La Crosse Encephalitis: A Persistent Arboviral Threat in North Carolina.

Mosquito-borne diseases remain a significant cause of economic, social, and health burdens in North Carolina. Although recently overshadowed by emerging threats such as chikungunya virus and Zika virus, La Crosse virus and other endemic arboviruses remain persistent environmental health hazards. Indeed, La Crosse virus, West Nile virus, and Eastern equine encephalitis virus accounted for more than 98% of the reported human arboviral diseases acquired in North Carolina in the past decade. Arbovirus infection is increasingly prevalent in Western North Carolina, with La Crosse encephalitis being endemic in this area. While infections are often asymptomatic and seldom fatal, the long-term neurologic sequelae of La Crosse encephalitis represent a significant burden.

A recombinant chimeric La Crosse virus expressing the surface glycoproteins of Jamestown Canyon virus is immunogenic and protective against challenge with either parental virus in mice or monkeys.

La Crosse virus (LACV) and Jamestown Canyon virus (JCV), family Bunyaviridae, are mosquito-borne viruses that are endemic in North America and recognized as etiologic agents of encephalitis in humans. Both viruses belong to the California encephalitis virus serogroup, which causes 70 to 100 cases of encephalitis a year. As a first step in creating live attenuated viral vaccine candidates for this serogroup, we have generated a recombinant LACV expressing the attachment/fusion glycoproteins of JCV. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. For the M segment, the open reading frame (ORF) of LACV is replaced with that derived from JCV and is flanked by the untranslated regions of LACV. The resulting chimeric virus retained the same robust growth kinetics in tissue culture as observed for either parent virus, and the virus remains highly infectious and immunogenic in mice. Although both LACV and JCV are highly neurovirulent in 21 day-old mice, with 50% lethal dose (LD50) values of 0.1 and 0.5 log10 PFU, respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 10³ PFU. Parenteral vaccination of mice with 10¹ or 10³ PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and induced neutralizing antibodies to JCV, LACV, and TAHV. When vaccinated monkeys were challenged with JCV, they were protected against the development of viremia. Generation of highly attenuated yet immunogenic chimeric bunyaviruses could be an efficient general method for development of vaccines effective against these pathogenic viruses.

DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells.

La Crosse virus (LACV)-mediated encephalitis is the most frequently reported arboviral disease in the United States, but to date no vaccine against this virus is available. We have established a new animal model, genetically targeted mice lacking a functional interferon type I receptor (IFNAR-1). These mice show an age-independent susceptibility to LACV and develop an acute encephalitis within 6 days of infection, thereby allowing the evaluation of vaccines against LACV. Taking advantage of this knockout mouse model, we have assessed the feasibility of DNA vaccination against this viral disease. Plasmid DNAs, encoding either the virus surface glycoproteins G1 and G2 or the internal nucleocapsid protein N, were used to immunize IFNAR-1-deficient mice. Mice vaccinated with DNA encoding the glycoproteins G1 and G2 produced neutralizing antibodies and exhibited a high degree of protection against challenge with high doses of LACV. Depletion of CD4+ T cells in mice vaccinated with DNA encoding G1/G2 reduced their capacity to control the infection. Virus titration and immunohistological analysis revealed that the protected mice showed no evidence of LACV particles in the brain. This indicates that the vaccine-induced immune response efficiently blocked viral spreading from the primary replication site to the brain. In contrast, immunization with DNA encoding protein N yielded only a partial protective effect that can be attributed to the cellular immune response. Taken together, this study shows that DNA vaccines can be designed to efficiently induce a protective immune response based on neutralizing antibodies and CD4+ T cells.

Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cytokines in mice.

