La Crosse Virus Research Articles

Histocompatibility antigens and La Crosse encephalitis.

Major histocompatibility complex antigens (HLA-ABC and -DR loci) on lymphocytes from 79 patients with La Crosse encephalitis were compared with those of 69 control subjects. Nine patients (11%) and none of the control population had HLA-B49 (P < .01, uncorrected for multiple comparisons). Of La Crosse encephalitis patients with acute-phase seizures, only 2 (7%) were positive for HLA-DR5, whereas 16 (37%) of nonseizure patients were positive for that antigen (P < .025). These findings suggest that susceptibility and complications associated with this disease may have an immunogenetic component.

Expression of the La Crosse M Segment Proteins in a Recombinant Vaccinia Expression System Mediates pH-Dependent Cellular Fusion

To study the expression of La Crosse virus (LAC) glycaproteins, G1 and G2, we constructed a cDNA copy of the open reading frame (ORF) of the middle RNA segment and expressed it in a recombinant vaccinia virus (VV.ORF). Cells infected with W.ORF expressed G1 and G2 at the cell surface and formed syncytia with a pH profile similar to that of LAC. These experiments provide a system of studying the biological functions of the LAC glycoproteins, including processing, targeting, fusion, receptor binding, and antigenicity.

Symptomatic La Crosse virus infections of the central nervous system: a study of risk factors in an endemic area.

In most years, La Crosse virus is the most common cause of reported mosquito-borne illness in the United States. The authors conducted a case-control study to determine if behavioral and environmental factors influenced the risk of La Crosse virus illness. Data were gathered on 31 serologically confirmed cases and 60 age-, sex-, and geography-matched controls in West Virginia in 1987 and 1988. Univariate analysis revealed minimal elevation of disease risk (odds ratios (ORs) less than 2.0) with increased time outdoors, non-use of insect repellent, non-use of air conditioning, lack of screened windows, and not wearing protective clothing. Univariate and multivariate analysis indicated that the presence of tree holes significantly increased disease risk (OR = 8.5 for greater than or equal to 1 tree hole vs. 0 tree holes). The following factors may also increase disease risk, although the findings were not statistically significant: discarded tires (OR = 3.2 for greater than or equal to 10 tires vs. 0-9 tires); non-tire artificial containers (OR = 4.1 for greater than or equal to 6 containers vs. 0-5 containers); and close proximity of the house to the forest edge (OR = 3.2 for 0-49 ft (0-14.9 m) vs. greater than or equal to 50 ft (greater than or equal to 14.9 m)). The authors conclude that the presence of natural breeding sites (tree holes) is an important risk factor for La Crosse virus illness. These results may be important in guiding future efforts aimed at preventing infection with La Crosse virus.

Genetics, infectivity and virulence of California serogroup viruses

The California serogroup of viruses includes 14 viruses which take their name from California encephalitis virus, the prototype of the serogroup, which was isolated in California in the 1950s (Hammon and Reeves, 1952). This serogroup is part of the Bunyauirus genus within the family Bunyaviridae (Gonzalez-Scarano and Nathanson, 1990; Kolakofsky, 1991; Whitman and Shope, 1962). La Crosse virus, the most medically significant member of the California serogroup, was isolated in 1964 from a fatal case of encephalitis diagnosed in La Crosse, Wisconsin (Thompson et al., 1965). Although California encephalitis virus was initially reported in association with a few cases of encephalitis, it is not an important human pathogen. On the other hand, La Crosse virus is a common cause of encephalitis in the pediatric age group, with an average of about 75 reported cases of encephalitis each year (Kappus et al., 1983). In years when other arboviruses are not epidemic, La Crosse virus is the most important cause of arbovirus encephalitis in the United States. The virus is estimated to cause more than 100,000 inapparent infections annually which are concentrated in the midwest (Grimstad et al., 1984), which is the principal habitat of Aedes triseriatus, its major mosquito vector. Bunyavirions are roughly spherical particles about 100 nm in diameter consisting of a lipid envelope containing nucleocapsids representing the three different RNA species (L segment, 6.8 kb; M segment, 4.5 kb; and S segment, 0.9 kb) which comprise the negative-sense genome (Elliott, 1990; Gonzalez-Scarano and Nathanson, 1990). The viral envelope contains two glycoproteins, Gl and G2, and

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.

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.

