La Crosse Virus Research Articles

California serogroup viruses in New York State: the role of subtypes in human infections.

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.

The replication of two California serogroup viruses in a cell line from the mosquito Aedes triseriatus (Diptera: Culicidae).

An Aedes triseriatus cell line was established from neonatal larval tissue. In the 60th passage, the cells have been characterized as to growth, chromosome number, absence of microbial contamination, and absence of La Crosse encephalitis virus (LAC) and other vertebrate viruses as determined by passage in suckling mice and electron microscopy. Isozyme analysis using polyacrylamide gel electrophoresis of lysed cell suspensions and suspensions of Aedes triseriatus larvae revealed that 4 enzyme phenotypes (isocitrate dehydrogenase, malic enzyme, phosphoglucoisomerase, and phosphoglucomutase) were identical. The Aedes triseriatus cells grow in plastic Falcon flasks in a medium (MM/VP12) composed of equal volumes of Mitsuhashi-Maramorosch and Varma-Pudney media with fetal bovine serum and antibiotics. The growth characteristics of 2 California serogroup viruses (La Crosse and trivittatus) in these cells are described

Human infections with La Crosse virus in Georgia, 1982.

Eight cases of central nervous system infection due to La Crosse (California serogroup) virus were detected in Georgia children in 1982. The patients were residents of the state, without histories of travel away from the vicinities of their homes during the 30 days before the onset of their illnesses. All were hospitalized with a diagnosis of aseptic meningitis or encephalitis; none had sequelae. Onset dates were from May 1 to Aug 30. Distribution of cases within the state suggested that active transmission of La Crosse virus occurred at least in the southeast quadrant of the state. The epidemiology and natural cycle of La Crosse in the southeastern United States are briefly discussed.

Dispersion of gravid Aedes triseriatus (Diptera: Culicidae) from woodlands into open terrain.

Activity of gravid Aedes triseriatus (Diptera: Culicidae) was monitored throughout the summer of 1981 in southwestern Wisconsin, USA, using ovitraps in open terrain such as fields between woodlots. Ovitraps were placed at various distances from the woods in various field types, including corn, alfalfa, pasture, mixed plantings, fallow old fields, and an open yard. Weekly ovitrap checks showed that Ae. triseriatus readily dispersed into all types of open areas; however, field type influenced the amount of oviposition activity observed. Dispersion of gravid females was lowest into alfalfa and corn fields, with only 4.3 and 7.6% of ovitrap observations positive for eggs, respectively. Dispersion was greater into wet meadow, old field, and open yard, as 17.9, 33.9, and 40% of ovitrap observations were positive for eggs, respectively. Distance between woodlots did not appear to influence dispersion rates, as a total of 14.5% of observations from all open-terrain ovitraps more than 100 m from woods were positive for eggs. In 1 pastureland site, ovitraps placed 273 m from the nearest woods were positive for eggs in 27.5% of trap observations. Ovitraps placed in fencerows or on isolated trees in open areas were more attractive to ovipositing mosquitoes than traps placed on wooden stakes in all open terrain habitats. Adult male and female Ae. triseriatus were observed in open terrain on a number of occasions. The effect of open-terrain dispersion by Ae. triseriatus on the epidemiology of La Crosse encephalitis virus is discussed.

Oral and Transovarial Transmission of la Crosse Virus by Aedes Atropalpus

A recently colonized strain of Aedes atropalpus was shown to be equally susceptible to oral infection with La Crosse (LAC) virus as the natural vector, Aedes triseriatus. La Crosse virus was shown to replicate and persist at high titers in orally infected Ae. atropalpus. In addition, orally infected females transmitted virus efficiently to suckling mice by mosquito bite. Transovarial transmission of LAC virus was demonstrated by recovery of virus from larval progeny after parental females were infected either orally or by intrathoracic inoculation. Virus was recovered from larval progeny that developed from eggs produced both autogenously and anautogenously. Transovarial transmission of LAC virus to F1 adults was also shown, and the filial infection rates were similar to that obtained with a strain of Ae. triseriatus used for comparison.

