Transovarial transmission of LaCrosse virus (California encephalitis group) in the mosquito, Aedes triseriatus.

LaCrosse virus (California encephalitis group) was recovered from F(1) eggs, larvae, and adults produced by experimentally infected Aedes triseriatus. The F(1) females transmitted the virus by bite to suckling mice and chipmunks. This, plus isolations of LaCrosse virus from larvae collected from their natural habitats in enzootic areas and from males and females reared from them, suggests that transovarial transmission is the overwintering mechanism for this arbovirus in northern United States.

Toxicant-disease-environment interactions associated with suppression of immune system, growth, and reproduction.

The effects of marginal malnourishment , infections, and environmental chemicals on growth and reproductive success in Swiss-Webster white mice and wild deer mice were studied with fractional factorial designs. Interaction effects were discovered. For example, malnourished mice were more sensitive to virus exposure and environmental chemicals (a plant growth regulator or polychlorinated biphenyls). Since several commercial plant growth regulators also appear to suppress the immune system, these results cast doubt on the adequacy of current toxicity testing procedures in which factors are studied individually and not in combination.

Identification and localization of conserved antigenic epitopes on the G2 proteins of California serogroup Bunyaviruses.

California (CAL) serogroup Bunyaviruses are significant agents of arboviral encephalitis in humans. They are maintained and transmitted in nature by mosquitoes to preferred vertebrate amplifying hosts. The G2 envelope glycoprotein of La Crosse virus (LAC) was proposed by Ludwig et al. to be a determinant for virus attachment to mosquito midgut cells. Monoclonal antibodies to G2 neutralize the infectivity of pronase-treated virus for mosquito cells. We determined the location of antigenic sites on the LAC G2. We showed that antigenic areas present on the LAC G2 protein are conserved among viruses in the California encephalitis and Melao subgroups of the CAL serogroup, but not in trivatattus virus, nor within the BUN serogroup. A comparison of the G2 exodomain amino acid sequences of eight CAL and three BUN viruses with monoclonal antibodies (MAb) binding data predicted the possible location of the antigenic sites. We used in vitro mutagenesis of the LAC G2 gene to construct a set of G2 genes with replacement sequences in the coding regions for the suspected MAb binding sites. The native and mutated proteins were expressed in Hela cells and the ability of MAbs to bind to the expressed proteins was tested. Four discontinuous amino acid sequences, conserved among eight CAL serogroup viruses, were identified as contributing to two conformational binding domains for neutralizing LAC G2 MAbs.

Transovarial transmission of LaCrosse virus in Aedes triseriatus.

As part of a continuing investigation on the ecology of LaCrosse virus in Wisconsin, field and laboratory studies were conducted to explore the possibility that the virus is transmitted transovarially in A. triseriatus mosquitoes. In laboratory experiments, A. triseriatus mosquitoes were infected by ingesting LaCrosse virus in defibrinated blood. LaCrosse virus was recovered from F1 eggs, larvae, and adults that originated from the infected parent mosquitoes. In a subsequent field study aimed at determining if transovarial transmission accounted for the survival of LaCrosse virus during the winter season, larvae that originated from overwintering A. triseriatus eggs were collected from a LaCrosse virus enzootic area in southwestern Wisconsin. LaCrosse virus was isolated from these larvae and from adult A. triseriatus that were reared from field-collected larvae. These findings strongly imply that A. triseriatus is the reservoir of LaCrosse virus and that transovarial transmission is the mechanism responsible for the maintenance of the virus during the winter season in the north central region of the United States.

Epidemiology of bluetongue viruses in the American tropics. Regional Bluetongue Team.

A study of the epidemiology of bluetongue viruses is in progress with the collaboration of 11 Central American and Caribbean countries. To date, over 200 bluetongue virus isolates have been obtained from cattle and sheep in sentinel groups distributed in the participating countries. Bluetongue serotypes identified include 1, 3, 6, and 12, virus types not previously recorded in the Western Hemisphere. Although the clinical impact of bluetongue virus infections in this hyperendemic environment appears to be minimal, the ubiquity of infection causes restrictions on the export of ruminant livestock and germ plasm. The stability of the Caribbean region ecosystem and the long-range implications of the interface with the northern temperate bluetongue virus ecosystem are reviewed.

