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Nucleotide sequences were determined for the 5' termini of La Crosse virus (LAC) S segment mRNA from persistently infected mosquito cell cultures (C6/36 from Aedes albopictus) and embryos (Aedes triseriatus). LAC primes transcription of its mRNA with "scavenged" 5' caps and adjacent oligonucleotides from host mRNAs, and these non-virus-encoded 5'-terminal extensions are heterogeneous in infected mammalian cells. The nature of mosquito host-derived primers has not been previously investigated. During early C6/36 cell infection, LAC mRNA 5'-terminal sequences were heterogeneous, but variability decreased as infection persisted. One predominant sequence, 5' CCACTCGCCACT (sequence 1), was observed throughout C6/36 cell infection but was more prevalent after 15 days postinfection. This LAC mRNA 5'-terminal sequence comprised 81% of the scavenged host oligonucleotides from vertically infected A. triseriatus eggs during embryogenesis. As these embryos progressed in the dormant overwintering stage (diapause), the predominant scavenged sequence became 5' AGGAAAAGATGGT (sequence 2), and sequence 1 became less prevalent. As the eggs emerged from diapause, the LAC mRNA 5' termini were more variable; 33% had sequence 1, and the remainder were heterogeneous. In post-diapausing eggs, 100% of viral mRNAs had sequence 1 at their 5' termini. Molecular analyses thus revealed continuous but selective LAC cap scavenging during persistent C6/36 cell infection and during embryogenesis and diapause in A. triseriatus eggs. The variety of host-derived sequences was limited in both biosynthetically active (embryonating) and dormant (diapausing) eggs.

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Neurotropic properties of Leiv 12724 Ax and Leiv 13004 Ax strains were demonstrated by inoculation of green monkeys, Syrian hamsters and white mice using different routes. The strain Leiv 13004 Ax showed more marked pathogenicity for monkeys and rodents producing lesions in all parts of the brain: temporal, frontal, occipital, cerebellar, medulla oblongata and spinal cord where productive vasculitis, perivascular infiltrations, hemorrhages, and dystrophy of nerve cells were observed. In hamsters, the strains Leiv 13004 Ax and Leiv 12724 Ax inoculated subcutaneously produced latent infection with long-term virus carrier state.

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Mosquitoes collected from coastal, inland valley, and alpine locations in California were evaluated for their experimental vector competence for two viruses in the California serogroup (Bunyaviridae:Bunyavirus). Aedes squamiger, a coastal salt marsh mosquito, was an efficient vector of a California encephalitis (CE)-like virus isolated from its habitat (89% of the pledget-fed females became infected and 61% transmitted virus). Aedes dorsalis, a coastal mosquito, and Ae. melanimon, an inland valley mosquito, were competent vectors of prototype CE virus (98% and 100% of the pledget-fed females became infected and 56% and 30%, respectively, transmitted virus). Aedes squamiger and Ae. dorsalis transmitted both viruses vertically to one or more of 20 of their progeny. Culiseta inornata was susceptible to infection with both viruses, but 5% or less transmitted virus perorally. Alpine mosquitoes, Ae. cataphylla, Ae. increpitus, and Ae. tahoensis, became infected with both CE and CE-like viruses, but 3% or less transmitted virus. All species of mosquitoes were more efficient vectors of both viruses following intrathoracic inoculation than following pledget feeding, suggesting the presence of mesenteronal barriers.

