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O'nyong-nyong (ONN) fever, caused by infection with a mosquito-borne central African alphavirus, is an acute, nonfatal illness characterized by polyarthralgia. During 1996-1997, south-central Uganda experienced the second ONN fever epidemic ever recognized. Among 391 persons interviewed and sampled, 40 cases of confirmed and 21 of presumptive, well-characterized acute, recent, or previous ONN fever were identified through active case-finding efforts or during a household serosurvey and by the application of clinical and laboratory criteria. Among confirmed cases, the knees and ankles were the joints most commonly affected. The median duration of arthralgia was 6 days (range, 2-21 days) and of immobilization was 4 days (range, 1-14 days). In the majority, generalized skin rash was reported, and nearly half had lymphadenopathy, mainly of the cervical region. Viremia was documented in 16 cases, primarily during the first 3 days of illness, and in some of these, body temperature was normal. During this epidemic, the combination of fever, arthralgia, and lymphadenopathy had a specificity of 83% and a sensitivity of 61% in the identification of cases of ONN fever and thus could be useful for surveillance purposes.

Aedes triseriatus (Say) population density patterns and La Crosse encephalitis virus infection rates were evaluated in relation to a variety of habitat parameters over a 14-wk period. Ovitraps and landing collections were used in a La Crosse virus-enzootic area in Nicholas County, WV. Study sites were divided into categories by habitat type and by proximity to the residences of known La Crosse encephalitis cases. Results demonstrated that Ae. triseriatus population densities were higher in sugar maple/red maple habitats than in hemlock/mixed hardwood habitats or in a site characterized by a large number of small red maple trees. Sites containing artificial containers had higher population densities than those without. La Crosse virus minimum infection rates in mosquitoes collected as eggs ranged from 0.4/1,000 to 7.5/1,000 in the 12 study sites, but did not differ significantly among sites regardless of habitat type or proximity to human case residences. La Crosse virus infection rates in landing Ae. triseriatus mosquitoes ranged from 0.0/1,000 to 27.0/1,000. La Crosse virus was also isolated from host-seeking Ae. canadensis (Theobald) in two study sites, at rates similar to those found in the Ae. triseriatus populations. The Ae. triseriatus oviposition patterns and La Crosse virus infection rates suggest that this mosquito species disperses readily in the large woodlands of central West Virginia. The La Crosse enzootic habitats in Nicholas County, WV, are contrasted with those studied in other geographic regions where La Crosse virus is found.

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-arboviral immunoglobulin G (IgG ELISAs) were developed for a comprehensive array of medically important arboviruses from the Alphavirus , Flavivirus , and Bunyavirus genera. Tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. MAbs were screened for suitability as capture vehicles for antigens from the three genera. The final test configuration utilized group-reactive MAbs eastern equine encephalitis virus 1A4B-6, dengue 2 virus 4G2, and La Crosse encephalitis virus 10G5.4 to capture the specific inactivated viral antigens. Serum IgG was detected by using alkaline phosphatase-conjugated anti-human IgG (Fc portion). A dilution of 1:400 was chosen as the universal screening serum dilution, with endpoint titrations of serum samples testing positive eliminating occasional false-positive results. IgG ELISA results correlated with those of the standard plaque-reduction neutralization assays. As expected, some test cross-reactivity was encountered within the individual genera, and tests were interpreted within the context of these reactions. The tests were standardized for laboratory diagnosis of arboviral infections, with the intent that they be used in tandem with the corresponding IgM antibody-capture ELISAs.