Global Climate Change and Infectious Diseases

The effects of global climate change on infectious diseases are hypothetical until more is known about the degree of change in temperature and humidity that will occur. Diseases most likely to increase in their distribution and severity have three-factor (agent, vector, and human being) and four-factor (plus vertebrate reservoir host) ecology. Aedes aegypti and Aedes albopictus mosquitoes may move northward and have more rapid metamorphosis with global warming. These mosquitoes transmit dengue virus, and Aedes aegypti transmits yellow fever virus. The faster metamorphosis and a shorter extrinsic incubation of dengue and yellow fever viruses could lead to epidemics in North America. Vibrio cholerae is harbored persistently in the estuaries of the U.S. Gulf Coast. Over the past 200 years, cholera has become pandemic seven times with spread from Asia to Europe, Africa, and North America. Global warming may lead to changes in water ecology that could enhance similar spread of cholera in North America. Some other infectious diseases such as LaCrosse encephalitis and Lyme disease are caused by agents closely dependent on the integrity of their environment. These diseases may become less prominent with global warming because of anticipated modification of their habitats. Ecological studies will help us to understand more fully the possible consequences of global warming. New and more effective methods for control of vectors will be needed.

Global climate change and infectious diseases.

The effects of global climate change on infectious diseases are hypothetical until more is known about the degree of change in temperature and humidity that will occur. Diseases most likely to increase in their distribution and severity have three-factor (agent, vector, and human being) and four-factor (plus vertebrate reservoir host) ecology. Aedes aegypti and Aedes albopictus mosquitoes may move northward and have more rapid metamorphosis with global warming. These mosquitoes transmit dengue virus, and Aedes aegypti transmits yellow fever virus. The faster metamorphosis and a shorter extrinsic incubation of dengue and yellow fever viruses could lead to epidemics in North America. Vibrio cholerae is harbored persistently in the estuaries of the U.S. Gulf Coast. Over the past 200 years, cholera has become pandemic seven times with spread from Asia to Europe, Africa, and North America. Global warming may lead to changes in water ecology that could enhance similar spread of cholera in North America. Some other infectious diseases such as LaCrosse encephalitis and Lyme disease are caused by agents closely dependent on the integrity of their environment. These diseases may become less prominent with global warming because of anticipated modification of their habitats. Ecological studies will help us to understand more fully the possible consequences of global warming. New and more effective methods for control of vectors will be needed.

Reassortment of La Crosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes

Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAH) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA; pLAC4.27: LAC M RNA; pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAH viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each blot was hybridized with 32P-labelled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTT, 259 (36%); TTL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%).

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.

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 LACY 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.

Effects of specific monoclonal antibodies on La crosse virus neutralization: Aggregation, inactivation by Fab fragments, and inhibition of attachment to baby hamster kidney cells

At high concentrations, several monoclonal antibodies to the G1 glycoprotein of La Crosse (LAC) virus aggregated the virus. To determine whether this accounted for the neutralization, the monoclonal antibodies were digested to make Fab fragments. With one exception, each monovalent antibody neutralized LAC virus to the same extent that bivalent antibody did, although higher concentrations were needed. Fab fragments of synergistic pairs of antibodies also exhibited enhanced binding in a competition binding assay but did not increase neutralization. To determine specific mechanisms for neutralization, the effects of polyclonal or monoclonal antibodies on virus attachment were examined. Polyclonal antibody to LAC virus reduced virus attachment by only 68% although it neutralized 99.99% of the virus. When virus was preincubated with a neutralizing monoclonal antibody to each of seven antigenic regions on G1, only antibody to one region reduced attachment of virus by as much as 92%. Antibodies to two regions that neutralize virus by 90–98% only inhibited attachment by 9 and 13%, respectively. The other antibodies showed intermediate degrees of neutralization and inhibition of attachment. Pairs of antibodies previously shown to be synergistic in neutralizing activity did not inhibit attachment any more than the single antibodies did.

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.

Heterologous Reassortment of Bunyaviruses in Aedes Triseriatus Mosquitoes and Transovarial and Oral Transmission of Newly Evolved Genotypes

Aedes triseriatus mosquitoes were orally infected with two different California serogroup bunyaviruses (La Crosse and snowshoe hare viruses) and high frequency reassortment occurred in these mosquitoes. Increased viral replication and subsequent gene segment reassortment was noted in the ovaries of mosquitoes that had ingested multiple blood-meals. To determine whether newly generated reassortant viruses could be transmitted transovarially to progeny mosquitoes, adult female mosquitoes were inoculated with the two temperature-sensitive (ts) parental viruses, and allowed to blood-feed and oviposit. Of 58 infected progeny mosquitoes assayed, six (10%) contained non-ts viruses, and three of these transmitted non-ts viruses to a susceptible host. Selected viruses of the non-ts phenotype, which were isolated from mosquitoes and from mice fed upon by the mosquitoes, were demonstrated to be reassortant viruses by oligonucleotide fingerprinting.