Natural La Crosse virus infection in the red fox (Vulpes fulva), gray fox (urocyon cinereoargenteus), raccoon (Procyon lotor), and opossum (Didelphis virginiana).

Natural infection of sentinel red foxes (Vulpes fulva) and free-ranging red foxes, gray foxes (Urocyon cinereoargenteus) and raccoons (Procyon lotor) with La Crosse (LAC) virus was demonstrated. One isolate of LAC virus was obtained from a sentinel red fox in an enzootic area. The viremia titer of the LAC virus-infected red fox was above the threshold of infection for Aedes triseriatus mosquitoes. Antibody responses were measured by the microneutralization test employing four California group viruses: LAC, snowshoe hare, trivittatus, and Jamestown Canyon. Four of six sentinel red foxes developed LAC virus neutralizing antibody. Antibody titers peaked between days 7 and 34 and were still detectable by dat 345 post-infection. Homologous LAC virus antibody titers were consistently 4-fold or greater than heterologous titers. Serological evidence for natural LAC virus infection was demonstrated in 33 of 57 (58%) free-ranging red foxes, 18 of 32 (57%) gray foxes, and 4 of 16 (25%) raccoons. Antibody titers were comparable in free-ranging foxes infected naturally and those infected experimentally by mosquito bite. The prevalence of infection was significantly different (P less than 0.001) for foxes trapped on three distinct areas within the enzootic region. Rates of infection in foxes by area coincided with LAC virus antibody prevalence observed in free-ranging chipmunks and with reported cases of human LAC encephalitis in Wisconsin.

La Crosse viremias in Mongolian gerbils (Meriones unguiculatis).

We examined the usefulness of mongolian gerbils (Meriones unguiculatus) as a new animal model for La Crosse virus (LACV) studies. Gerbils were exposed to LACV by either intramuscular (im) inoculation or exposure to transovarially infected Aedes triseriatus. Our studies indicate that gerbils may be a suitable animal model for LACV infection. Gerbils were susceptible to LACV, survived viral infection, and developed viremias and neutralizing antibody titers following exposure by im injection and by the bite of infected mosquitoes. Moreover, they are attractive to mosquito vectors. Gerbils have other advantages as laboratory vertebrate hosts for LACV; they are inexpensive, breed in captivity, and are usually mild-mannered and easy to handle. Thus, gerbils are a suitable model in the study of LACV pathogenesis as well as of transplacental and vector transmission.

Viremia and antibody response to La Crosse virus in sentinel gray squirrels (Sciuris carolinensis) and chipmunks Tamias striatus).

Six isolates of La Crosse (LAC) virus were obtained from sentinel gray squirrels (Sciuris carolinensis) and four from sentinel chipmunks (Tamias striatus) in an endemic area. Viremia titers were measured by plaquing on Vero cells. Antibody responses of the animals were measured by a microneutralization test employing four California group viruses: LAC, snowshoe hare (SSH), trivittatus, and Jamestown Canyon. In both species LAC antibody titers peaked at approximately 21 days and were still detectable in all animals at 256 days post-viremia. In chipmunks, homologous LAC virus antibody levels were consistently higher than heterologous antibody responses throughout the period recorded. However, in squirrels, homologous LAC virus and heterologous SSH virus antibody responses were initially comparable. This heterologous SSH titer rapidly declined while LAC antibody levels remained relatively high. Data indicate that antibody response persists from one summer season to the next. Viremia titers in both species indicate that these two species are capable of infecting Aedes triseriatus, the principal vector of LAC virus. This is the first reported field isolation of LAC virus from the squirrel.

Aedes triseriatus and La Crosse virus: lack of infection in eggs of the first ovarian cycle following oral infection of females.

