Induction of apoptosis by La Crosse virus infection and role of neuronal differentiation and human bcl-2 expression in its prevention.

La Crosse virus causes a highly cytopathic infection in cultured cells and in the murine central nervous system (CNS), with widespread neuronal destruction. In some viral infections of the CNS, apoptosis, or programmed cell death, has been proposed as a mechanism for cytopathology (Y. Shen and T. E. Shenk, Curr. Opin. Genet. Dev. 5:105-111, 1995). To determine whether apoptosis plays a role in La Crosse virus-induced cell death, we performed experiments with newborn mice and two neural tissue culture models. Newborn mice infected with La Crosse virus showed evidence of apoptosis with the terminal deoxynucleotidyl transferase-mediated nicked-end labeling (TUNEL) assay and, concomitantly, histopathological suggestion of neuronal dropout. Infection of tissue culture cells also resulted in DNA fragmentation, TUNEL reactivity, and morphological changes in the nuclei characteristic of apoptotic cells. As in one other system (S. Ubol, P. C. Tucker, D. E. Griffin, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 91:5202-5206, 1994), expression of the human proto-oncogene bcl-2 was able to protect one neuronal cell line, N18-RE-105, from undergoing apoptosis after La Crosse virus infection and prolonged the survival of infected cells. Nevertheless, expression of bcl-2 did not prevent eventual cytopathicity. However, a human neuronal cell line, NT2N, was resistant to both apoptosis and other types of cytopathicity after infection with La Crosse virus, reaffirming the complexity of cell death. Our results show that apoptosis is an important consequence of La Crosse virus infection in vivo and in vitro.     

Protection from La Crosse virus encephalitis with recombinant glycoproteins: role of neutralizing anti-G1 antibodies.

La Crosse virus, a member of the California serogroup of bunyaviruses, is an important cause of pediatric encephalitis in the midwestern United States. Like all bunyaviruses, La Crosse virus contains two glycoproteins, G1 and G2, the larger of which, G1, is the target of neutralizing antibodies. To develop an understanding of the role of each of the glycoproteins in the generation of a protective immune response, we immunized 1-week-old mice with three different preparations: a vaccinia virus recombinant (VV.ORF) that expresses both G1 and G2, a vaccinia virus recombinant (VV.G1) that expresses G1 only, and a truncated soluble G1 (sG1) protein prepared in a baculovirus system. Whereas VV.ORF generated a protective response that was mostly directed against G1, VV.G1 was only partially effective at inducing a neutralizing response and at protecting mice from a potentially lethal challenge with La Crosse virus. Nevertheless, a single immunization with the sG1 preparation resulted in a robust immune response and protection against La Crosse virus. These results indicate that (i) the G1 protein by itself can induce an immune response sufficient for protection from a lethal challenge with La Crosse virus, (ii) a neutralizing humoral response correlates with protection, and (iii) the context in which G1 is presented affects its immunogenicity. The key step in the defense against central nervous system infection appeared to be interruption of a transient viremia that occurred just after La Crosse virus inoculation.     

Human MxA Protein Protects Mice Lacking a Functional Alpha/Beta Interferon System against La Crosse Virus and Other Lethal Viral Infections

The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-alpha/beta). MxA inhibits the multiplication of several RNA viruses in cell culture. However, its antiviral potential in vivo has not yet been fully explored. We have generated MxA-transgenic mice that lack a functional IFN system by crossing MxA-transgenic mice constitutively expressing MxA with genetically targeted (knockout) mice lacking the beta subunit of the IFN-alpha/beta receptor (IFNAR-1(-/-) mice). These mice are an ideal animal model to investigate the unique antiviral activity of human MxA in vivo, because they are unable to express other IFN-induced proteins. Here, we show that MxA confers resistance to Thogoto virus, La Crosse virus, and Semliki Forest virus. No Thogoto virus progeny was detectable in MxA-transgenic mice, indicating an efficient block of virus replication at the primary site of infection. In the case of La Crosse virus, MxA restricted invasion of the central nervous system. In contrast, Semliki Forest virus multiplication in the brain was detectable in both MxA-expressing and nonexpressing IFNAR-1(-/-) mice. However, viral titers were clearly reduced in MxA-transgenic mice. Our results demonstrate that MxA does not need the help of other IFN-induced proteins for activity but is a powerful antiviral agent on its own. Moreover, the results suggest that MxA may protect humans from potential fatal infections by La Crosse virus and other viral pathogens.     