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae , Flaviviridae , and Bunyaviridae . Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of ≥2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

Chronic heart failure (CHF) may be considered a state of immune activation and persistent inflammation expressed by increased circulating levels of pro- and anti-inflammatory cytokines. The purpose of the study was to investigate the immune status in patients with CHF compared to normal individuals. We measured serum cytokine levels as well as cytokine production after ex vivo LPS stimulation of whole blood taken from 14 patients with CHF and 14 healthy volunteers. We used 500 pg/ml of LPS for an incubation period of 4 h to stimulate 100 μL of whole blood. Patients with CHF had significantly higher levels of TNF-RI, and TNF-RII in serum compared to normal individuals. TNF-α, IL-6, and IL-10 did not differ significantly. After LPS stimulation, patients with CHF had significantly higher levels of TNF-α and IL-10, and significantly lower IL-6 levels compared to normal individuals. TNF-α receptors did not differ significantly. Patients with CHF may be found in a pro- as well as an anti-inflammatory state. They also do not develop endotoxin tolerance in an ex vivo laboratory model using whole blood stimulated with LPS. They may have increased TNF-α and IL-10 production after LPS stimulation of whole blood, which may contribute to a worsening of heart function, more severe disease presentation and a worse outcome during infections. © 2007 Elsevier Ltd. All rights reserved.

In the fall of 1995, a 20-day-old female ostrich chick, 1 of a group of 20, was presented live with clinical signs of 2 days duration characterized by unsteady gait, circling to the left, and walking backward. Another bird with similar clinical signs had died and another had recovered. The bird was euthanized and examined at necropsy. Twenty-five milliliters of serous fluid was in the abdominal cavity and there was increased pericardial fluid. Histopathology of the brain revealed mild to moderate nonsuppurative encephalitis characterized by mild multifocal malacia, perivascular cuffing by lymphocytes, and gliosis. The heart had multifocal infiltrations of lymphocytes mixed with macrophages and a few plasma cells throughout the myocardium. Cytopathic effects were observed in primary chicken embryo liver cells following inoculation with a tissue homogenate prepared from the brain of the affected ostrich. Virus particles the size and morphology of the family Bunyaviridae were observed in cell culture lysate by negative-stain electron microscopy. Viral characterization demonstrated that the virus isolate is a previously unknown serotypic variant (subtype) of Turlock virus. Twelve of 65 sera collected over a 3-year period from ostriches aged from 1 month to 4 years were positive for neutralizing antibody to both the Turlock prototype strain and the new subtype of Turlock virus described in this report.

Arboviruses isolated and identified from mosquitoes in South Carolina (USA) are described, including new state records for eastern equine encephalitis virus (EEE), St. Louis encephalitis virus (SLE), Flanders virus, Tensaw virus (TEN), and a variant of Jamestown Canyon virus (JC). Mosquitoes were collected at 52 locations in 30 of 46 South Carolina counties beginning in June 1996, and ending in October 1998, and tested for arboviruses. Of 1,329 mosquito pools tested by virus isolation (85,806 mosquitoes representing 34 mosquito species or complexes), 15 pools were positive. Virus isolations included EEE from 1 pool each of Anopheles crucians complex and Culex erraticus; a variant of JC from 1 pool of An. crucians complex; a California serogroup virus from 1 pool of Aedes atlanticus/tormentor; TEN from 5 pools of An. crucians complex and 1 pool each of Culex salinarius and Psorophora ciliata; Flanders virus from 1 pool of Culiseta melanura; and Potosi virus from 1 pool each of Aedes vexans, Coquillettidia perturbans, and Psorophora columbiae. Of 300 mosquito pools tested by antigen-capture assay for EEE and SLE (14,303 mosquitoes representing 16 mosquito species or complexes), 21 were positive for EEE and I was positive for SLE. Positive EEE mosquito pools by antigen-capture assay included An. crucians complex (14 pools), Anopheles punctipennis (1 pool), Anopheles quadrimaculatus (1 pool), Cq. perturbans (4 pools), and Cs. melanura (1 pool). One pool of Cx. salinarius was positive for SLE by antigen-capture assay. Arbovirus-positive mosquito pools were identified from 12 South Carolina counties, all located in the Atlantic Coastal Plain, and from 4 of 8 Carolina bays surveyed.