La Crosse (LAC) virus filial infection rates were 0% for 279 first ovarian cycle larvae, 43% for 380 second ovarian cycle larvae, and 58% for 363 third ovarian cycle larvae from orally infected mosquitoes representing 14 Wisconsin populations of Aedes triseriatus. LAC virus was not detected in 72 pools representing 2,250 first ovarian cycle larvae, while 35 pools and 16 pools each containing 30 second and third ovarian cycle larvae, respectively, were all positive for LAC virus. Similar results were obtained when the extrinsic incubation period temperature was 25 degrees C, 27 degrees C, or variable (17, 23, and 29 degrees C for 8 hours each). LAC virus was not detected in 240 second ovarian cycle larvae in which the bloodmeal for the first ovarian cycle was non-infectious. Infection was not detected in 337 first ovarian cycle larvae from female mosquitoes that had been injected intrathoracically with LAC virus concomitantly with receiving a non-infectious bloodmeal. After an extrinsic incubation temperature of 25 degrees C, LAC virus was discovered in dissected mosquito ovarian tissue 7 days postfeeding on an infectious bloodmeal. The epidemiological implications of these findings are discussed.

Effects of La Crosse virus infection on pregnant domestic rabbits and mongolian gerbils.

The transplacental transmission of La Crosse virus (LACV) was evaluated in domestic rabbits (Oryctolagus cuniculus) and Mongolian gerbils (Meriones unguiculatis) as a potential mechanism for the maintenance of the virus. Rabbits were infected with LACV at different times of gestation by injection of viral suspensions or by exposure to LACV transovarially (TO) infected Aedes triseriatus. Pregnant gerbils were exposed between 16-24 days of gestation to LACV TO- infected Ae. triseriatus. Our results indicate that LACV can infect gerbils in utero. The LACV was isolated from the brain of suckling gerbils that died 3-5 days after birth from LACV-exposed mothers, representing the first evidence of LACV transplacental transmission. Microgliosis was found histologically in the cerebral cortex. In addition, LACV infection of both pregnant gerbils and rabbits resulted in in utero and neonatal mortality. La Crosse virus was not detected in surviving young of infected rabbits even after immunosuppression by administration of cyclophosphamide. Thus, there was no evidence of persistent infection of rabbits following in utero exposure. Surprisingly, some of the infected pregnant gerbils developed progressive paralysis 9-14-days postexposure, and LACV was isolated from the brains of these animals. Histopathologic studies of these tissue samples showed acute meningoencephalitis. The effects of natural LACV infection should be studied in pregnant amplifying hosts, such as chipmunks and squirrels, and in pregnant women.

La Crosse viremias in juvenile, subadult and adult chipmunks (Tamias striatus) following feeding by transovarially-infected Aedes triseriatus.

Viremia and antibody responses to La Crosse (LAC) virus were determined for juvenile, subadult and adult chipmunks (Tamias striatus). Viremia was detected in 16 of 16 juveniles, 13 of 17 subadults and 21 of 29 adults fed upon by transovarially (TO)-infected Aedes triseriatus. Mean viremia titers for juvenile, subadult and adult chipmunks responding to LAC infection were 3.0, 2.9 and 3.2 log10SMICLD50/0.025 ml, respectively. The average duration of viremia with LAC virus was 2.4, 2.3 and 2.4 days for juveniles, subadults and adults, respectively. Mean viremia titers and durations did not differ significantly among chipmunk age-classes. Neutralizing antibodies to LAC virus were detected in viremic chipmunks at day 5 and day 20 post-infection. Observations of TO-infected Ae. triseriatus and their sibling controls refeeding on restrained chipmunks indicated that a significant number of infected (34/54) and uninfected (37/59) females probed multiple times to obtain a second bloodmeal. Data indicate that chronological age or time between successive bloodmeals affects feeding behaviors. No relationship between probing or ability to refeed and infection was found.

Experimental La Crosse virus infection of red fox (Vulpes fulva), raccoon (Procyon lotor), opossum (Didelphis virginiana), and woodchuck (Marmota monax).