La Crosse virus infection of mammalian cells induces mRNA instability.

La Crosse virus infection of BHK cells leads to a dramatic shutoff of not only host protein synthesis but also viral protein synthesis later in infection. This shutoff can be accounted for by the loss of the cytoplasmic cellular and viral mRNAs. The induction of mRNA instability requires extensive virus replication, since when cycloheximide is added early in infection the preexisting viral and cellular mRNAs do not decrease upon incubation of the cultures. Pretreatment of the cultures with actinomycin D does not affect the ability of La Crosse virus infection to induce mRNA instability, and examination of the rRNAs shows no evidence of specific degradation due to activation of the interferon-associated latent RNase. The induction of mRNA instability therefore does not appear to operate through an interferon pathway. Viral mRNA synthesis, on the other hand, is not turned off during infection, and the cap-dependent endonuclease involved in viral mRNA initiation may be responsible for the mRNA instability.     

Anti-mRNAs in La Crosse bunyavirus-infected cells.

Unlike some members of the family Bunyaviridae which contain ambisense genomes, all La Crosse virus reading frames are translated from antigenome sense mRNAs. Nevertheless, La Crosse virus genome sense mRNAs or anti-mRNAs are initiated from antigenome templates. These are characterized by the same range of capped, nontemplated sequences at their 5' ends as mRNAs, but their 3' ends are presumed to be heterogenous, as they were not seen on RNA blots. The anti-mRNAs are estimated to be 15 to 30 times less abundant than mRNAs, but remarkably, this ratio is similar to that of functional genome sense mRNAs made from other bona fide ambisense segments. A role for these anti-mRNAs during infection is unclear.     

An avirulent G1 glycoprotein variant of La Crosse bunyavirus with defective fusion function.

La Crosse virus, a member of the California serogroup of the family Bunyaviridae, causes encephalitis in humans and laboratory rodents. A variant virus (V22) selected with a monoclonal antibody against the large (G1) glycoprotein showed diminished neuroinvasiveness after peripheral inoculation. This variant has an alteration in its fusion function, requiring a lower pH for the activation of fusion and demonstrating reduced efficiency of cell-to-cell fusion of BHK-21 cultures. V22 was studied in detail following the infection by intraperitoneal or intracerebral routes in suckling, weanling, or adult CD-1 mice. It exhibited a marked reduction in its ability to replicate in striated muscle and to produce viremia; however, after intracerebral injection V22 virus replicated almost as rapidly in brain as its parent, La Crosse virus. V22 virus thus represents an example of reduced neuroinvasiveness associated with an alteration at a specific epitope of the G1 glycoprotein. This same epitope also influences the fusion activity of the glycoprotein.     

Electron microscopy of the segmented RNA genome ofLa Crosse virus: absence of circular molecules.

The three species of single-stranded RNA present in La Crosse virus were examined in the electron microscope. Because large amounts of contaminating cellular DNA are copurified with the virus despite extensive attempts to purify the virus, it was necessary to use procedures that eliminated the bulk of this DNA before the viral RNA was analyzed. When this was done, the modal lengths of La Crosse virus RNA were 0.4, 2.0, and 3.1 mum. These lengths correspond well to their known molecular weights of 0.4 x 106, 1.8 x 106, and 2.9 x 106. Under the denaturing conditions used to permit complete spreading of these single-stranded RNA molecules, no single-stranded circular molecules are observed. Therefore, the circular nucleocapsids present in La Crosse virus and some other bunyaviruses do not appear to be due to convalent linkage of the ends of the RNA genome.     

Three unique viral RNA species of snowshoe hare and La Crosse bunyaviruses.

Two-dimensional gel electrophoreses of RNase T1-derived oligonucleotides of the three individual RNA segments of the bunyavirus snowshow hare virus indicate that its three RNA segments possess distinct nucleotide sequences. The fingerprints of the RNA species of snowshoe hare virus differ from those of the antigenically closely related La Crosse virus. Three viral RNA species have been identified in preparations of Melao and Trivittatus as well as snowshoe hare, Lumbo, and La Crosse bunyaviruses.     