A St. Louis encephalitis (SLE) epidemic in Florida during 25 weeks in 1990–1, resulted in 222 laboratory-diagnosed cases, an attack rate in the 28 affected counties of 2·25/100000. Disease risk rose with advanced age, to 17·14/100000 in persons over 80 years, and all 14 fatal cases were in persons over 55 years (median, 70 years). Community serosurveys in Indian River County, the epicenter of the outbreak (attack rate 21/100000), showed acute asymptomatic infections in 3·6% of the persons surveyed, with higher rates in persons with outdoor occupational exposure (7·4%) and in clients of a shelter for the indigent (13·3%). A matched case-control study found that evening outdoor exposure for more than 2 h was associated with an increased risk for acquiring illness (odds ratio [OR] 4·33, 95% CI 1·23–15·21) while a number of recommended personal protective measures were protective. Four SLE patients were dually infected with Highlands J virus, the first reported cases of acute infection with this alphavirus. The case-control study provided the first evidence that a public education campaign to reduce exposure had a protective effect against acquiring the disease.

A virus isolate, ThCAr105/92, from a pool of mosquitos, Culex tritaeniorhynchus, collected in Chiang Mai, Thailand in 1992, appeared to be a member of the genus Flavivirus of the family Flaviviridae, based on the reverse transcription polymerase chain reaction (RT-PCR) using flavivirus cross-reacting primer pairs, electron microscopic examination, and serological tests. However, RT-PCR using Japanese encephalitis (JE) virus-specific primers showed that the isolate was different from JE virus. Sucrose density gradient sedimentation of the virus replicated in C6/36 cells indicated that the virus is relatively unstable in the infected culture fluids at 37 degrees C. Antibody prepared against this virus and a virus seed for the isolate were tested by cross neutralization against a panel of flaviviruses and the results showed that the new isolate was a distinct subtype of Tembusu virus.

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.

Outbreaks of yellow fever (YF) have never been recorded in Kenya. However, in September 1992, cases of hemorrhagic fever (HF) were reported in the Kerio Valley to the Kenya Ministry of Health. Early in 1993, the disease was confirmed as YF and a mass vaccination campaign was initiated. Cases of suspected YF were identified through medical record review and hospital-based disease surveillance by using a clinical case definition. Case-patients were confirmed serologically and virologically. We documented 55 persons with HF from three districts of the Rift Valley Province in the period of September 10, 1992 through March 11, 1993 (attack rate = 27.4/100,000 population). Twenty-six (47%) of the 55 persons had serologic evidence of recent YF infection, and three of these persons were also confirmed by YF virus isolation. No serum was available from the other 29 HF cases. In addition, YF virus was isolated from a person from the epidemic area who had a nonspecific febrile illness but did not meet the case definition. Five patients with confirmed cases of YF died, a case-fatality rate of 19%. Women with confirmed cases of YF were 10.9 times more likely to die than men (P = 0.010, by Fisher's exact test). Of the 26 patients with serologic or virologic evidence of YF, and for whom definite age was known, 21 (81%) were between 10 and 39 years of age, and 19 (73%) were males. All patients with confirmed YF infection lived in rural areas. There was only one instance of multiple cases within a single family, and this was associated with bush-clearing activity. This was the first documented outbreak of YF in Kenya, a classic example of a sylvatic transmission cycle. Surveillance in rural and urban areas outside the vaccination area should be intensified.

Prospective studies were conducted at used-tire sites in Illinois during 1994–1995 in an effort to isolate arboviruses from mosquitoes, particularly Aedes alhopictus (Skuse) and Aedes triseriatus (Say). Three isolates of Potosi virus were obtained from Ae. albopictus collected at a waste tire site in Jasper County during 1994 and 1995. Also, a single isolate of Cache Valley virus was obtained from Ae. albopictus collected at the Jasper County site during 1995. These are the first records of arbovirus isolations from Ae. albopictus in Illinois and the first isolate of Cache Valley virus from this mosquito species. During 1994, two isolates of La Crosse virus were made from Ae. triseriatus collected at a used-tire site in Peoria County in proximity to the residence of a human La Crosse encephalitis case. This is the first evidence in Illinois that indicates increased risk to humans living near used-tire sites, which may serve as foci for production of Ae. triseriatiis , the vector of La Crosse virus. Tire removal and improved environmental sanitation at such sites may greatly reduce the abundance of vector mosquitoes, and, therefore, the risk of arbovirus transmission.