Susceptibility to infection, resulting viremia and antibody responses, and potential to provide infectious blood meals for Aedes triseriatus were determined and compared for the red fox (Vulpes fulva), raccoon (Procyon lotor), and opossum (Didelphis virginiana) exposed to La Crosse (LAC) virus transmitted by mosquitoes, Ae. triseriatus. Woodchucks (Marmota monax) were infected with LAC virus by needle and syringe. All 5 red foxes became viremic following the bite of a single LAC virus-infected female Ae. triseriatus. Maximum viremia titers were at or above the threshold of infection for Ae. triseriatus in 4 of 5 red foxes for 1-3 days. Biological transmission of LAC virus from infected red foxes to chipmunks (Tamias striatus) was accomplished by Ae. triseriatus. Neutralizing antibody titers in red foxes peaked between day 13 and 27 and were still detectable 3 months post-infection. Woodchucks appear to be efficient amplifiers of LAC virus. Three of 4 inoculated woodchucks became viremic. Maximum viremia titers were consistently above the experimentally determined threshold of infection for Ae. triseriatus. Raccoons and opossums were not as susceptible to LAC virus infection as were red foxes or woodchucks. Only 1 of 5 raccoons became viremic. The viremia titer was low and was detected on only 1 day. Four of 5 raccoons developed LAC virus-neutralizing antibody titers, however. None of the opossums became viremic and only 2 developed LAC virus-neutralizing antibody titers.

Oral infection and transmission of La Crosse virus by an enzootic strain of Aedes triseriatus feeding on chipmunks with a range of viremia levels.

The susceptibility of Aedes triseriatus to oral infection with La Crosse (LAC) virus resulting from feeding on chipmunks with viremia titers of 0.6 to 4.6 log10SMICLD50/0.025 ml was determined. Results indicated that viremia titers must exceed 3.2 log10SMICLD50/0.025 ml before a significant proportion (greater than or equal to 50%) of mosquitoes are infected and capable of transmitting LAC virus. Mosquitoes which fed on chipmunk blood-LAC virus mixtures through a membrane feeder had significantly lower infection rates at virus titers of 1.8 to 4.4 log10SMICLD50/0.025 ml and transmission was also significantly reduced. Application of these data to LAC viremia titers measured in chipmunks in an earlier study indicate that viremias sufficiently high to ensure transmission by the mosquitoes becoming orally infected average only about 1 day per infective bite delivered to the susceptible portion of the amplifier population. Oral infection and transmission rates were also determined for Ae. triseriatus feeding on chipmunk blood containing LAC virus neutralizing (N) antibodies and for Ae. triseriatus feeding on deer blood containing Jamestown Canyon (JC) virus N antibodies. Infection rates were similar to those observed in mosquitoes imbibing blood free of N antibody at the virus titers tested, but, oral transmission was reduced in females feeding on chipmunk blood-LAC virus mixtures containing LAC N antibodies and there was no transmission by females feeding on deer blood-LAC virus mixtures containing JC N antibodies. These data suggest that high LAC antibody prevalences in chipmunk populations and high LAC or JC antibody prevalences in deer populations may be antagonistic to horizontal LAC virus transmission.

Studies on the transmission of Venezuelan equine encephalitis virus by Colombian simuliidae (Diptera).

The ability of Simulium mexicanum and Simulium metallicum to serve as biological or mechanical vectors of an enzootic and an epizootic strain of Venezuelan equine encephalitis (VEE) virus was examined. Guinea pigs were inoculated with the epizootic Cordoba strain or the enzootic RPVP407 strain of VEE virus. Wild-caught adult Simuliidae were fed on the viremic guinea pigs and the virus content of groups of flies was determined at daily intervals post-engorgement to test for viral replication. Flies were refed on suckling mice at greater than or equal to 8 days post-engorgement to test for biological transmission. Other flies were interrupted while feeding on viremic guinea pigs and refed on suckling mice to test for mechanical transmission. Neither S. mexicanum nor S. metallicum appear to be efficient vectors of either strain of VEE virus, although occasional mechanical transmission was obtained. Titers of virus in flies decreased rapidly after engorgement and from 3-12 days post-engorgement virus was detected only in 5%-25% of both species of flies. Although earlier field evidence implicated both S. mexicanum and S. metallicum as vectors of epizootic VEE, we conclude that it is highly unlikely that they play an important role as vectors of the virus in nature.

