Virus-vector-host relationships of Aedes stimulans and Jamestown Canyon virus in a northern Indiana enzootic focus.

Collections of hematophagous Diptera at the Kingsbury State Fish and Wildlife Area in northern Indiana between 1982 and 1984 yielded 118,972 mosquitoes from which 5 isolates of Jamestown Canyon virus and 3 isolates of trivittatus virus were obtained. All Jamestown Canyon isolates were from Aedes stimulans, including 1 from a pool of newly emerged males and 2 from pools of newly emerged females. These 3 isolates suggest that Jamestown Canyon virus is transovarially transmitted by Ae. stimulans. All isolates of trivittatus virus came from pools of Ae. trivittatus. No isolates were obtained from greater than 4,000 tabanids collected along with the mosquitoes those years. Transmission trials with field-collected newly emerged adult female Ae. stimulans demonstrated a mean midgut infection rate of 44%, a disseminated infection rate of 16%, and an oral transmission rate of 12% to suckling mice. Precipitin tests of field-collected bloodfed female mosquitoes indicated that white-tailed deer were the preferred host for numerous mosquito species including Ae. stimulans. The results of this study suggest that Ae. stimulans is a primary vector of Jamestown Canyon virus and that transovarial transmission is the probable overwintering mechanism for this California group virus.

Cache Valley and Potosi viruses (Bunyaviridae) in white-tailed deer (Odocoileus virginianus): experimental infections and antibody prevalence in natural populations.

Cache Valley virus (CVV) and Potosi virus (POTV) are two closely related mosquito-borne viruses (Bunyaviridae: Bunyamwera group) that appear to circulate in several regions of the United States, especially the Midwest. We determined the prevalence of specific neutralizing antibodies to both viruses in Indiana white-tailed deer and conducted infection experiments to assess whether deer could serve as an vertebrate-amplifying host. Cross-infection experiments also were carried out to investigate the level of antibody cross-reactivity and cross-protection between the two viruses. The seroprevalence rate was high for both CVV (> 66%) and POTV (> 43%) in adult deer statewide. Antibodies neutralizing CVV were more common among deer harvested in the northern part of Indiana whereas the prevalence of POTV antibodies suggested a more southern distribution for this virus. Experimental infections of captive deer showed that they may serve as amplifying hosts for either virus. Deer infected with CVV or POTV developed a 1-3-day viremia with 3.0 and 4.1 log10 plaque-forming units/ml mean peak titers, respectively. However, significant levels of antibody cross-reactivity between the two viruses were observed. Viremia was lower and shorter when animals immune to either CVV or POTV were cross-infected with the alternate virus and antibody responses following cross-infections resembled original antigenic sin with higher titers of antibodies against the primary agent.

Transmission of dengue virus by orally infected Aedes triseriatus.

Transmission of dengue type 1 was demonstrated for 3 strains of Aedes triseriatus mosquitoes after oral infection. Rates of infection were similar to those observed in a control strain of Aedes aegypti. Three additional species belonging to the subgenus Protomacleaya (Aedes brelandi, Aedes hendersoni, and Aedes zoosophus) were also susceptible to oral infection with dengue type 1 virus but transmission could not be demonstrated although virus was detected in the salivary glands of infected mosquitoes. Virus transmission was demonstrated for Ae. hendersoni following parenteral infection. The results of this study support the view that non-Stegomyia mosquitoes may become involved in the transmission of dengue virus to humans.

Identification of Culex species by electrophoresis.

This paper extends the usefulness of polyacrylamide gel electrophoresis to studies of mosquito taxonomy and mosquito vector ecology. Using a newly developed double staining technique, it is possible to unambiguously separate several species of Culex mosquitoes found in northern Indiana. These include Culex restuans, Culex pipiens pipiens, and Culex territans. Preliminary work indicates that Culex pipiens quinquefasciatus and Culex salinarius can also be identified by this method. This technique enables precise taxonomic identification of individual mosquitoes or pooled samples whether intact or damaged beyond identification by standard taxonomic methods. The small sample required (20 microliter) per pool leaves sufficient material for virus isolation techniques.