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.

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.

Summary Background Japanese encephalitis is a major cause of death and disability throughout Asia, including the Indian subcontinent. Although an effective vaccine for Japanese encephalitis is available, hundreds of millions of susceptible individuals remain unimmunised because of the vaccine's cost. In 1988, an inexpensive live-attenuated vaccine (SA14-14-2) was licensed in China. We have measured the effectiveness of this vaccine. Methods In a case-control study in rural Sichuan Province, China, the 56 cases consisted of children admitted to hospital with acute Japanese encephalitis, and were confirmed serologically. 1299 village-matched and age-matched controls were identified, and vaccination histories obtained from pre-existing written records. Findings The effectiveness of one dose was 80% (95% Cl 44 to 93%); that of two doses was 97·5% (86 to 99·6%). Controlling for multiple potential confounders did not alter these results. Interpretation We conclude that a regimen of two doses of live-attenuated Japanese encephalitis vaccine, administered 1 year apart, is effective in the prevention of clinically important disease. Subsequent study is needed to assure the safety of this vaccine.

Mosquitoes were collected for virus isolation tests from 36 sites in Bradford, Lake, Leon, Manatee, Orange, Osceola, Pasco, Putnam, and Sarasota counties, FL, from April 6 through October 11, 1993. A total of 158,129 adult specimens were collected in 726 trap nights using CDC light traps, usually baited with dry ice. At least 35 species were represented, although 60% of the collections was made up of 3 species (Aedes infirmatus, 6.5%; Anopheles crucians, 14.4%; and Culex nigripalpus, 39.4%). Four of the 36 collecting sites were located at waste-tire sites, where 254 trap nights yielded 27,455 specimens (17.4% of 9-county total). Forty-three virus strains were isolated from 2,812 mosquito pools consisting of 158,129 specimens. The viruses isolated include eastern equine encephalitis (EEE), 5 strains; Everglades (EVE), 2 strains; Keystone (KEY), 6 strains; Tensaw (TEN), 21 strains; trivittatus (TVT), one strain; Shark River (SR), one strain; and Flanders (FLA), one strain. In addition, 2 strains that are either KEY or Jamestown Canyon (JC) virus, and 4 ungrouped viruses remain to be identified. Twenty-one (48.8%) of the 43 virus strains were isolated from mosquitoes collected at waste-tire sites as follows: EEE (1), KEY (5), KEY/JC (1), TEN (13), and one ungrouped virus. The vector relations of the viruses are discussed and the potential importance of waste-tire sites as breeding habitats and harborages for vector and nuisance species is emphasized.

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).

Potosi (POT) virus, a recently characterized Bunyamwera serogroup virus, was discovered when it was isolated from Aedes albopictus collected at a waste-tire site in Potosi, Washington County, Missouri, during 1989. During the following year, POT virus was not isolated from 39,048 mosquitoes, including 17,519 Ae. albopictus, collected in Washington County. In 1991, mosquito collections from South Carolina, Ohio, and Michigan yielded 8 strains of POT virus: 6 from Coquillettidia perturbans and one each from Culex restuans and Psorophora columbiae. Additional collections of Ae. albopictus from several states during 1990-93 failed to yield further isolates of POT virus. In 1994, POT virus was isolated from Ae. albopictus and Anopheles punctipennis in North Carolina and from Ae. albopictus in Illinois. These represent the first virus isolations of any type for Ae. albopictus in those states. Thus far, POT virus has been isolated from 5 mosquito species in different genera in 6 states. The known geographic range of POT virus, based on virus isolations, has been extended from Missouri to the upper Midwest and the Atlantic seaboard. Potential vector relationships and possible transmission cycles of POT virus are discussed.

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.

A total of 4,169 adult mosquitoes were aspirated from 3 tire disposal sites in North Carolina for virus assays. Aedes albopictus was the dominant species, with a relative abundance of approximately 99%. Potosi virus was isolated from one pool of 68 female Ae. albopictus. Priorities for future Potosi virus research and the implications of the North Carolina isolate are discussed.