BACKGROUND: DNA vaccines have been shown to induce protective immunity against viral infections in different animal models. We have recently demonstrated that DNA vaccine induced protective immunity against influenza A virus and La Crosse virus (LACV) is primarily mediated by humoral immune response. OBJECTIVE: The goal of this study was to investigate whether administration of DNA coding for cytokines such as interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF) could increase the protective immune response induced by vaccination with DNA coding for viral antigens. STUDY DESIGN: For the influenza A virus or LACV model, C57BL/6 or interferon-alpha/beta receptor (IFNAR-1)-deficient mice, respectively, were vaccinated once or twice with 100 micrograms of DNA encoding viral antigens. At the same time plasmid DNAs (100 micrograms) coding either for mouse GM-CSF or mouse IL-12 were administered. The mice were subsequently challenged with a lethal dose of influenza A virus or LACV and monitored for clinical symptoms (weight loss) and survival. RESULTS: To achieve a high degree of protection (70% survival) two injections of DNA encoding the influenza A virus surface protein hemagglutinin (HA) were required. Intriguingly, administration of DNA coding for IL-12 alone also led to a pronounced protective effect against virus challenge. Co-administration of DNAs encoding IL-12 and HA significantly increased the protective immunity against influenza A virus, while IL-12 expression did not improve protection upon vaccination with DNA coding for the internal nucleocapsid protein N of LACV. Co-injection of DNA coding for mouse GM-CSF and HA also showed an adjuvant effect. CONCLUSIONS: The data clearly indicate that co-administration of DNA encoding cytokines such as IL-12 and GM-CSF with DNA coding for viral antigens has adjuvant effects on the protective immune response against different viral pathogens.

Experimental infection of vertebrates of the Pocomoke Cypress Swamp, Maryland with Keystone and Jamestown Canyon viruses.

Experimental studies were conducted to assess the susceptibility of white-tailed deer (Odocoileus virginianus), gray squirrels (Sciurus carolinensis), and cottontail rabbits (Sylvilagus floridanus) to Jamestown Canyon (JC) and/or Keystone (KEY) virus infection. Viremia occurred in 5 of 6 deer inoculated with JC virus; however, all deer developed KEY virus neutralizing antibody. Based on the observation that antibody elicited by primary infection of deer with either KEY or JC virus exhibited partial heterologous neutralization in vitro, cross-challenge experiments were performed in these animals. Keystone virus failed to infect deer 30 days post primary JC virus infection; however, deer became infected when challenged with KEY virus 80 days after the initial JC virus infection as indicated by a substantial increase in antibody titer. Similarly, JC virus failed to produce viremia in immune animals infected with KEY virus 80 days previously, although 2 of the 3 animals challenged had serological evidence of infection. Three field-collected cottontail rabbits with no evidence of KEY antibody were readily susceptible to KEY virus infection and developed viremias of 1-4 days' duration; rabbits with KEY virus antibody did not develop viremia upon KEY virus challenge. Eight antibody-negative field-collected gray squirrels became viremic following injection with KEY virus; however, a comparable group of squirrels did not become viremic when injected with JC virus.

Using administrative medical claims data to supplement state disease registry systems for reporting zoonotic infections.

OBJECTIVE: To determine what, if any, opportunity exists in using administrative medical claims data for supplemental reporting to the state infectious disease registry system. MATERIALS AND METHODS: Cases of five tick-borne (Lyme disease (LD), babesiosis, ehrlichiosis, Rocky Mountain spotted fever (RMSF), tularemia) and two mosquito-borne diseases (West Nile virus, La Crosse viral encephalitis) reported to the Tennessee Department of Health during 2000-2009 were selected for study. Similarly, medically diagnosed cases from a Tennessee-based managed care organization (MCO) claims data warehouse were extracted for the same time period. MCO and Tennessee Department of Health incidence rates were compared using a complete randomized block design within a general linear mixed model to measure potential supplemental reporting opportunity. RESULTS: MCO LD incidence was 7.7 times higher (p<0.001) than that reported to the state, possibly indicating significant under-reporting (∼196 unreported cases per year). MCO data also suggest about 33 cases of RMSF go unreported each year in Tennessee (p<0.001). Three cases of babesiosis were discovered using claims data, a significant finding as this disease was only recently confirmed in Tennessee. DISCUSSION: Data sharing between MCOs and health departments for vaccine information already exists (eg, the Vaccine Safety Datalink Rapid Cycle Analysis project). There may be a significant opportunity in Tennessee to supplement the current passive infectious disease reporting system with administrative claims data, particularly for LD and RMSF. CONCLUSIONS: There are limitations with administrative claims data, but health plans may help bridge data gaps and support the federal administration's vision of combining public and private data into one source.