Aedes triseriatus and LA crosse virus: geographic variation in vector susceptibility and ability to transmit.

In laboratory tests, 20 strains of Aedes triseriatus differed widely in response to La Crosse virus. Infection rates ranged from 40% to 93%, and rate of transmission ranged from 20% to 90%. A control strain, tested in seven different trials, showed no significant variation in susceptibility (71--77%) or transmission ability (54--68%). A distinct geographic pattern was evident. The susceptibility and transmission rates of strains from areas where La Crosse virus is endemic were lower than that of strains from non-endemic regions, showing 71% vs. 87% for susceptibility to infection, and 46% vs. 74% for ability to transmit. Similar results have been observed in other mosquito-parasite systems, leading to the hypothesis that A. triseriatus in the upper Midwest is evolving resistance to La Crosse virus. Laboratory colonization had diverse and unpredictable effects on both transmission and infection, as might be expected with small populations and genetic drift.

A case of encephalitis in a human associated with a serologic rise to Jamestown Canyon virus.

An 8-year-old girl living in rural southwestern Michigan experienced sudden onset of symptoms beginning with headache, dizziness and fever which rapidly progressed to central nervous system involvement with seizures and coma. Following 27 days of hospitalization her recovery was uneventful, with no apparent sequelae 15 months after discharge. Serologic studies of paired sera showed a rise in antibody to Jamestown Canyon virus, a member of the California serogroup (family Bunyaviridae). Specific IgM anti-Jamestown Canyon virus antibody was detected in sera drawn 9 days after onset. A concomitant rise in complement fixation antibody to herpesvirus was also noted. We believe this is the first reported case of encephalitis associated with Jamestown Canyon virus infection. Reasons are presented for the current inability to routinely detect infection and clinical illness caused by this virus.

Distribution and prevalence of Mermet virus infections in the central United States.

Tests were run on 3,198 bird sera for neutralizing antibody of Mermet virus. The birds were mostly House Sparrows (Passer domesticus) captured in the central U.S. Antibody was detected in birds from Texas, Mississippi, Tennessee, Ohio, Indiana, Illinois, and Wisconsin, but not Kentucky or Missouri. Antibody prevalence differed by location and between years in similar locations. These results confirmed the widespread activity of Mermet virus in the central U.S., suggested irregular activity of the virus, and provided the first evidence that Mermet virus activity occurs in Mississippi, Indiana, and Wisconsin. No antibody to Mermet virus was found in paired sera from 966 humans with suspected arboviral infection.

Jamestown Canyon virus (California serogroup) is the etiologic agent of widespread infection in Michigan humans.

In a sample population of 780 Michigan residents tested for neutralizing antibodies to California serogroup viruses, 216 (27.7%) had specific neutralizing antibody to Jamestown Canyon virus. An additional eight (1.0%) had specific neutralizing to trivittatus virus; none had specific neutralizing antibody to La Crosse virus. Significantly more male residents than female residents of the Lower Peninsula had antibody to Jamestown Canyon virus. The frequency of neutralizing antibody titers fits the Poisson distribution, suggesting that Jamestown Canyon virus infections occur endemically in residents of Michigan. Among 128 sera with specific neutralizing antibody to Jamestown Canyon virus, only two (1.6%) were found to have significant hemagglutination-inhibiting antibody titers with La Crosse virus, while 23 of 44 (52%) had significant titers with Jamestown Canyon virus; a single serum had significant antibody by complement fixation tests with both La Crosse and Jamestown Canyon viruses. This study confirms earlier speculation that complement fixation and hemagglutination-inhibition tests with La Crosse virus (the only tests for California serogroup virus infections performed by most state diagnostic laboratories) fail to detect antibody to Jamestown Canyon virus. ASPEX computer-drawn maps demonstrated that the distribution of persons with antibody to Jamestown Canyon virus and residing in Michigan's Lower Peninsula is closely correlated with the estimated distribution of white-tailed deer in that part of the state, further supporting the hypothesis that white-tailed deer are the primary vertebrate host for Jamestown Canyon virus.