Enhanced neutralization of La Crosse virus by the binding of specific pairs of monoclonal antibodies to the G1 glycoprotein

Monoclonal antibodies specific for eight antigenic sites on the G1 glycoprotein of La Crosse virus were studied in a kinetics of neutralization assay. When examined at the same protein concentration, there were differences in the rates and degree of neutralization induced by these antibodies. The maximum amount of neutralization detected in antibody excess ranged from 1.5 to 3 logs. Another 2 to 3 logs of viral infectivity could be neutralized by the addition of polyclonal anti-G1 antibody or a second monoclonal antibody. However, only certain pairs of monoclonal antibodies enhanced the neutralization the additional 2 to 3 logs. The antibody pairs enhancing neutralization were those also found to be synergistic in a competition binding assay ( L. Kingsford, L. D. Ishizawa, and D. W. Hill, 1983, Virology, 129, 443–455) as well as any neutralizing antibody used in combination with antibodies to epitopes within one particular site on the glycoprotein. The data indicate that antibody binding to two different specific sites on G1 is needed to neutralize La Crosse virus to the same extent that is observed with polyclonal antibody.

Aedes Triseriatus (Diptera: Culicidae) and La Crosse Virus III. Enhanced Oral Transmission by Nutrition-Deprived Mosquitoes1

Adult Aedes triseriatus , reared as larvae under a dietary regimen that produced 3 distinct body size classes [small (S), normal (N), and large (L)], were orally infected with La Crosse (LAC) virus. In all trials female mosquitoes that had endured the severest nutritional deprivation as larvae (S class) transmitted LAC virus at rates considerably higher than the mosquitoes from the moderately deprived N and nondeprived L classes (mean transmission rates of 82, 54, and 52%, respectively). While the S class mosquitoes ingested a proportionally larger infectious blood meal in relation to dry weight than the other 2 classes, the actual amount of virus ingested varied only about 0.4 log10 plaque-forming units/0.1 mg dry body weight among the 3 size classes. Thus, while enhanced oral transmission may be dose related to some extent, it is also apparent that adult mosquitoes nutritionally deprived as larvae may be physiologically less resistant to infection by LAC virus than less deprived individuals. These results indicate that virus transmission experiments conducted with adult mosquitoes reared on optimal larval diets in the laboratory may not reflect the vector capacity found in field populations. Consequently, epidemiologically important variables such as oral transmission rates may have been seriously underestimated in many previous studies.

Characterization of La Crosse virus small-genome transcripts.

With restriction fragments from DNA clones of the La Crosse virus S genome segment, the 3' end of the S mRNA was located by S1 nuclease mapping near a polyuridine tract, approximately 100 nucleotides, from the end of the S genome. Genome replication in La Crosse virus-infected cells was abolished by the drug cycloheximide, similar to other negative-strand RNA viruses. However, the synthesis of S mRNA could not be detected in cells pretreated with cycloheximide, suggesting that ongoing protein synthesis is required for La Crosse virus genome transcription and replication. Primer extension experiments in the presence of chain-terminating nucleoside triphosphates demonstrated that the 5' end of the La Crosse virus S mRNA begins 10 to 14 nucleotides before the 3' end of the S genome segment, suggesting that the La Crosse virus S mRNA is initiated on a host primer. A hypothesis consistent with these unexpected findings is presented.

La crosse bunyavirus can mediate pH-dependent fusion from without

Lipid binding properties which are dependent on exposure to acid pH are an important mechanism for the cellular entry pathway for some enveloped viruses and for other macromolecules. Cell-to-cell fusion can be used to demonstrate this function. La Crosse virus, a member of the family Bunyaviridae, fused BHK-21 cells from without (FFWO) upon exposure of the absorbed virus to pH 6.3 or below. A high multiplicity of infection and temperature of 37° were necessary for optimum fusion. The pH requirement was similar to that reported for Semliki forest virus, an alphavirus, but higher than that required for the demonstration of cell fusion by members of the Orthomyxoviridae. Virus inactivated by ultraviolet light also mediated FFWO. This is the first report of pH-mediated fusion for this family of viruses and it suggests, by analogy with other viruses, that their entry pathway includes exposure to acid.