Larval habitat characterization for Aedes triseriatus (Say), the mosquito vector of LaCrosse encephalitis in West Virginia.

OBJECTIVE: The principal aim of this investigation was to provide quantitative information on the frequency of occurrence for Aedes triseriatus (Say) larvae in West Virginia at 3 broad elevation categories, in different months of the year, and in specific types of larval habitats. METHODS: Larvae were collected in 54 West Virginia counties over a 6-year period, and their occurrence was recorded for defined habitat categories. RESULTS: Aedes triseriatus larvae were collected at 206 of the 587 sites examined and in 48 of the 54 West Virginia counties sampled. CONCLUSION: Aedes triseriatus larvae are distributed equally across all elevation categories from March through October and are predisposed to shaded habitats.

La Crosse encephalitis: occurrence of disease and control in a suburban area.

Fifteen (6.4%) of 233 residents sampled in State Road Coulee (SRC) during 1972-73 had antibodies neutralizing La Crosse (LAC) virus, 9 (3.9%) trivittatus (TVT), 12 (5.2%) Jamestown Canyon (JC) and 2 (0.9%) Bunyamwera serogroup virus. Six of seven youths who had antibodies to LAC virus had been ill: three were serologically confirmed as cases of LAC encephalitis and three had possibly related illnesses. The other reported no illness. Possibly related illnesses were reported by 1 of 10 adults with antibody to LAC; also by 3 of 6 youths who had antibodies to TVT or JC viruses, and by 4 of 39 youths who did not have these antibodies. In prospective studies, 4 of 132 sampled again during 1977 had acquired antibodies, including 2 to LAC and 2 to JC virus. The LAC disease-infection rate was 50%. One of those with antibody to LAC virus was a girl with encephalitis who had a serologically confirmed infection a year after she had moved away from SRC. Her exposure to mosquitoes was not in SRC. The other was a boy who remained a resident but did not have encephalitis or other severe illness. The two who had acquired antibody to JC virus were boys who reported histories of undiagnosed febrile illnesses while attending summer camps in central Wisconsin. Control measures, including closure of treeholes, removing old tires and education, were applied. LAC infections in SRC were reduced from six during 1965 thru 1972, to only one during 1973 thru 1977. No cases have been found in SRC since 1977. Following expansion to a county-wide control program, LAC encephalitis in La Crosse County has been reduced from seven and eight cases during 1978 and 1979 to only one and two during 1980 and 1981. No cases have been found in La Crosse County so far during 1982.

DNA vaccination against La Crosse virus.

For the development of effective conventional vaccines or DNA vaccines against viruses, the availability of suitable animal models is an essential prerequisite. For many recently emerging zoonotic viruses, suitable animal models are still missing. We have established a novel small animal model for DNA vaccines using mice lacking a functional interferon-alpha/beta receptor (IFNAR-1). IFNAR-1-deficient mice are highly susceptible to many different viruses despite their ability to mount a normal humoral and cellular immune response. Taking advantage of this animal model, we show that mice can be completely protected from lethal challenge with a single injection of plasmid DNA encoding the viral envelope proteins G1 and G2. By contrast, vaccination with a plasmid encoding the internal nucleocapsid protein N had little effect. In an effort to enhance the protective immune response to N we assessed the efficacy of vaccination with plasmid DNA encoding N in combination with a plasmid encoding the cytokine IL-12 as adjuvant. IL-12 enhanced the survival of mice following viral challenge, but the effect was independent of N indicating the involvement of components of the innate immune system such as NK cells.

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.