Aedes triseriatus (Diptera: Culicidae) and La Crosse virus. IV. Nutritional deprivation of larvae affects the adult barriers to infection and transmission.

Groups of Aedes triseriatus (Say) were reared either as nutritionally deprived (two regimens) or well fed (one regimen) throughout larval development, and the vector competence of resulting small, normal, and large females was assessed for La Crosse virus. When fed a high dose of virus (4.6 log10/0.025 ml in Vero cell culture), 90% of small Ae. triseriatus females transmitted La Crosse virus to suckling mice compared with 70% of normal and 42% of large females. Among small females, 100% had disseminated infections as did 86% of normal females, whereas only 69% of large females had disseminated infections. All females had infected mesenterons (midguts). When fed a low dose of virus (2.2 log10/0.025 ml in Vero cell culture) in a second experiment, 15% of small females transmitted compared with 0% of large females; 50% of small females developed disseminated infections compared with 16% of large females. mesenteronal infection occurred in 70% of small but only 32% of large females. Electron microscopy of mesenteronal tissues from large and small females revealed physical differences in the basement membranes (basal laminae). The mesenterons of small females had 3-6 laminae (mean thickness of the basement membrane = 0.14 microns) compared with 9-16 laminae (mean thickness of the basement membrane = 0.24 microns) in large females. These morphological differences indicated that the mesenteronal escape barrier, which accounted for the difference in the percentage of small and large females with disseminated infections, may be, in part, a physical barrier that was modified by nutritional deprivation in the larval instars.

Replication and dissemination of La Crosse virus in the competent vector Aedes triseriatus and the incompetent vector Aedes hendersoni and evidence for transovarial transmission by Aedes hendersoni (Diptera: Culicidae).

The time course and pattern of the replication and dissemination of La Crosse virus was studied in orally infected Aedes triseriatus (Say) and Ae. hendersoni Cockerell. Development of La Crosse virus was approximately the same in both species when plaque assay titers of intact mosquitos or dissected tissues were compared. The mosquitoes were equally susceptible to infection; all Ae. hendersoni and 99% of the Ae. triseriatus tested showed detectable midgut infections. Virus was first detected in hemolymph, salivary glands, and ovaries 10-13 d after infection in both species. The pattern of infection suggests virus dissemination beyond the midgut to be via the hemolymph. By 21 d after infection, 100% (10 of 10) of Ae. triseriatus and 70% (7 of 10) of Ae. hendersoni had infected salivary glands, and the geometric mean titer of Ae. hendersoni salivary glands was 10 times higher than the geometric mean titer of those of Ae. triseriatus, However, when tested for transmission 22 d after infection by refeeding on suckling mice, only 9% (2 of 22) of the Ae. hendersoni with disseminated infections transmitted virus versus 71% (12 of 17) of the Ae. triseriatus. A salivary gland escape barrier was shown to be primarily responsible for the failure of Ae. hendersoni to orally transmit La Crosse virus. However, eight parenterally infected Ae. hendersoni females transovarially transmitted the virus to 25% (5 of 20) of their progeny.

Role of Anopheles quadrimaculatus and Coquillettidia perturbans (Diptera: Culicidae) in the transmission cycle of Cache Valley virus (Bunyaviridae: Bunyavirus) in the midwest, USA.

