Throw out the Map: Neuropathogenesis of the Globally Expanding California Serogroup of Orthobunyaviruses.

The California serogroup (CSG) comprises 18 serologically and genetically related mosquito-borne orthobunyaviruses. Of these viruses, at least seven have been shown to cause neurological disease in humans, including the leading cause of pediatric arboviral encephalitis in the USA, La Crosse virus. Despite the disease burden from these viruses, much is still unknown about the CSG viruses. This review summarizes our current knowledge of the CSG viruses, including human disease and the mechanisms of neuropathogenesis.

Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

Emerging arboviruses in Quebec, Canada: assessing public health risk by serology in humans, horses and pet dogs.

Periodic outbreaks of West Nile virus (WNV), Eastern equine encephalitis virus (EEEV) and to a lesser extent, California serogroup viruses (CSGV), have been reported in parts of Canada in the last decade. This study was designed to provide a broad assessment of arboviral activity in Quebec, Canada, by conducting serological surveys for these arboviruses in 196 horses, 1442 dogs and 485 humans. Sera were screened by a competitive enzyme linked immunosorbent assay and positive samples confirmed by plaque reduction neutralisation tests. The percentage of seropositive samples was 83·7%, 16·5%, 7·1% in horses, 18·8%, 0·6%, 0% in humans, 11·7%, 3·1%, 0% in adult dogs and 2·9%, 0·3%, 0% in juvenile dogs for CSGV, WNV and EEEV, respectively. Serological results in horses and dogs appeared to provide a meaningful assessment of risk to public health posed by multiple arboviruses.

The Seasonal Timing of Snowshoe Hare Virus Transmission on the Island of Newfoundland, Canada.

Arthropod-borne diseases negatively affect humans worldwide. Understanding the biology of the arthropod vectors and the pathogens they harbor, the arthropods are moving targets as a result of climate change, ecosystem degradation, species introductions, and increased human travel. Viruses within the California serogroup of the genus Orthobunyavirus (family Bunyaviridae) are among the mosquito-borne viruses of concern owing to their zoonotic potential. Two of these, snowshoe hare virus (SSHV) and Jamestown Canyon virus, were shown, using a combination of serology and virus isolations, to circulate on the Island of Newfoundland, Canada, in the 1980s. More recently, serological analysis demonstrated that these two viruses continue to circulate on the Island in several domesticated and wild animals. Here, we detected the seroconversion to SSHV in wild snowshoe hares and in a single sentinel rabbit. The seroconversion in the sentinel rabbit occurred in early August (2011), which corresponded to the weeks of peak mosquito collections and the timing of the detection of SSHV in suspected mosquito vectors. A portion of the SSHV S segment sequence was generated from mosquito pools collected at sites near the sentinel rabbits and phylogenetically analyzed using the neighbor-joining method with other available California serogroup virus sequences. This analysis validated the SSHV identification but showed that the Newfoundland sequence fell outside the other SSHV sequences available, which originated from the United States between 1959 and 2005.

Four emerging arboviral diseases in North America: Jamestown Canyon, Powassan, chikungunya, and Zika virus diseases.

Arthropod-borne viruses, or arboviruses, are viruses that are transmitted through the bites of mosquitoes, ticks, or sandflies. There are numerous arboviruses throughout the world capable of causing human disease spanning different viral families and genera. Recently, Jamestown Canyon, Powassan, chikungunya, and Zika viruses have emerged as increasingly important arboviruses that can cause human disease in North America. Unfortunately, there are currently no proven disease-modifying therapies for these arboviral diseases, so treatment is largely supportive. Given there are also no commercially available vaccines for these four arboviral infections, prevention is the key. To prevent mosquito or tick bites that might result in one of these arboviral diseases, people should wear long-sleeved shirts and pants while outside if feasible, apply insect repellant when going outdoors, using window screens or air conditioning to keep mosquitoes outside, and perform tick checks after being in wooded or brushy outdoor areas.

[Arborviruses of the California encephalitis serogroup 1N Russia and their contribution to infectious pathology].

This review focuses on arborviral infections associated with California serocomplex (Bunyaviridae, Orthobunyavirus). Results of relevant eco-epidemiological and clinical studies in Russia are presented suggesting the ubiquitious nature of diseases caused by viruses of the California encephalitis serocomplex (Inko, Tahyna, Snowshoe Hare). The etiologic structure of these diseases in taiga and mixed woods of the European part and Western Siberia is dominated by the Inco virus and in southern regions by Tahina. The diseases have a well apparent seasonal pattern (July-August) in agreement with the peak summer activity of the pathogens. Two clinical forms of pathology are distinguished, influenza-like and generalized, the latter affecting lungs, kidneys, liver, and CNS. The Inco virus plays a key role in pathogenesis of the generalized form affecting CNS.

[Biological and phylogenetic analysis of first isolate of Tahyna virus in China].