A new Phlebovirus provisionally named Tunis virus has been isolated from Argas reflexus hermanni ticks parasitizing domestic pigeons. It is the first isolation of an arbovirus from Tunisia and the fourth tick-borne virus to be isolated from the Moghreb following Soldado, Essaouira and Kala Iris in Morocco. The pathogenic potential of this virus is briefly discussed according to the behaviour of its vector and previous serosurveys in the country.

Arbovirus surveillance was conducted during an epizootic of western equine encephalitis (WEE) during 1987 and during a nonepizootic year, 1991, in the same area in northern Colorado. Mosquitoes were collected in Larimer County, CO, during weeks 33-37 (10 August to 7 September) in 1987 and during weeks 26-35 (24 June to 26 August) in 1991. In total, 13,099 mosquitoes in 694 pools collected during 1987 and 8,672 mosquitoes in 242 pools collected during 1991 were tested for virus. WEE virus was isolated in both years from Culex tarsalis Coquillett and from Cx. pipiens L. in 1987. Infection rates and population levels of Cx. tarsalis were not significantly different in the 2 yr during weeks 33, 34, and 35 (12-26 August). St. Louis encephalitis virus was isolated in 1987 from Cx. tarsalis. Other viruses isolated included Hart Park, Turlock, and Jerry Slough, a variety of Jamestown Canyon virus.

The clinical records of 15 children admitted to Hospital Infantile de México Federico Gómez with diagnosis of viral meningitis were reviewed. They were part of 19 patients admitted with this diagnosis during a 5 week period (March 22 to April 30, 1992) and represent a significant increase of aseptic meningitis compared with the same periods of previous years at Hospital Infantile de Mexico and in Mexico City where there is an ongoing epidemic outbreak of this entity. All the patients studied had spinal fluid findings consistent with viral meningitis and in 4 of them on ECHO virus type 30 was isolated at the Enterovirus Section of the CDC, Atlanta Georgia USA.

Orungo and Lebombo orbivirus isolates were examined for their intra- and intergroup genetic relatedness by blot hybridization and gene reassortment; blot hybridization was also used to examine the relatedness of selected Orungo and Lebombo isolates to known orbiviruses. Among the Orungo isolates, greater than 74% sequence similarity was shown in the majority of their genes. Gene 2 was the most divergent gene, with four unique types identified, and genes 5, 6 and 10 were variant among the isolates. Plaque reduction neutralization tests revealed at least four serotypes, a result which correlated with the hybridization data. Gene reassortment was shown between two representative Orungo isolates. Among the Lebombo isolates, two hybridization types were identified between which gene reassortment was demonstrated. Unique genes were not shown, whereas genes 2, 5 and 10 exhibited minor sequence variability. Geographic distribution correlated with relatedness among the Lebombo isolates, which was not the case among the Orungo isolates. Orungo and Lebombo viruses did not cross-hybridize or reassort their genes in vitro, in intergroup studies. In blot hybridization tests of Orungo and Lebombo isolates with known orbivirus serogroups and ungrouped orbiviruses, no strong cross-hybridization was seen. These results demonstrate that Orungo and Lebombo are distinct from each other and from other orbiviruses, and should therefore be recognized as two new Orbivirus serogroups.

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 norther 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.

Adult mosquitoes were collected for virus isolation from two sites in Indian River County, FL, from May 5 through August 13, 1998 using dry ice-baited CDC light traps (81 trap-nights) and CDC gravid traps (254 trap-nights). A total of 46,150 female mosquitoes (923 mosquito-pools, 50 females/pool) were processed for virus isolation. These females represented 18 species of mosquitoes, with Culex nigripalpus comprising 77.4% of all mosquitoes collected, followed by Aedes infirmatus (4.9%), Ae. vexans (4.0%) and Cx. erraticus (2.4%). No St. Louis encephalitis (SLE) and eastern equine encephalitis (EEE) virus isolates were obtained. Keystone (KEY) and Tensaw (TEN) viruses were isolated from Ae. albopictus (one isolate of KEY); Anopheles crucians (two isolates of TEN); Cx. nigripalpus (one isolate of TEN and 2 isolates of KEY); Coquilletidia perturbans (two isolates of TEN); and Wyeomyia vanduzeei (one isolate of TEN). All isolates were obtained from mosquitoes collected in CDC light traps, except for the KEY virus isolate from Ae. albopictus, which was collected in a CDC gravid trap. The isolation of TEN virus from Wy. vanduzeei is a first record for 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.