Quantitative genetics of vector competence for La Crosse virus and body size in Ochlerotatus hendersoni and Ochlerotatus triseriatus interspecific hybrids

La Crosse virus is a leading cause of pediatric encephalitis in the United States. The mosquito Ochlerotatus triseriatus is an efficient vector for La Crosse virus, whereas the closely related O. hendersoni transmits only at very low rates. Quantitative trait loci (QTL) affecting the ability to orally transmit this virus and adult body size were identified in 164 F(2) female individuals from interspecific crosses of O. hendersoni females and O. triseriatus males using a combination of composite interval mapping (CIM), interval mapping (IM) for binary traits, and single-marker mapping. For oral transmission (OT), no genome locations exceeded the 95% experimentwise threshold for declaring a QTL using IM, but single-marker analysis identified four independent regions significantly associated with OT that we considered as tentative QTL. With two QTL, an increase in OT was associated with alleles from the refractory vector, O. hendersoni, and likely reflect epistatic interactions between genes that were uncovered by our interspecific crosses. For body size, two QTL were identified using CIM and a third tentative QTL was identified using single-marker analysis. The genome regions associated with body size also contain three QTL controlling OT, suggesting that these regions contain either single genes with pleiotropic effects or multiple linked genes independently determining each trait.     

M viral RNA segment of bunyaviruses codes for two glycoproteins, G1 and G2.

Tryptic peptide digests of the two viral glycoproteins (G1 and G2) of snowshow hare (SSH) virus, La Crosse, La Crosse (LAC) virus, and an SSH/LAC recombinant virus which has a large (L)/medium (M)/small (S) RNA segment genome composition of SSH/LAC/SSH were analyzed by ion-exchange column chromatography. The analyses prove that the M RNA species of bunyaviruses codes for the two viral glycoproteins.     

Midgut and salivary gland barriers to La Crosse virus dissemination in mosquitoes of the Aedes triseriatus group

Vector competence for La Crosse virus (LACV) was compared for four species in the Aedes (Protomacleaya) triseriatus group: Ae. triseriatus (Say), Ae. hendersoni Cockerell, Ae. zoosophus Dyar and Knab and Ae. brelandi Zavortink (Diptera: Culicidae). Rates of replication and dissemination of virus in the mosquito hosts were compared and rates of oral transmission of virus to suckling mice were determined. Barriers to virus dissemination which limited the ability of each species to transmit virus were identified. Ae. zoosophus displayed the highest vector competence for LACV. Both infection and transmission rates were high: 99% and 85% respectively; no significant barriers to LACV were found. Disseminated infection of Ae. triseriatus with LACV was controlled primarily be a midgut escape barrier. When virus was introduced directly into the haemocoel, transmission rates were significantly increased (37% v. 79%). Ae. hendersoni showed high susceptibility to LACV infection but a very low rate of oral transmission (7%). Ae. brelandi was also highly susceptible to infection by LACV and transmitted virus at an intermediate rate (27%). Modulation of vector competence in both Ae. hendersoni and Ae. brelandi resulted from a salivary gland escape barrier. As these four species of mosquitoes comprise a closely related monophyletic series, their differences of vector competence for LACV provide an excellent model for studying the genetic basis of the barriers involved.     

Natural products as leads to potential mosquitocides

Mosquitoes are the crucial vectors for a number of mosquito-borne infectious diseases i.e. dengue, yellow fever, chikungunya, malaria, Rift Valley fever, elephantiasis, Japanese Encephalitis, and Murray Valley encephalitis etc. Besides, they also transmit numerous arboviruses (arthropod-borne viruses) for example West Nile virus, Saint Louis encephalitis virus, Eastern equine encephalomyelitis virus, Everglades virus, Highlands J virus, and La Crosse Encephalitis virus. The emergence of widespread insecticide resistance and the potential environmental issues associated with some synthetic insecticides (such as DDT) has indicated that additional approaches to control the proliferation of mosquito population would be an urgent priority research. The present review highlights some natural product mosquitocides that are target-specific, biodegradable, environmentally safe, and botanicals in origin.     

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 μM) and La Crosse virus (EC(50)=0.27 μM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).     