Rapid Diagnosis of Viral Infections in the Central Nervous System

Rapid diagnosis of viral infections in the central nervous system has become increasingly important. Antiviral treatment, prevention of spread of disease and differentiation from infections caused by agents sensitive to antibiotics may be the important consequences of a virus specific diagnosis gained early in the disease. The diagnosis can be obtained by detection of virus or viral antigen in the human specimen: herpes simplex virus by electron microscopy, immunofluorescence or immunosorbent assays in brain biopsies; rabies virus by immunofluorescence in corneal cells or skin and mucous membranes. The presence of measles or influenza antigens in nasopharyngeal secretions, shown by immunofluorescence or enzyme immunoassays, may diagnose an encephalitis caused by either of these viruses. Where suitable material is not available the detection of virus-specific IgM in a single serum specimen may be used for diagnosis. Mumps specific IgM activity is detected by enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence techniques; tick-borne encephalitis (TBE) specific IgM by immunosorbent assays or by reduction of hemagglutination-inhibition (HI) titer by 2-mercaptoethanol treatment of serum. Reports have been given on the detection of IgM activity by ELISA also in other arboviral infections such as Japanese and LaCrosse encephalitis. The demonstration of an intrathecal production of virus-specific immunoglobulins may reveal the type of virus causing the infection in the central nervous system.

Localized conserved regions of the S RNA gene products of bunyaviruses are revealed by sequence analyses of the Simbu serogroup Aino virus

The complete nucleotide sequence has been determined for the S RNA of Aino virus, a member of the Simbu serogroup ( Bunyavirus genus, family Bunyaviridae). The S RNA is 850 nucleotides long (2.76 × 10 5 dallons) and in the viral complementary sequence has a short 5' non-coding region of 34 nucleotides and a more extensive 3' non-coding region of 117 nucleotides. The 3'-5' complementarity of the Aino S RNA is about 25 residues long, depending on the arrangement. The Aino sequence predicts that, like snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses (Bishop D.H.L., et al. (1982) Nucleic Acids Res., 10, 3703–3713; Akashi H. and Bishop D.H.L. (1983) J. Virol. 45, 1155–1158), there are two S coded gene products, a nucleoprotein N, and a non-structural protein, NS S, that are read from overlapping reading frames in the viral complementary sequence. The Aino N primary gene product is composed of 233 amino acids (26.2 × 10 3 daltons) and is 45% homologous in sequence with that of LAC virus. The NS S protein of Aino virus is composed of 91 amino acids (10.5 × 10 3 daltons) and is 35% homologous in sequence with the LAC NS S protein. Unlike those viruses there are no uridylate tracts longer than 4 residues in the 5' non-coding region of the S viral RNA that could function as a template for polyadenylation of Aino S mRNA species.

A variant of La Crosse virus attenuated for Aedes triseriatus mosquitoes.

A plaque-purified variant was cloned from prototype La Crosse (LAC) virus. This variant (PP-31) was lethal to suckling mice by the intracerebral route, produced "wild-type" plaques in Vero and BHK-21 cells, and grew to high titers (greater than 10(7) PFU/ml) in suckling mice and in cell culture. The variant was able to orally infect the vector, Aedes triseriatus; however, it was unable to escape infected midgut cells and disseminate to secondary target organs. Large, atypical, focal accumulations of viral antigen were detected in these midguts by immunofluorescence. Orally infected mosquitoes were unable to transmit virus by bite to suckling mice or vertically to their progeny. Even after inoculation of the variant virus into mosquitoes, there appeared to be a restriction on cell to cell virus movement. The role such variants may play in the modulation of infection in an arthropod vector is discussed.