This is the last of three papers describing the use of electron microscopy and antigenic tests intended to characterize and place in taxa more than 60 previously unclassified viruses. The first paper describes the viruses we classified as members of the families Arenaviridae, Paramyxoviridae, or Poxviridae. The second paper describes those classified as members of the virus family Bunyaviridae. Another paper, published separately, discusses viruses classified as members of the family Rhabdoviridae. In this paper we report that electron microscopy provided sufficient evidence to provisionally place 14 viruses in the family Reoviridae. By using serologic methods, we placed Minnal virus in the Umatilla serogroup and established new antigenic groups for seven other viruses (Ieri, Picola, Arkonam, Tembe, Fomede, Wongorr, and Gomoka). No antigenic relationships were determined for six other viruses (Andasibe, Itupiranga, Kammavanpettai, Lake Clarendon, Matucare, and Ndelle) provisionally placed in the family Reoviridae.

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.

Summary Comparison of seven strains of WEE virus by neutralization, hemagglutination-inhibition and complement-fixation indicated considerable antigenic differences among these strains. Four strains (RI-Y, RI-C, A-42 and McM) maintained for several passages in the laboratory were indistinguishable by any of the serologic methods employed. Three other strains (Florida, Massachusetts, and HJ), representing relatively recent isolates, showed significant differences from the four laboratory-maintained strains in at least two or all three of the serological tests. In addition, significant one-way cross-differences were recognizable among these three strains by one or more of the serologic methods utilized. These antigenic variations were further reflected by differences in the prevalence of HI antibodies to WEE in approximately 1000 bloods screened with RI-Y and HJ strains. The number of positive HI reactors was twice as high with the HJ strain as with the RI-Y strain.

Summary A method for monitoring variations in antigenic constitution of arbovirus strains has been developed. The technique is based upon the magnitude of cross neutralization of virus strains by antisera prepared against virus fractions obtained from gradient sedimentation of a single strain. At a specific point in time, antigenically distinct Western encephalitis viral populations were found to coexist within various tissues of an infected mouse. The antigenic nature of such populations was found to be unstable and in a continuous state of flux. Eastern encephalitis virus strains had distinct antigenic properties when stocks were examined directly. The antigenic properties also varied when strains were carried from one passage to another in mice or in tissue culture, or when passaged in parallel in mice and tissue culture. The phenomenon of antigenic variation of strains was explained in part by the differential selection and ultimate accumulation of certain antigen subpopulations over others during virus replication in various tissue or cell types.

Summary Genetron-treated fluids of chick embryo cultures infected with Eastern and Western equine encephalomyelitis viruses were found, after freezing and thawing, to possess hemolytic activity over a wide temperature range (4 to 37°C). The virus hemolysin and hemagglutinin were inactivated by heat, whereas treatment with trypsin, ether and Ca++ tended to cause a selective suppression of the hemolysin activity of both viruses. Certain changes in the pH at which assays were performed resulted in a reduction of hemolytic activity which was greater than any change in hemagglutinating activity. Treatment of chick red blood cells with RDE and trypsin failed to remove the receptors for either of the activities. However, trypsin treatment significantly increased the hemagglutinating activity of both viruses. The hemolysins were inhibited by specific viral antibody and were sedimentable with the hemagglutinins.