Midwestern populations of Coquillettidia perturbans (Walker) and Anopheles quadrimaculatus (Say) were tested for their ability to transmit Cache Valley virus (CV), a recognized human and animal pathogen. Field-collected mosquitoes were fed artificial blood meals containing 5.2-6.2 log10 pfu/ml of CV. After 9-23 d at 28 degrees C, 75-93% of blood-fed Cq. perturbans had disseminated infections and 6-62% transmitted the virus to suckling mice. However, when infected with a lower virus titer (3.3 log10 pfu/ml), only 10-36% of the mosquitoes had disseminated infections and 0-10% transmitted the virus to suckling mice. A similar infection rate (21%) was observed in Cq. perturbans fed on viremic (3.2 log10 pfu/ml) hamsters. An. quadrimaculatus were infected (81-100%) by both doses used, with transmission rates ranging from 13-67% after 16-23 d of incubation. Transmission rates for the laboratory strain An. quadrimaculatus SAVANNAH ranged from 20 to 33% after 7-14 d of incubation. Our data show that although An. quadrimaculatus is more susceptible to CV infections than Cq. perturbans, both mosquito species could be involved in the midwestern transmission cycle of the virus.

Vector competence of Aedes hendersoni (Diptera: Culicidae) for La Crosse virus: lack of impaired function in virus-infected salivary glands and enhanced virus transmission by sporozoite-infected mosquitoes.

Previous research has shown Aedes hendersoni Cockerell to be an incompetent vector of La Crosse (LAC) virus because of a salivary gland escape (SGE) barrier; that is, the salivary glands are infected but the mosquito fails to transmit the virus orally. Intradermal probing behavior and ability to locate blood were studied in infected mosquitoes as indicators of salivary gland impairment to determine if the SGE barrier was due to virus-induced pathology of the salivary glands. No evidence of salivary gland impairment as a result of virus infection was detected in infected Ae. hendersoni. This was also true for Aedes triseriatus (Say), a competent vector of LAC virus, which was used as a control. However, coinfection of Ae. hendersoni with Plasmodium gallinaceum and LAC virus dramatically increased virus transmission (72 versus 8%), whereas transmission by coinfected Ae. triseriatus was not significantly affected. Possible causes for the SGE barrier in Ae. hendersoni are discussed.

Variation in the efficiency of vertical transmission of dengue-1 virus by strains of Aedes albopictus (Diptera: Culicidae).

Five geographical strains of Aedes albopictus (Skuse) were compared for their ability to transmit vertically a dengue-1 isolate from Jamaica. The OAHU strain of Ae. albopictus and a strain of Aedes aegypti (L.) from the United States were included as controls. The offspring of orally infected females were assayed individually for vertical infection. Vertical transmission rates among strains ranged from 11 to 41%, and filial infection rates of strains ranged from 0.5 to 2.9%. Filial infection rates of individual positive families within strains ranged from 1.4 to 17.4%. These rates were higher than those previously recorded for Ae. albopictus. The observed differences in rates of vertical transmission among the strains were not statistically significant, because 95% of the measured variation was attributed to families within strains. The most significant source of variation in vertical transmission of dengue-1 by Ae. albopictus was at the individual level.

Serological evidence of California group and Cache Valley virus infection in Minnesota white-tailed deer.

Blood samples were obtained from 138 white-tailed deer (Odocoileus virginianus) harvested at three sites surrounding the greater Minneapolis-St. Paul, Minnesota, metropolitan area (USA) and tested for neutralizing antibody to Cache Valley virus and three California serogroup (Jamestown Canyon, La Crosse, trivittatus) viruses (Bunyaviridae). Deer at each site had neutralizing antibody to one or more California serogroup viruses and/or Cache Valley virus. The majority of adult deer (85%) had antibody to both a California serogroup virus and Cache Valley virus. Antibody prevalence varied significantly with age of the deer. Fawns had a significantly lower prevalence of antibody to either a California serogroup (17%) or Cache Valley virus (39%) than did older (greater than 1-yr-old) deer (89% for a California serogroup virus and 91% for Cache Valley virus). The geometric mean titers of antibody in fawns to California serogroup (1:6) and Cache Valley viruses (1:17) were also less than that seen in older animals (1:11 and 1:28 for California serogroup and Cache Valley viruses, respectively). Of 76 older deer with antibody to the California serogroup, 91% had antibody specific for Jamestown Canyon virus. Jamestown Canyon is the primary California serogroup virus circulating in the suburban/rural Minneapolis-St. Paul area. Transmission occurs in an enzootic pattern similar to that documented in Indiana and Michigan. Cache Valley virus also appears to be enzootically transmitted in this area. However, the impact on domestic or wild animal populations is unknown.

