envelope (E) glycoprotein gene from RO97-50 and a Romanian WN virus strain obtained from a human cerebrospinal fluid sample, RO96-1030, were sequenced. Phylogenetic analyses of 23 WN virus strains and one KUN virus strain using the amino acid and nucleotide sequences for a small portion of the E gene suggest the existence of two large lineages of viruses. Bootstrap analysis of the nucleotide alignment indicated strong support (95%) for a lineage composed of WN virus strains from northern Africa, including isolates from Egypt and Algeria, and west, central, and east Africa, all of the European isolates, those from France and Romania, an Israeli isolate, and an isolate of KUN virus from Australia. The nucleotide sequence of RO97-50 was identical to the sequence of a WN virus isolate obtained from Cx. neavei mosquitoes from Senegal and Cx. univittatus mosquitoes from Kenya. The phylogenetic analyses were compatible with the introduction of virus into Romania by birds migrating from sub-Saharan Africa, to northern Africa, and into southern Europe.

Isolation of Tete serogroup bunyaviruses from Ceratopogonidae collected in Colorado.

Two viruses were isolated from ceratopogonid midges collected in northern Colorado. Electron microscopy indicated that both isolates were bunyavirus-like. Indirect fluorescent antibody and serum dilution-plaque reduction neutralization tests showed that these isolates were members of the Tete serogroup, most closely related antigenically to Tete and Batama viruses but distinguishable from both and from each other. We suggest the name Weldona virus for these isolates. Antibody in both waterfowl and passerine birds in northern Colorado indicates the enzootic presence of these viruses in northern Colorado and raises unanswered questions about the introduction and establishment of Tete serogroup viruses in the Americas.

Epidemiologic aspects of a St. Louis encephalitis epidemic in Jefferson County Arkansas, 1991.

In 1991, the first epidemic of St. Louis encephalitis (SLE) ever reported in Arkansas resulted in 25 cases in Pine Bluff (attack rate: 44 per 100,000; 95% confidence interval [CI] 28-65). To identify risk factors for SLE viral infection and risk factors for neuroinvasive illness, we conducted a community-based, cross-sectional study of noninfected and asymptomatically infected persons and a case-control study of asymptomatically and symptomatically infected persons. The SLE viral infection rate was similar in all age groups and in all studied census tracts. Risk factors for asymptomatic infection included: living in a low income household (relative risk [RR] = 2.6, 95% CI 1.1-6.0), sitting outside in the evening (RR = 2.1, 95% CI 1.0-4.8), and living in homes with porches (RR = 2.9, 95% CI 0.9-9.3) or near open storm drains (RR = 2.2, 95% CI 1.0-4.9). Compared with asymptomatically infected persons, symptomatic persons were older (odds ratio [OR] for age > or = 55 years = 13.0, 95% CI 1.2-334) and more likely to have a previous history of hypertension (OR = 8.5, 95% CI 1.1-72). Our results indicate that advanced age is the most important risk factor for developing encephalitis after infection with SLE virus. Hypertension and vascular disease may predispose to neuroinvasive disease, but this epidemiologic study has not ruled out the confounding effects of age.