Summary Examination of Navarro, Kwatta,and Mossuril viruses by electron microscopy revealed typical rhabdovirus morphology. They are longer than most other rhabdoviruses. All three viruses were observed in sections of infected mouse brain while Navarro and Kwatta viruses were also observed in infected Vero cells. The three viruses were unrelated antigenically to each other and to other rhabdoviruses of animals.

SummaryA total of 25 arboviruses representing 5 different serogroups (A, B, C, Bunyamwera and California) were studied and found to possess hemolytic activity. With the exception of Japanese B virus, special treatment of viral antigens was necessary to demonstrate hemolytic activity by the group B viruses. The hemolytic properties appeared to be a common property of the arboviruses.

SummaryNariva virus induced syncytium formation in BHK-21 and Vero cultures. Such infected cultures showed positive hemadsorption for guinea pig red blood cells (RBC). A hemagglutinin for guinea pi...

A procedure was developed for producing a hemagglutinin for the California serogroup (family Bunyaviridae, genus Bunyavirus) virus Jamestown Canyon, a human pathogen. Serum samples from humans putatively infected with this virus or with La Crosse virus were tested by hemagglutination inhibition. Each antigen detected antibody to the respective virus, with little cross-reactivity. These results suggest that both antigens should be used when the hemagglutination inhibition test is applied to the diagnosis of human infections with California serogroup viruses in North America.

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.

Among a population of 4.436 Bassa, Kpelle and Mano people in the Gbawein and Wroughbarh Clan region of Grand Bassa Country, Liberia, 123 cases of epilepsy could be documented. In 38% of these cases infections involving the central nervous system precipitated the onset of seizures. Sera from 67 epilepsy patients, 50 direct healthy relatives and 22 geographically matched controls were tested for antibodies to 16 arboviruses of the Togaviridae and Bunyaviridae known to occur in Africa. Antibodies to arboviruses were found in 16.5% of the epilepsy patients, 36% of the mostly older family members, and in 22% of the controls. Males and females were equally affected as were the different clans and language groups. Although meningoencephalitis with sequelae, like seizures, are known to result from arbovirus infections, no evidence for a correlation between epilepsy in this are of Central Liberia and previous arbovirus infection could be established.

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).

Eight viral isolates were recovered from Eidolon helvum bats collected in Nigeria and Cameroon in 1971 and from the Central African Republic in 1974. Subsequent studies showed that the agents from the three countries were similar and were strains of a new virus of the Orbivirus genus, family Reoviridae. This new virus was characterized with respect to its sensitivity to lipid solvents and to pH, its relative size and morphology by electron microscopy, its effect on mice experimentally infected by various routes, the histopathology found in infected mice, and its antigenic relationship or lack of a relationship to other known orbiviruses.

Summary A probing ecologic study was conducted in south-central Florida during May, June and July, 1960, and August, 1961, to determine the nature and distribution of arthropod-borne viruses in this area. Collections for virus and/or antibody examination included 1380 specimens composed of arthropod pools, birds, small mammals, reptiles and sentinels (mice and chicks). EEE virus was isolated from two pools of Culex mosquitoes (mixed species) and from a Common Grackle. WEE was recovered from a pool of Aedes taeniorhynchus, and the Highlands J strain of WEE from the blood of two Blue Jays and the brain of a sentinel mouse. Antibody rates in birds which were high for EEE (21%) and WEE (23%) in the 1960 collection and indicated recent infections, were markedly lower (2.7% and 3.4%, respectively) in 1961. Bird plasma reactors to SLE were similar (8% in 1960, 5% in 1961), except positives were found with blood from juvenile birds in 1961 but not in 1960, indicating probably recent activity of SLE or a related group B arthropodborne virus in the 1961 bird population sampled. HI antibody rates in wild-caught rodents in 1960 were: WEE, 10%; SLE, 30%; EEE, 0%. There were marked differences in two WEE strain antigens when used to measure antibody levels in birds. Forty-three or 10% of the samples were positive using the RI strain and 95 or 23% using the HJ strain of WEE. Moreover, a variance in the level of antibody was noted in bird plasmas examined by the HI and neutralization tests. These differences were attributed to the asynchronous development and/or disappearance of these antibody types.