Seroconversion rates to Jamestown Canyon virus among six populations of white-tailed deer (Odocoileus virginianus) in Indiana.

The annual seroconversion of fawns, yearlings, and adult white-tailed deer (Odocoileus virginianus) to Jamestown Canyon virus (California group) was followed at six Indiana sites from 1981 through 1984. In all, sera from 1,642 deer (515 fawns, 618 yearlings, and 509 adults) were tested for neutralizing antibody to three California serogroup viruses: Jamestown Canyon, La Crosse, and trivittatus. Virtually all deer with specific neutralizing antibody showed evidence of a prior infection with Jamestown Canyon virus; only three deer showed evidence of a prior infection with only La Crosse virus and none showed evidence of an infection with only trivittatus virus. While there were no significant differences in antibody prevalence to Jamestown Canyon virus between yearling and adult deer at any site, fawns had significantly lower antibody prevalences than either of the two older age groups. Significant differences in antibody prevalence were found between northern versus southern populations of white-tailed deer in Indiana, however, no significant differences were found among the four northern populations or between the two southern populations. The mean antibody prevalences in the two southern fawn, yearling, and adult populations were 15%, 38%, and 41% respectively, while the prevalences in the four northern fawn, yearling, and adult populations were 5%, 67%, and 67% respectively. These different prevalences (northern vs. southern) correlate with the higher Jamestown Canyon virus antibody prevalence in human residents of northern Indiana (2-15%) compared to residents of southern Indiana (less than 2%) found in other studies. The significantly lower prevalence of antibody to Jamestown Canyon virus in fawns is attributed to maternal antibody protecting them from a primary infection their first summer. Yearling deer showed high rates of seroconversion following their second summer of life. These results suggest that infection of white-tailed deer in Indiana with Jamestown Canyon virus is a common phenomenon.

Prevalence of neutralizing antibody to Jamestown Canyon virus (California group) in populations of elk and moose in northern Michigan and Ontario, Canada.

Blood samples were collected from free-ranging elk (Cervus elaphus) harvested in Michigan's northern Lower Peninsula, from moose (Alces alces) relocated from Ontario's Algonquin Provincial Park to Michigan's Upper Peninsula, and from moose from Michigan's Isle Royale National Park. Sera were tested by serum dilution neutralization tests in Vero cell culture for neutralizing antibody to California serogroup viruses, in particular Jamestown Canyon (JC), La Crosse/snowshoe hare (LAC/SSH), and trivittatus (TVT) viruses. Specific neutralizing antibody to JC virus was detected in 71% of 31 and 65% of 20 moose from Algonquin and Isle Royale, respectively. An additional six moose from Algonquin and five from Isle Royale showed evidence of multiple infection. One juvenile moose from Isle Royale had specific neutralizing antibody to TVT virus. Specific neutralizing antibody to JC virus was detected also in 54% of 50 elk from Michigan; 20 of the 50 elk showed evidence of multiple infection. While no single serum sample showed specific neutralizing antibody only to LAC/SSH virus, its presence in sera from some animals may have been masked by the high prevalence of antibody to JC virus.

Infection of white-tailed deer (Odocoileus virginianus) in Michigan with Jamestown Canyon virus (California serogroup) and the importance of maternal antibody in viral maintenance.