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Jamestown Canyon (JC) virus of the California (CAL) serogroup has been isolated in 12 American states and 6 Canadian provinces. A study was undertaken to produce monoclonal antibodies (MAbs) to JC virus and to use these MAbs to assay for possible heterogeneity among naturally occurring JC topotypes in Canada. MAbs were produced to the prototype strain of JC virus using BALB/c mice. Twenty-seven secreting MAbs were obtained and three of these MAbs were propagated and studied. All three MAbs, M1 (IgG1), M2 (IgG2b), and M3 (IgG2a), were reactive by immunofluorescent antibody assay against JC-infected vero cells and by ELISA against JC antigen. MAb M2 reacted with all members of the Melao complex, MAb M1 reacted only with Keystone virus, while MAb M3 exhibited no reactivity with other CAL serogroup viruses. Only MAb M3 possessed neutralization and hemagglutination inhibition activities against JC virus. The MAbs were also tested by ELISA and for neutralizing activity against 13 JC topotypes isolated in 5 provinces from Newfoundland to Saskatchewan. ELISA confirmed closer identity of the Canadian topotypes to JC as opposed to the closely related South River virus. The MAbs verified all Canadian topotypes to be JC virus but revealed different patterns of reactivity between these topotypes and prototype JC virus.

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The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.

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Aedes triseriatus and Culiseta inornata mosquitoes were compared in their ability to transmit vertically La Crosse (LAC) and snowshoe hare (SSH) viruses. LAC virus was transovarially transmitted by 53% of Ae. triseriatus, the natural vector, and by 22% of Cs. inornata mosquitoes. SSH virus was transovarially transmitted by 89% of Cs. inornata, a proposed natural vector, and by 29% of Ae. triseriatus. A genetic approach, using LAC, SSH, and LAC/SSH reassortant viruses was then used to elucidate viral genetic determinants of transovarial transmission of bunyaviruses by Ae. triseriatus mosquitoes. Viruses containing the LAC medium sized (M) RNA segment were most efficiently transovarially transmitted by Ae. triseriatus mosquitoes. LAC, SSH, and Tahyna (TAH) viruses were compared in their ability to be venerally transmitted. All three viruses replicated in the reproductive tract of male Aedes triseriatus and were venereally transmitted to female mosquitoes. LAC and TAH viruses infected previously blood fed (BF) but not non-blood fed (NBF) Aedes triseriatus female mosquitoes.

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A general account is given in this paper of history, incidence, pathogen properties, morphogenesis, isolation, and culturing of Tahyna virus. Reference is also made to methods for detection, host spectrum, immunity, epizootiology, pathogenesis, and clinical symptoms in man and animals and also to aspects relating to pathological anatomy as well as to regular and differential diagnosis.

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The results are presented of comparative studies over the course of experimental Tahyna infection of viremia in the blood and organs, localization and patterns of lesions in the same organs after different routes of inoculation of white mice and hamsters with a new strain LEIV 9843 Mur of Tahyna virus. The results of these experiments are compared with similar studies in monkeys with asymptomatic infection, including humoral antibody detection. The pattern of lesions in the organs of animals infected with Tahyna virus (LEIV 9843 Mur strain), the mode of virus spread, tissue tropism of the virus were established. The virus thermostability and sensitivity to different pH values of the medium were determined.

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The California serogroup viruses are important human pathogens, originally discovered in the United States, but now recognized to occur and cause disease in many parts of the world. In addition to their significance to public health, study of their natural history has resulted in a better understanding of the way in which arboviruses are maintained in nature. Viruses of the California serogroup have also been carefully investigated on the molecular level, resulting in a better appreciation of how viruses are constructed, organized, and replicate. William C. Reeves, in whose honor this symposium is held, has contributed throughout his career to each of these fields of endeavor. His pioneering work on these viruses, beginning with the discovery of the prototype California encephalitis virus, has helped to establish the foundations upon which our current understanding of these viruses is built.

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Two squirrels aged 16 weeks and three muskrats aged 24 weeks were subcutaneously infected with a dose of 400 SMicLD50 of the extraneurally passaged "236" strain of Tahyna virus. Viremia was detected in one squirrel (48 and 96 hours post infection) and in two muskrats (24 and 48 hours p.i.). Seroconversion was demonstrated by plaque-reduction neutralization test (PRNT) 21 days p.i. in all animals.