Preparations of eastern encephalitis virus were disrupted by treatment with sodium deoxycholate. Tween 80 and ether, and sodium lauryl sulfate. The effect of these treatments on the viral specific activities of eastern encephalitis virus were studied. Untreated and disrupted preparations of eastern encephalitis virus were physically characterized by buoyant density centrifugation in cesium chloride density gradients and by rate zonal sedimentation in sucrose density gradients. Disruption by both sodium deoxycholate and Tween 80-ether resulted in the release of hemagglutinating subunits of higher density than the hemagglutinin associated with the intact virion. The hemagglutinating subunits released by both treatments were similar in density and size. A dense complement-fixing component, believed to represent released viral nucleoprotein core, was detected in cesium chloride gradients after sodium deoxycholate treatment of the virus. It was not detected in untreated preparations or in preparations treated with Tween 80-ether. Even at low concentrations, sodium lauryl sulfate was destructive of all viral activities save complement-fixation activity. The hemolytic activity of eastern encephalitis virus was physically closely associated with infectivity and hemagglutinating activity of the virus and was apparently dependent on the structural integrity of the virion.

Antigenic relations among viruses isolated from bats were studied by complement-fixation, hemagglutination-inhibition, and neutralization testing. This information would help to determine which of these viruses could be identified as strains and which could be recognized as antigenically distinct agents. The viruses were also characterized with respect to their comparative infectiousness for several host systems. Most of the viruses isolated from bats have been previously placed in serogroup B; two were considered ungrouped. The group B viruses included Rio Bravo, Burns, Dakar, BP 180, Ib An 8646, Bukalasa, and Entebbe virus. Antigenic studies indicated that BP 180 and Ib An 8646 were strains of Dakar virus. Bukalasa virus was shown to be closely related to the Dakar virus strains. Rio Bravo and Burns viruses were indistinguishable and were distantly related to MML virus, which, by certain tests, was also distantly related to the Dakar virus strains. The two ungrouped viruses, Kern Canyon and Lagos, were not related to one another nor to any of the other viruses. Plaque formation on LLC-MK2 or Vero cell cultures, or both, and the production of cytopathic effects (CPE) in BHK-21 cell cultures were also studied. Lagos bat and Bukalasa viruses did not produce plaques and Lagos bat virus did not produce CPE in BHK-21 cell cultures. A comparison of the in vitro assays with assays performed in suckling mice indicated that the intracerebral inoculation of suckling mice provided a slightly more sensitive assay system than any of the in vitro systems. Neutralization in LLC-MK2 cell cultures was significantly enhanced by the presence of “fresh” normal serum in the neutralization mixtures, although in certain instances, the enhancement of neutralization was accompanied by a lessening in the specificity of the neutralization reaction.

Two complement fixation (CF) components were detected in all of the various preparations (mouse liver, brain, serum, and BHK-21 cell culture) of Oriboca examined by equilibrium density centrifugation. The CF components possessed a buoyant density of 1.21 and 1.18 g/ml, and they were precipitated by treatment with protamine. Analysis of Oriboca virus by rate zonal sedimentation separated three components. The most rapidly sedimenting component was the infectivity which cosedimented with a peak of hemagglutinating (HA) activity designated VHA. A second, more slowly sedimenting, noninfectious HA component (HA-2) was followed by a peak of CF activity which barely moved from the top of the gradient. The single rate zonal CF peak and the two density gradient CF components were broadly cross-reactive serologically with Murutucu antiserum. They could not be identified as Oriboca on the basis of their CF reactions with Murutucu and Oriboca antisera. Oriboca-infective virions possessed an equilibrium density of 1.20 g/ml if derived from BHK-21 cell culture fluid concentrates or mouse serum. Infective virions prepared from mouse brain or liver banded at a buoyant density of 1.17 to 1.18 g/ml. These density differences, along with observed differences in reactivity with protamine and in rate of neutralization, are consistent with the interpretation that these are related to the problem of whether the source preparations of Oriboca represented primarily intracellular or extracellular particles.