Experimental infection of chipmunks and snowshoe hares with La Crosse and snowshoe hare viruses and four of their reassortants.

The infection of chipmunks (Tamias striatus), the vertebrate host of La Crosse (LAC) arbovirus, or snowshoe hares (Lepus americanus), the host of snowshoe hare (SSH) virus, was analyzed following subcutaneous inoculation with either LAC or SSH or certain LAC-SSH reassortant viruses. After inoculation, no adult hares or chipmunks developed signs of illness. Another reassortant, genotype LAC/LAC/SSH, has exhibited a marked inability to infect chipmunks and was avirulent for mice, probably because of a mutation in its L RNA segment. In chipmunks inoculated with reassortant viruses, neither the durations of viremias nor the maximum titers attained were demonstrably different from those found after inoculation with the parent LAC or SSH viruses. However, viruses with a SSH M RNA induced a higher viremia in snowshoe hares than viruses with a LAC M RNA. Also, the amount of virus needed to produce a detectable infection of chipmunks or hares was lower for viruses in which the M RNA came from the SSH parent than for those viruses having a LAC M RNA. Convalescent phase chipmunk and hare sera from animals infected with SSH virus or reassortant viruses having a SSH M RNA, neutralized parental SSH virus more effectively than did LAC virus. Conversely, the sera of animals infected with LAC virus or reassortants having a LAC M RNA, neutralized LAC virus more efficiently than did SSH virus.

California serogroup virus infections in Wisconsin domestic animals.

A serologic survey and experimental virus transmission studies were done to assess the role of domestic animals as amplifier hosts of La Crosse (LACV) and Jamestown Canyon (JCV) viruses. Serum from 319 cows, 88 dogs, 122 equines, 47 swine, 10 goats, and 4 cats were tested for neutralizing antibody to LACV, JCV, trivittatus (TVTV), and snowshoe hare (SSHV) viruses. Antibody prevalences of LACV, TVTV, and SSHV were less than 10% in all species. Antibody to JCV was detected in all species except cats. Prevalence ranged from 10% in goats and swine to 29% in dogs. No age-associated trends in JCV prevalence were noted. Two of 6 adult dogs, and 2 of 4 pigs inoculated with 6.3-6.5 log10 suckling mouse intracerebral 50% lethal doses (SMICLD50) of LACV developed viremias ranging of less than 1.0-2.9 log10 SMICLD50/ml 1-3 days after inoculation. Of 4 puppies inoculated with LACV, 3 developed fatal infections. Viremias were not detected in 4 cows, 4 ponies, 7 cats, or 6 sheep. Two cats fed LACV infected suckling mice shed virus from the oropharynx for 1 day each. All animals except 1 cow, 1 cat, and 1 sheep had greater than or equal to 4-fold rise in antibody titers. Five additional dogs fed upon by LACV-infected Aedes triseriatus mosquitoes did not develop viremias or antibody and uninfected Ae. triseriatus engorging on the dogs 1-5 days after feeding by infected mosquitoes failed to become infected. Five ponies, 6 calves, 2 ewes, 6 dogs, and 5 piglets were inoculated with 3.6-7.3 log10 SMICLD50 of JCV. None developed detectable viremias, although greater than or equal to 4-fold rises in antibody titers developed in 60% of the ponies, 17% of the calves, 50% of the dogs, and 1 of 2 ewes. None of the pigs developed corresponding rises in antibody titers. We conclude that juvenile and adult animals of the species tested are not efficient amplifier hosts of LACV or JCV, but may be useful sentinels of local virus transmission.

Experimental transmission of trivittatus virus (California virus group) by Aedes trivittatus.