Serologic evidence of arboviral infections in white-tailed deer from central Wisconsin

A survey conducted during 1979-1980 on white-tailed deer (Odocoileus virginianus) in central Wisconsin revealed serological evidence of infection by selected arboviruses. Among sera from 41 deer, antibody was detected for Jamestown Canyon virus (56%) and Bunyamwera group virus (80%), demonstrating their continuing endemic activity. Antibody for La Crosse virus, not found previously in sera from deer in central Wisconsin, also was detected (5%) in this study.     

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.     

Impacts of climate, land use, and biological invasion on the ecology of immature aedes mosquitoes: implications for la crosse emergence

Arthropod-borne viruses (arboviruses) cause many diseases worldwide and their transmission is likely to change with land use and climate changes. La Crosse virus (LACV) is historically transmitted by the native mosquito Aedes triseriatus (Say) in the upper Midwestern US, but the invasive congeners Aedes albopictus (Skuse) and A. japonicus (Theobald), which co-occur with A. triseriatus in water-holding containers, may be important accessory vectors in the Appalachian region where La Crosse encephalitis is an emerging disease. This review focuses on evidence for how climate, land use, and biological invasions may have direct abiotic and indirect community-level impacts on immature developmental stages (eggs and larvae) of Aedes mosquitoes. Because vector-borne diseases usually vary in space and time and are related to the ecology of the vector species, we propose that the ecology of its mosquito vectors, particularly at their immature stages, has played an important role in the emergence of La Crosse encephalitis in the Appalachian region and represents a model for investigating the effects of environmental changes on other vector-borne diseases. We summarize the health effects of LACV and associated socioeconomic costs that make it the most important native mosquito-borne disease in the US. We review of the transmission of LACV, and present evidence for the impacts of climate, land use, and biological invasions on Aedes mosquito communities. Finally, we discuss important questions about the ecology of LACV mosquito vectors that may improve our understanding of the impacts of environmental changes on LACV and other arboviruses.     

Structural proteins of La Crosse virus.

Preparations of La Crosse virus, a member of the California encephalitis group of bunyaviruses, were found to possess three major virion proteins. Two of the proteins were glycosylated (G1 and G2) and were located on the surface of the virus particles. These two glycoproteins were present in equimolar amounts and possessed apparent molecular weights of 120 X 10(3) and 34 X 10(3). Virion nucleocapsids, isolated by a nonionic detergent and salt treatment, contained another major protein, N (molecular weight = 23 X 10(3)). A large, but minor, protein species L (molecular weight = 180 X 10(3)) was also found in virus preparations. The approximate number of protein molecules per virion has been determined. Electron microscopy of purified La Crosse virus indicated that the virus particle (mean diameter, 91 nm) is enveloped and possesses irregular surface projections (length, 10 nm).     

Virulence of La Crosse virus is under polygenic control.

To identify which RNA segments of the California serogroup bunyaviruses determine virulence, we prepared reassortant viruses by coinfecting BHK-21 cells with two wild-type parents, La Crosse/original and Tahyna/181-57 viruses, which differed about 30,000-fold in virulence. The progeny clones were screened by polyacrylamide gel electrophoresis to ascertain the phenotype of the M and S RNA segments, and RNA-RNA hybridization was used to determine the genotype of selected clones. Two or three clones of each of the six possible reassortant genotypes were characterized quantitatively for neuroinvasiveness by determining the PFU/50% lethal dose (LD50) ratio after subcutaneous injection into suckling mice. The reassortants fell into two groups. (i) Six of seven reassortants with a La Crosse M RNA segment were as virulent as the parent La Crosse virus (about 1 PFU/LD50); the one exception was strikingly different (about 1,000 PFU/LD50) and probably represents a spontaneous mutant. (ii) The seven reassortants with a Tahyna M RNA segment were about 10-fold more virulent than the parent Tahyna virus (median 1,600 PFU/LD50 for reassortants and 16,000 PFU/LD50 for Tahyna virus). A comparative pathogenesis study in suckling mice of one reassortant virus and the parent Tahyna virus confirmed the greater neuroinvasiveness of the reassortant virus. From these data it was concluded that the M RNA segment was the major determinant of virulence, but that the other two gene segments could modulate the virulence of a nonneuroinvasive California serogroup virus.