Monoclonal antibodies specific for the G1 glycoprotein of La Crosse virus that react with other California serogroup viruses

Various degrees of cross-reactivity have been observed among members of the California serogroup of bunyaviruses. Since the G1 glycoprotein of La Crosse (LAC) virus, a member of this serogroup, has been shown to contain the antigenic determinants involved in neutralization, monoclonal antibodies to this glycoprotein were used in enzyme immunosorbent and kinetics of neutralization assays with the other California viruses to detect cross-reactive sites that may be involved in neutralization. The results first indicated that five of the antigenic regions previously mapped on the LAC G1 glycoprotein contained overlapping epitopes within them. In all, a total of 16 epitopes within eight antigenic regions were identified on the G1 glycoprotein of LAC virus. One epitope was found on all of the California viruses and is therefore a group-specific antigenic determinant. A LAC-specific epitope was also identified. Different patterns of reactivity were observed among the California viruses for the rest of the antibodies. When the monoclonal antibodies (MAbs) were assayed with the uncloned reference strain of LAC and with two New York isolates, differences were found for epitopes within two antigenic regions. With one exception (snowshoe hare virus, a variety of LAC), pairs of antibodies shown to enhance neutralization of LAC virus did not increase neutralization of the other California viruses having the corresponding pairs of epitopes.

Regulation of Viral and Cellular RNA Turnover in Cells Infected by Eukaryotic Viruses including HIV-1

The following reviews the role of mRNA stability in the regulation of both viral and cellular gene expression in virus-infected cells. Indeed, several eukaryotic viruses, including the human immunodeficiency virus, HIV-1, regulate cellular protein synthesis via such control mechanisms. The following systems will be discussed: (i) the degradation of viral and cellular mRNAs in cells infected by herpes simplex virus (HSV) and advances made using the HSV virion host shutoff mutant; (ii) the degradation of viral and cellular mRNA and ribosomal RNA in cells infected by vaccinia virus and the possible role of the oligoadenylate synthetase-RNase L pathways; (iii) the turnover of RNAs in cells infected by encephalomyocarditis virus, reovirus, and La Crosse virus; and finally (iv) recent studies from our laboratory on the degradation of cellular mRNAs in cells infected by HIV-1.

The translational requirement for complete La Crosse virus mRNA synthesis is cell-type dependent

The translational requirement to prevent premature termination during La Crosse virus S mRNA synthesis was found to be cell-type dependent. This requirement was present in the BHK, HEL, and Vero cell lines we examined, but not in C6/36 mosquito cells. The cell-dependent translational requirement could be reproduced in vitro by using either cell extracts or purified virions of BHK and C6/36 cells. In the BHK reactions, the polymerase terminated predominantly at nucleotide 175 in the absence of concurrent translation and required translation to read through this position. In the C6/36 reactions, however, the polymerase reads through nucleotide 175 efficiently independent of translation. Reconstitution studies suggested that the translational requirement was due to a factor(s) present in BHK, but not in C6/36, cells.

La Crosse virus nucleocapsid protein controls its own synthesis in mosquito cells by encapsidating its mRNA

Within 24 to 48 h of La Crosse virus infection of mosquito cells, greater than 75% of the S mRNA was found to band in CsCl density gradients at the position of genome or antigenome nucleocapsids. The encapsidation of the S mRNA correlates with the repression of N protein synthesis in vivo, and the encapsidated S mRNA cannot be translated in vitro. Unlike genome and antigenome assembly, S mRNA assembly is a relatively slow process, which is not coupled to its synthesis. Within the encapsidated S mRNA population, three forms could be distinguished, those with intact primers which were or were not also assembled with N protein and those in which the primer and up to 3 template bases had been lost. We suggest that genome replication, but not transcription, is down regulated with time in mosquito cells for reasons that are unclear. The pool of unassembled N protein then increased to the point at which it began to interact with its own mRNA, as this mRNA also contains what is considered to be the assembly site, i.e., the conserved sequences at the 5' ends of all genome and antigenome chains. This lead to the assembly of the entire mRNA, except for the nontemplate primer. Some of the primers were then also assembled with N protein, whereas others were digested to produce truncated mRNAs.

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.

Mechanism of La Crosse virus inhibition by ribavirin.

The effect of ribavirin on the growth and replication of La Crosse virus was examined. The data suggest that low concentrations of ribavirin have a marked effect on the initial steps of La Crosse virus transcription. The therapeutic potential of ribavirin in the treatment of human California encephalitis serotype infections is discussed in light of these findings.