Thomas M. Yuill

Role of La Crosse virus glycoproteins in attachment of virus to host cells.

Data presented in this report demonstrate that the initial event of La Crosse virus (LACV) infection of cells is probably the interaction of viral glycoproteins with specific cellular receptor sites. We have shown that LACV glycoprotein G1 binds, in a dose-dependent manner, to continuous vertebrate and mosquito cell lines, but not to mosquito midguts isolated ex vivo. This binding can be inhibited by the pretreatment of cells with excess homologous glycoprotein but not with excess heterologous LACV glycoprotein. In contrast, we have shown that LACV glycoprotein G2 binds to the continuous mosquito cell line and vector midgut cells, but not to vertebrate cells. LACV infection of vertebrate cells can be inhibited by treatment of cells with purified G1, while infection in mosquito cells can be reduced by treatment of cells with a combination of G1 and G2. The results suggest that G1 is the viral attachment protein (VAP) for vertebrate cells, and that G2 serves the same purpose for mosquito midgut cells. We speculate that the protease-resistant G2 molecule may have evolved to serve as the VAP in the midgut under conditions in which G1 might be altered or removed from the virus envelope, and thus is essential to the evolution and maintenance of vertebrate-invertebrate transmission cycles.

Enzyme processing of La Crosse virus glycoprotein G1: A bunyavirus-vector infection model

Efficient transmission, amplification, and dissemination of arboviruses require viral replication in vertebrate and invertebrate hosts. As a result, virions are exposed to two significantly different environments. Exposure of LaCrosse virus (LACV) to proteolytic enzymes, such as those that may be found in the mosquito midgut, increases virus affinity for mosquito cells. These enzymes remove the major envelope glycoprotein (G1) while leaving the second glycoprotein (G2) intact. Processing of LACV glycoproteins in the mosquito midgut may be necessary to expose attachment proteins on the virion surface before attachment to, and infection of, midgut cells can occur. This model may suggest answers to questions regarding the molecular basis for midgut infection barriers and species susceptibility to arbovirus infection in nature.

PREVALENCE OF HELMINTHS IN A CYCLIC SNOWSHOE HARE POPULATION

Five species of helminths were monitored in a population of snowshoe hares (Lepus americanus) near Rochester, Alberta, during 1961-1977. Prevalence of both Obeliscoides cuniculi and Protostrongylus boughtoni among young hares averaged about 50% by age 2 mo, then tended to level off. Prevalence of Taenia pisiformis (cysticerci) and Dirofilaria scapiceps rose more slowly, but continued to increase steadily beyond their mean levels of 8% and 1% at age 2 mo. There were well denned seasonal (within-year) cycles in prevalence of O. cuniculi and P. boughtoni that were generated evidently to a major degree by arrested development of larvae in fall and renewed development in late winter. It was hypothesized that renewed larval development was triggered (in February) in O. cuniculi by the seasonal rise of circulating pituitary gonadotropins, and (in April) in P. boughtoni by the seasonal rise of gonadal androgens and estrogens. Indices to gonadal hormone levels in hares indicated that these increased most rapidly among males, and may have accounted for the higher prevalences of P. boughtoni in males during April-May. Neither T. pisiformis nor D. scapiceps exhibited conspicuous seasonal changes in prevalence. Maximum prevalence of T. pisiformis was attained at about 1 yr of age, whereas D. scapiceps increased among adult snowshoes through age 2 yr before stabilizing. Long-term (between-year) changes in prevalence of O. cuniculi, T. pisiformis, and D. scapiceps were correlated significantly with the cyclic hare population which declined from a peak in fall 1961 to a low in 1965–1966, rose to another peak by fall 1970, and declined again to a low in 1975. There was no detectable time lag between this “10-yr” cycle in hare density and the cycles of parasite prevalence among juveniles (<1 yr of age). Among adult hares, the cycle of O. cuniculi prevalence was likewise synchronous with that of the hare population, but the cycles of D. scapiceps and T. pisiformis lagged by approximately 1 and 2 yr, respectively. This lag in T. pisiformis prevalence was largely inexplicable to us. Our data on P. boughtoni were not suitable for analyses of between-year trends; nor were those for the fifth helminth, Taenia serialis (coenuri), because mean prevalence was less than 1% among both juveniles and adults. An apparent decline in T. serialis after the early 1950s, and its continued scarcity thereafter, paralleled a major change in numbers of one important definitive host—the red fox (Vulpes vulpes). Lighter weights of young hares at age 37–96 days, and of adults and fully grown juveniles, were associated with P. boughtoni infections. There was no demonstrable relationship between snowshoe hare reproductive parameters and helminth parasitism.