Sera collected from a captive population of white-tailed deer (Odocoileus virginianus) penned in the lower peninsula of Michigan were assayed over a 29-mo period for neutralizing antibody to California serogroup viruses. In all, 130 individual white-tailed deer were bled one to 22 times between June 1983 and November 1985. Of the 130 sampled after active transmission had ceased, or passage of maternal antibody in colostrum had occurred, only one (0.8%), a newborn fawn, had no serum neutralizing antibody to California group viruses. All 18 1-yr-old does sampled acquired specific neutralizing antibody to Jamestown Canyon (JC) virus within a 6-wk period in 1984 and within a 10-wk period in 1985 indicating the prevalence of infection in this nonimmune age group was 100% for 2 successive yr. All 32 2- to 7-yr-old adult does and eight bucks sampled between June 1983 and June 1985 had specific neutralizing antibody to JC virus. No white-tailed deer had specific neutralizing antibody to trivittatus or La Crosse/snowshoe hare viruses at this study site. In 1984 and 1985, 78% and 63% of the adult does respectively exhibited significant anamnestic responses; all 19 adult does sampled over two seasons (between October 1983 and June 1985) showed a significant anamnestic response during at least 1 of the 2 yr. One-third of adult does with significant springtime antibody titer increases apparently experienced reexposure prior to the emergence of aedine mosquitoes, suggesting an alternate vector may overwinter at this site and transmit viruses in early spring. Specific neutralizing antibody was detected in 98% (66/67) of nursing fawns bled within 5 wk of birth in May-June 1984 and 1985, including three of three nursing fawns bled within 24-96 hr of birth. Of the 66 newborn fawns with specific neutralizing antibody to JC virus in June 1984 and 1985, 95% (54/57) of the surviving fawns lost maternal antibody and had no measurable titer when sampled 20-24 wk after birth, however. Serum antibody titers in 25 newborn (1984-cohort) fawns and their mothers and titers in 38 newborn (1985-cohort) fawns and their mothers were significantly correlated at the 5% and 1% levels respectively, suggesting that maternal antibody rather than a naturally acquired infection was the source of immunity in these suckling fawns.(ABSTRACT TRUNCATED AT 400 WORDS)

Changing patterns in mosquito-borne arboviruses.

Research leading to the current state of knowledge on the epidemiology of La Crosse virus (LACV), Jamestown Canyon virus (JCV) and dengue (DEN) viruses is summarized in relation to the generally recognized criteria for incriminating vectors. The importance of vector biology and local ecological conditions is emphasized as is the necessity of a good balance between laboratory and field-based studies. The influence of human activity in shaping the epidemiological patterns of all three of these arboviruses is readily apparent.

Laboratory transmission of Jamestown Canyon and snowshoe hare viruses (Bunyaviridae: California serogroup) by several species of mosquitoes.

The ability of 14 species of mosquitoes to biologically transmit Jamestown Canyon virus was tested. Four species not previously described as vectors of that virus transmitted to suckling mice. Among membrane-fed mosquitoes with disseminated infections, field-collected Aedes canadensis (1/3), Anopheles punctipennis (1/12), Coquillettidia perturbans (2/14) and a laboratory strain of Ae. epactius (19/67) transmitted virus. Two species were tested for their ability to transmit snowshoe hare virus: field-collected Ae. provocans (4/20) and Ae. abserratus-punctor (2/20) successfully transmitted to suckling mice. Evidence regarding the role of these species as field vectors is summarized.

Isolation of Jamestown Canyon virus (California serogroup) from Aedes mosquitoes in an enzootic focus in Michigan.

Twenty isolates of Jamestown Canyon virus were obtained from adult females of 5 Aedes species collected at the Houghton Lake Wildlife Research Area, Missaukee County, in north-central Michigan between 1985 and 1989. Fourteen were from Aedes provocans, and 6 were from 4 other snowmelt Aedes species. One isolate of trivittatus virus and one Cache Valley-like virus were also obtained. Seasonal succession patterns for numerous mosquito species were recorded over 4 years. The temporal association of adult mosquito emergence, virus isolations, and infection and seroconversion of sentinel deer suggest that Ae. provocans is a primary enzootic vector of Jamestown Canyon virus in that focus. We hypothesize that Ae. provocans provides an overwintering reservoir for Jamestown Canyon virus at the study site. A large dry ice-baited "tent trap" was the most productive method for collecting numerous aedine and other mosquito species.