The mosquito, Aedes trivittatus, when fed through a membrane a trivittatus virus dosage of 10(3.4) to 10(5.5) suckling mouse LD50/0.03 ml of blood, transmitted the virus to suckling mice. Virus multiplication indicative of a biological vector occurred in this species. When Aedes vexans and A. triseriatus ingested similar doses of trivittatus virus, both the infection and transmission rates were low and virus multiplication was poor. These results, added to evidence based on virus isolations from mosqito populations in nature, indicate that A. trivittatus is the primary vector of trivittatus virus in the north central United States.

Infection rates of Aedes triseriatus following ingestion of La Crosse virus by the larvae.

Infection rates ranged from 0-2.1% in adults of Aedes triseriaus reared from groups of larvae that had ingested La Crosse (LAC) virus (Clifornia encephalitis group) at dosages of 7.0-8.3 log 10 SMICLD50/ml. Females form orally infected larvae transmitted the virus to suckling mice. Larvae that devoured carcasses of transovarially infected larvae containing 3.0 log 10 SMICLD 50/ml failed to become infected. Ingestion by larvae of infected carcasses appears, therefore, to be unimportant as a method of horizontal amplification of LAC virus.

Serologic and virologic evidence of a Prospect Hill-like hantavirus in Wisconsin and Minnesota.

The purposes of this study were to determine if hantaviruses were present in the Great Lakes port areas of Wisconsin and Minnesota and if so, to identify which virus and which rodent host species were involved. Rodents were trapped in Duluth, Minnesota, Superior, Green Bay, and Milwaukee, Wisconsin, all ports of call for international maritime shipping. A total of 675 wild rodents were captured and tested, including 310 meadow voles (Microtus pennsylvanicus), 173 Norway rats (Rattus norvegicus), 179 Peromyscus spp., (including white footed mice [P. leucopus] and deer mice [P. maniculatus gracilis and P. maniculatus bairdii]), and 13 house mice (Mus musculus). Twenty percent of the rats, 17% of the meadow voles, 8% of the house mice, and 3% of the Peromyscus spp. had antibody to a hantavirus by immunofluorescent antibody assay (IFA). By the plaque-reduction neutralization test (PRNT), nine of 36 meadow voles, one of 4 P. leucopus, and one of 34 rats had hantavirus antibody, with the highest titers to Prospect Hill (PH) virus. All of the PRNT-seropositive individuals were from the twin cities of Superior and Duluth. Hantavirus antigen was detected in lung tissue by IFA in M. pennsylvanicus and Peromyscus spp., but not in rats. Two hantaviruses, designated SD-1 and SD-2, were isolated from M. pennsylvanicus captured in Duluth and found to be very similar to prototype PH virus by cross-IFA and cross-PRNT. Virus isolation attempts were unsuccessful from tissues of the Peromyscus spp. and the rats.

La Crosse viremias in white-tailed deer and chipmunks exposed by injection or mosquito bite.

To further understand the role of wild mammals in the maintenance of La Crosse virus (LACV) in nature, we investigated the effects of inoculation method and virus source on the duration and amplitude of LACV viremia in vertebrate hosts. Earlier work suggested that deer are not sufficiently susceptible to LACV to play an important role in its maintenance. We re-evaluated the susceptibility of deer since subsequent studies showed that they constitute 65% of Aedes triseriatus blood meals, and thus would be exposed frequently to the virus. In our study, deer developed higher and longer viremia following exposure to LACV by infected Ae. triseriatus than those previously reported by inoculation with needle and syringe. However, susceptible Ae. triseriatus that fed on these viremic animals did not become infected. Because a large number of uninfected mosquitoes can feed upon a viremic deer in nature, we believe that deer should not be disregarded completely as a possible amplifier in the LACV transmission cycle. We also infected chipmunks to determine if there were significant differences in viremia response from mosquito delivery of virus to the chipmunk host, compared with artificial exposure by injection. Chipmunks exposed to infected mosquitoes had higher and longer viremias than the ones produced by intramuscular injection of an LACV suspension. These findings show the importance of using LACV infected mosquitoes for transmission experiments in mammals.