The effect of proteolytic cleavage of La Crosse virus G1 glycoprotein on antibody neutralization.

The envelope of the bunyavirus La Crosse contains two glycoproteins, G1 (120 000 mol. wt.) and G2 (38 000 mol. wt.). When incubated with trypsin or plasmin, the G1 glycoprotein of virus grown in cell culture was cleaved, leaving two different sized polypeptides in the envelope (67 000 and 95 000 mol. wt.). Chymotrypsin cleaved G1 leaving polypeptides of 70 000 and 100 000 mol. wt. G2, however, was not altered by these enzymes. When used in antibody neutralization studies, these proteolytically modified viruses were neutralized approximately 1 to 2 log10 units in 60 min while control virus was neutralized by over 4 log10 units in 20 min. Because antibody to G1, but not G2, was involved in La Crosse virus neutralization, cleavage of G1 appeared to be directly responsible for these altered kinetics of neutralization. Antibody did bind to the polypeptides remaining associated with the envelope resulting in infectious virus-antibody complexes. This indicated that a critical site in terms of antibody neutralization was removed from G1 by proteolytic enzymes.

Quantitation of La Crosse virus in venereally infected Aedes triseriatus.

The replication of two strains of La Crosse (LAC) virus, prototype and an unpassaged field isolate, was followed in intrathoracically inoculated male and venereally infected female Aedes triseriatus mosquitoes. The venereal infection rate following induced mating was 13.6% of 44 females inseminated by males inoculated with prototype virus, and 26.3% of 38 females inseminated by males inoculated with an unpassaged field strain. The males consistently contained concentrations of virus in reproductive tracts equal to or greater than those in salivary glands (SG). In contrast, venereally infected females generally contained the highest concentrations of virus in SG. Prototype LAC virus was demonstrable in nearly all organs of the infected females by 10 days following mating; the unpassaged field strain was detectable in all organ systems tested by day 6. There appeared to be no significant differences in replication between the prototype and field isolate viruses.

Biological activities of monoclonal antibodies reactive with antigenic sites mapped on the G1 glycoprotein of la crosse virus

Monoclonal antibodies have been prepared which are specific for the G1 glycoprotein of La Crosse virus. By competitive radioimmunoassay, 20 IgG-producing clones were found to map in eight antigenic sites; three distinct and five which showed individual patterns of partial competition indicating they may be in close proximity. Unique in situ trypsin cleavage sites on G1 have helped in orienting these defined epitopes relative to the viral membrane. Antibody molecules belonging to one epitope (H) mapped on the trypsin-resistant part of G1 and had negative or extremely low neutralizing and hemagglutination inhibition activities. Seven epitopes were located on the trypsin-sensitive part of G1, a 25,000-Da region which is probably the amino terminus of the protein. Antibodies binding to six of these seven epitopes (A, B, D, E, F, and G) were positive for neutralization and inhibition of hemagglutination, but exhibited a wide range of activities. Epitopes A, F, and G seem to be in an immunodominant region containing the primary site(s) for attachment to cell receptors. Antibody specific for the remaining epitope (C) was unique in that it bound to a site closely adjacent to neutralizing antibody sites, enhanced antibody binding to epitopes A and G, but lacked the capacity to neutralize viral infectivity or inhibit hemagglutination. Enhancement of antibody binding also occurred between two other closely adjacent sites (B and D) and one other distinct epitope (G). In addition, antibody from an IgM-producing clone competed with antibodies to these same four epitopes (B, C, D, and G), indicating they are in close proximity. These data have been used to construct an antigenic map that may now be used as a working model for the study of virus neutralization.

Monoclonal antibodies against La Crosse virus.

Monoclonal antibodies have been used to show that an epitope is present on the G1 glycoprotein of prototype La Crosse virus that is absent or significantly altered on several isolates of La Crosse virus made in New York State, U.S.A. The portion of the G1 protein where this epitope is located plays a role in both virus neutralization and haemagglutination. Additional experiments revealed that under the appropriate assay conditions the monoclonal antibodies permitted discrimination between representatives of the North American members of the California serogroup.