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Tahyna virus (Bunyaviridae, Bunyavirus, the California encephalitis complex) was isolated from Aedes communis complex mosquitoes collected at the border of the north-taiga landscape zone (in latitude 68 degrees North and longitude 33 degrees East) at the Kolsky peninsula (the Murmansk region). The LEIV-9843 Mur strain was isolated from 2.4 thousand mosquitoes collected there (altogether 3.8 thousand mosquitoes had been collected in the Murmansk region). This is the first isolation in the USSR of a California complex virus in the Arctic and the northernmost site of Tahyna virus isolation in the world. 18% of the human population residing near the site of the virus isolation had virus-neutralizing antibody to Tahyna virus.

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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.

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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.

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The roles of various subtypes of the California serogroup viruses as infectious agents and as neuropathogens were evaluated by using the plaque reduction neutralization test. Sera from 394 patients with central nervous system (CNS) infections during 1971-1982 and from 501 persons without CNS manifestations were studied. Jamestown Canyon (JC) and La Crosse (LAC) viruses were found to have been common infectious agents in New York State for at least 16 years. JC virus was the prevalent indicated agent in patients with antibody to California serogroup viruses in screening tests (62 of 93 cases), followed by LAC virus (11 cases), snowshoe hare (2 cases), and trivittatus (1 case). In the remaining 17 patients the subtype was undetermined. LAC virus appears to be more pathogenic for children and to produce more serious illness, as judged by the frequent clinical diagnosis of encephalitis. JC virus affects mainly adults, and meningitis was the most common diagnosis. JC virus appears to cause a stronger neutralizing antibody response than does LAC virus, with a longer persistence of high levels of antibody. Some cases of JC virus infection may have been missed in the past due to the choice of a LAC-like isolate from New York State as the sole antigen in hemagglutination-inhibition (HI) screening tests. Comparison of the HI test and a single-dilution neutralization assay for screening for the two major subtypes, JC and LAC, indicated that the latter procedure is more broadly reactive and is less likely to miss cases if only one test antigen is used.

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To analyze mechanisms of virulence in the California serogroup bunyaviruses, the virulent La Crosse/original (LAC/original) strain was compared with the avirulent Tahyna/181-57 strain. In suckling mice, both viruses were lethal upon intracerebral injection but differed markedly in their neuroinvasiveness following subcutaneous injection; 20 and 20,000 plaque-forming units, respectively, were equivalent to 1 subcutaneous LD50. The sequential course of infection was followed after subcutaneous injection of 700 plaque-forming units; LAC/original replicated in striated muscle, caused a high titer plasma viremia, invaded the central nervous system, and killed all mice; the same dose of avirulent Tahyna/181-57 failed to replicate in extraneural tissues, did not invade the central nervous system, and caused no apparent illness. Immunofluorescent examination of peripheral and central nervous system tissues showed the same distinctions between virulent and avirulent viruses and pinpointed striated muscle as the major extraneural target of virulent LAC/original virus. Paradoxically, after intracerebral injection of suckling or adult mice, Tahyna/181-57 virus killed more quickly than LAC/original. This difference was correlated with replication differences; Tahyna/181-57 multiplied marginally faster in the brain than did LAC/original virus.

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Transmission rates of La Crosse (LAC) virus observed in Aedes triseriatus females that had engorged on chipmunks with antibody to LAC and had been mated by infected males 5-11 days later (24%, 69/288) were 40% lower than in those fed on chipmunks without antibody (38%, 112/293). Similar results were obtained in three separate trials using males infected 1) by inoculation with prototype LAC virus, 2) transovarially with a field strain, or 3) transovarially with the field strain following passage through a viremic chipmunk. Similar rates were also observed in trials with F2 and F3 progeny of several strains of Ae. triseriatus collected from LAC-endemic and non-endemic areas. Reduction of oral transmission by venereally infected females mated by transovarially infected males following engorgement of antibody in chipmunks or other vertebrates could be important in the natural control of LAC virus, since most adult chipmunks sampled in endemic areas have antibodies neutralizing LAC. Ten-fold higher rates of venereal infection found in females mated by infected males 5 or more days after engorgement on LAC antibody-negative chipmunks than in those mated without prior engorgement extend previous findings of higher rates of transmission after engorgement on laboratory mice to include the natural vertebrate host.

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