Oil and related toxicant effects on mallard immune defenses

A crude oil, a petroleum distillate, and chemically dispersed oil were tested for their effects on resistance to bacterial infection and the immune response in waterfowl. Sublethal oral doses for mallards were determined for South Louisiana crude oil, Bunker C fuel oil, a dispersant--Corexit 9527, and oil/Corexit combinations by gizzard intubation. Resistance to bacterial challenge (Pasteurella multocida) was significantly lowered in mallards receiving 2.5 or 4.0 ml/kg of Bunker C fuel oil, 4.0 ml/kg of South Louisiana crude oil, and 4.0 ml/kg of a 50:1 Bunker C fuel oil/Corexit mixture daily for 28 days. Ingestion of oil or oil/Corexit mixtures had no effect on mallard antibody-producing capability as measured by the direct spleen plaque-forming assay.

Virulence of six strains of duck plague virus in eight waterfowl species.

Susceptibility of New World waterfowl to the Lake Andes strain of duck plague virus (DPV) was assessed by intramuscular inoculation of adult muscovies (Cairina moschata), mallards (Anas platyrhynchos), Canada geese (Branta canadensis), wood ducks (Aix sponsa), redheads (Aythya americana), gadwalls (Anas strepera), blue-winged teal (Anas discors), and pintails (Anas acuta). The relative virulence of DPV strains isolated from five United States and one Canadian location was established in muscovies, mallards, and Canada geese. Differences in DPV strain virulence were detected by formation of plaques in cell culture. Two strains that consistently formed plaques killed adult mallards while non-plaque forming strains killed hatchling but not adult mallards. Based on mortality after exposure to the Lake Andes strain, blue-winged teal, then wood ducks and redheads were highly susceptible, muscovies and gadwalls moderately susceptible, mallards and Canada geese less susceptible, and pintails the least susceptible. Mean death times were significantly (P < 0.01) different between adult muscovies (4.5 days) versus mallards and Canada geese (5.8 days each). Mean death time of the virulent Lake Andes and Minnesota strains were shorter (P < 0.05) than for the other four, less virulent DPV strains. Four of the less virulent strains killed hatchling but not adult mallards. Susceptibility to mortality was dependent upon age and route of inoculation. The intramuscular route of inoculation required the least amount of virus to kill mallard and muscovy ducks, the intranasal and conjunctival routes required more virus, and the oral route the most virus. This study was conducted from 1974 to 1977 between the months of September and April, with the exception of two titrations conducted in early May at the University of Wisconsin Department of Veterinary Science and the Charmany research facility of the University of Wisconsin-Madison.

SEASONAL PREVALENCE OF CLOSTRIDIUM BOTULINUM TYPE C IN SEDIMENTS OF A NORTHERN CALIFORNIA WETLAND

The prevalence of Clostridium botulinum type C (% of positive sediment samples) was determined in 10 marshes at Sacramento National Wildlife Refuge (SNWR), located in the Central Valley of California (USA), where avian botulism epizootics occur regularly. Fifty-two percent of 2,200 sediment samples collected over an 18-mo period contained C. botulinum type C (both neurotoxic and aneurotoxic) which was present throughout the year in all 10 marshes. The prevalence of C. botulinum type C was similar in marshes with either high or low botulism losses in the previous 5 yr. Marshes with avian botulism mortality during the study had similar prevalences as marshes with no mortality. However, the prevalence of C. botulinum type C was higher in marshes that remained flooded all year (permanent) compared with marshes that were drained in the spring and reflooded in the fall (seasonal). The prevalence of C. botulinum type C declined in seasonal marshes during the dry period. Similar declines did not occur in the permanently flooded marshes.