Evaluation of Immunofluorescence and Cell Culture Techniques for Detecting La Crosse Virus Infection in Individual Aedes Triseriatus (Diptera: Culicidae)1

Two methods, (1) the indirect immunofluorescent antibody technique (IFAT) using head squashes, and (2) the method of tissue trituration and inoculation of supernatant into a susceptible assay system, were evaluated for detection of La Crosse (LAC) virus infection of individual Aedes triseriatus . Twenty-one days after fully engorging on an infectious blood meal containing LAC virus, 63% (119/189) of the control group females transmitted virus to suckling mice; virus infection persisted in 97% (184/189) of the control group females. The test group females were cut into 3 parts (head, anterior thorax, abdomen) to determine the extent of viral infection using IFAT on head squashes and the trituration method on thoracic and abdominal tissues. Head-squash I FAT indicated 71% (52/73) of the test group had disseminated infections; 21% (15/73) had nondisseminated infections. Exclusive use of head-squash IFAT would have indicated a 3 to 24% lower infection rate than the trituration method. However, judicious use of the IFAT will save time and reduce the cost of assay, since only mosquitoes with negative head squashes need be further assayed; in this study a 70% reduction in individual trituration assays would have been realized. A comparison of test and control groups indicated disseminated infections could not be directly equated with the probability of virus transmission.

Multiple leader RNAs and messenger RNAs are transcribed from the la crosse virus small genome segment

Nucleotide sequencing has demonstrated that the Small genome segment of Bunyaviruses contains the genetic information for two viral proteins (N and NS s) in overlapping reading frames ( Akashi and Bishop, 1983; Cabradilla et al., in press). Using 3′ end-labeled genome probes, La Crosse virus (LAC) infected cells were shown to contain three leader RNAs, which start at position 1 and terminate at approximate positions 74, 95, and 115 from the 3′ end of the genome. Primer extension and S1 mapping studies have shown that LAC-infected cells contain seven mRNAs, five of which initiate at positions 1, 74, 101, 117, and 123 and two more which initiate at approximate positions 147 and 159 from the 3′ end of the Small genome segment. The existence of multiple leader RNAs and mRNAs from the Small genome segment may allow access to both overlapping reading frames by ribosomes that still initiate only at 5′ proximal AUGs, without the need to splice the mRNA.

Occurrence of La Crosse (California Serogroup) Encephalitis Viral Infections in Illinois *

La Crosse (LAC) virus (California encephalitis serogroup) was identified as the etiologic agent of disease in 61 cases of California encephalitis (CE) that occurred in Illinois between 1976 and 1980. This group represented 54% of all 114 laboratory-diagnosed cases of CE between 1966 and 1980. Key epidemiologic observations included: 88% of the 61 cases had onset in July, August, or September; the 5- to 9-year-old age group represented 48% of the cases; the male:female sex ratio was 2:1; 97% of the cases occurred in the northern half of the state; and 61% of the cases occurred in Peoria County. Four instances of probable common source of exposure were reported. Average length of hospitalization was 8.3 days (range 3-31 days). Hospitalization costs were estimated at $3,967-$5,750 per case. An "average" year in Illinois yields 12 cases of LAC encephalitis with direct hospitalization costs of $47,604-$69,000.

Molecular cloning and sequencing of the La Crosse virus S RNA

Complete double-stranded DNA copies of the La Crosse virus (LAC) S genome have been synthesized and cloned into plasmid pBR322. The cloned genome was characterized and sequenced. The LAC S genome consisted of 981 nucleotides and contained two overlapping open reading frames. The first reading frame begins at nucleotide 82 and encodes a protein of 235 amino acids. A polypeptide of 92 amino acids can be translated in a +1 reading frame 16 nucleotides downstream from the start of the first reading frame. This second reading frame is initiated with two AUG codons followed by the serine codon UCG, the same serine codon which immediately follows the AUG of the first reading frame.