Assay of arbovirus neutralizing antibody by micro methods

Abstract Efficient, reproducible and specific neutralizing antibody assays are needed, particularly for use in arboviral studies in areas where antigenic cross reactions make serological interpretation difficult. Accordingly, 2 methods were developed for this purpose. A micrometabolic inhibition (MMI) test with BHK-21 cells in disposable “U”-bottomed microtitre plates was used with Japanese encephalitis, Wesselsbron, Sindbis, chikungunya and Batai viruses. The MMI test was highly specific, of efficiency comparable with the haemagglutination-inhibition (HI) test, and was very sparing of reagents and sera. Although equal to or better in sensitivity than conventional cell culture neutralization tests, it was not as sensitive as HI or plaque-reduction neutralization (PRN) tests. The LLC-MK2 cell microculture PRN test was devised for use with the dengue viruses or for use when a combination of precision of antibody measurement and efficiency of testing was required. The microculture and 1 oz. bottle culture PRN tests were of equal specificity. Although not as precise as the bottle PRN test, the microculture PRN test is suitable for routine serology and has the added advantages that it is more efficient and requires less serum and reagents.

PREVALENCE OF NEUROTOXIC CLOSTRIDIUM BOTULINUM TYPE C IN THE GASTROINTESTINAL TRACTS OF TILAPIA (OREOCHROMIS MOSSAMBICUS) IN THE SALTON SEA

Tilapia (Oreochromis mossambicus) have been implicated as the source of type C toxin in avian botulism outbreaks in pelicans (Pelecanus erythrorhynchos, Pelecanus occidentalis californicus) at the Salton Sea in southern California (USA). We collected sick, dead, and healthy fish from various sites throughout the Sea during the summers of 1999 through 2001 and tested them for the presence of Clostridium botulinum type C cells by polymerase chain reaction targeting the C1 neurotoxin gene. Four of 96 (4%), 57 of 664 (9%), and five of 355 (1%) tilapia tested were positive for C. botulinum type C toxin gene in 1999, 2000, and 2001, respectively. The total number of positive fish was significantly greater in 2000 than in 2001 (P<0.0001). No difference in numbers of positives was detected between sick and dead fish compared with live fish. In 2000, no significant relationships were revealed among the variables studied, such as location and date of collection.

Selected microbial agents in snowshoe hares and other vertebrates of Alberta.

Serologic surveillance of populations of snowshoe hares and other vertebrate species of north-central Alberta from 1961 to 1969, revealed activity of one bacterial and eight viral agents. The most prevalent agents infecting the snowshoe hare were California encephalitis and Silverwater viruses, while in other vertebrates California encephalitis and Western equine encephalomyelitis viruses were the most common. The role of the snowshoe hare in the natural history of the agents is considered as is the effect of the agent on the hare ten-year cycle of abundance.

Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus

Neutralizing monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus (LACV) were prepared. Two antibodies immunoprecipitated the 120 kDa virus attachment protein for vertebrate cells, G1, while five immunoprecipitated the 35 kDa G2 protein, whose function is currently unknown. Two monoclonal antibodies were obtained that specifically precipitated both G1 and G2 from [35S]cysteine labeled LACV infected cell lysates. The G2 specific monoclonal antibodies had high neutralizing titers when assayed in mosquito cells but limited ability to neutralize virus in mammalian cells. The G1/G2 specific antibodies neutralized virus infectivity in both vertebrate and invertebrate cells at high titers. These results suggest that G2 is involved in the interaction of virus with mosquito cells and that G1 and G2 may share a common structural epitope relevant to their role as attachment proteins in vertebrate and mosquito cells. Monoclonal antibodies directed against G2 or G1/G2 have not previously been reported and should be useful tools for characterizing the biological functions of these molecules in the divergent micro-environments of vertebrate and invertebrate hosts.