In 2006, the first Chinese Tahyna virus isolate (XJ0625) was obtained in Xinjiang province and human infection were found in the same region. In this study, cell culture, animal experiments, electron microscopy, immunofluorescence assay and cross neutralization tests were performed to see the cell susceptibility, animal pathogenicity, morphology and antigenic and other biological characteristics of XJ0625. In addition, molecular biology software was used to analyze the characteristics of molecular evolution. The results showed that BHK-21 cell line was susceptible to XJ0625 and the virus was lethal to suckling mice when injected by intracranial ways. Similar to the other Bunyavirus, Tahyna virus is spherical enveloped virus under electron microscopy. XJ0625 infected cells showed strong fluorescent signal and could be neutralized by immune asities fluid with immnity to protype Tahyna virus Bardos 92. The sequence of the S and M segments showed 91.8% and 81.9% homology with Bardos 92.

[Investigation of Tahyna virus infection among unknown fever cases in Xinjiang, China].

To investigate the infection status and the spatial distribution of Tahyna virus infection among unknown fever cases in Xinjiang, China. Sera samples of unknown fever cases from Kashi in southern Xin-jiang and Yili in northern Xinjiang were tested against Tahyna virus by IFA. Partial positive cases were tested against Tahyna virus/Snowshoe hare virus/Inkoo virus parrelled. Finally, 742 sera samples of unknown fever cases were collected from Kashi, Southern Xinjiang in 2007-2008, the positive rate of IgM antibody against Tahyna virus was 5.3%, the positive rate of IgG antibody against Tahyna virus was 18.3%. 222 sera samples of unknown fever cases were collected from Yili, Northern Xinjiang in 2008, no positive case of IgM antibody against Tahyna was found. 10 cases showed antibody neutralization against Tahyna virus by plaque reduction neutralization test. Our results demonstrate that there is current infection and past infection of Tahyna virus among Southern Xinjiang residents.

Bunyavirus superinfection and segment reassortment in transovarially infected mosquitoes.

Rapid evolution of bunyaviruses may occur by RNA segment reassortment between closely related viruses. Reassortment between viruses occurs in dually infected mosquitoes when two different viruses are simultaneously ingested or when the second virus is ingested within 2 days of the first virus. By 3 days after oral infection, interference to superinfection occurs, thus limiting the potential for evolution. Aedes triseriatus mosquitoes can also be transovarially infected (TI+) with LaCrosse (LAC) virus. In these studies, the potential for oral superinfection of TI+ mosquitoes was assessed. Approximately 20% of mosquitoes TI+ with either a temperature-sensitive LAC virus or wild-type (wt) LAC virus became superinfected by ingesting blood meals containing wt LAC or snowshoe hare (SSH) viruses. LAC virus TI+ mosquitoes superinfected with SSH virus were detected by blot hybridization or RT-PCR. Viruses from these mosquitoes were plaque purified and genotyped using RT-PCR. Reassortant genomes were detected in 2.3% of the viruses genotyped, and 4.0% of the genomes tested were diploid for one genome segment.

Adsorptive endocytosis of California encephalitis virus into mosquito and mammalian cells: a role for G1.

The G1 glycoprotein of California encephalitis (CE) virus plays a critical role in the infection of mosquito and mammalian cells. We found that CE virus enters baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cells by the endocytic pathway. Ammonium chloride, a lysosomotropic amine that prevents release of virus from endosomes, inhibited infection of both cell types when added within 10 min after viral adsorption. In addition, infected cells formed polykaryons when the extracellular pH was lowered to 6.3; optimal fusion occurred at pH 5.8 and 6.0 (C6/36 and BHK-21 cells, respectively). Two neutralizing G1 MAba, 6D5.5 and 7D4.5, inhibited low pH-induced syncytia formation without affecting viral attachment, suggesting a role for G1 in viral entry. Since viral fusion proteins have been demonstrated to undergo conformational changes at low pH, acid-induced changes in G1 and G2 were assessed. While both G1 and G2 demonstrated low pH-induced alterations in detergent binding, only G1 displayed an altered protease cleavage pattern at the fusion pH. These results indicate that the G1 protein of CE virus undergoes conformational changes necessary for low pH-mediated entry into both mosquito and mammalian cells.

Tropism of bunyaviruses: evidence for a G1 glycoprotein-mediated entry pathway common to the California serogroup.

The California serogroup is composed of antigenically and biologically related viruses within the Bunyavirus genus of the Bunyaviridae. We used a large panel of murine cells to study their tissue tropisms and found virtually identical patterns of viral replication among all of the members of this serogroup, in contrast to other members of the family (Bunyamwera, Cache Valley, and Punta Toro viruses). By analyzing the nonpermissive infections with both an RNA dot-blot and a virus binding assay, we determined that tropism for cultured cells was determined at the level of entry. A truncated soluble form of the La Crosse G1 glycoprotein (sG1) was expressed in a baculovirus system and, despite slight differences in glycosylation, was shown to resemble native G1 by immunoprecipitation with six monoclonal antibodies. sG1 bound to permissive but not to nonpermissive cell lines, as demonstrated by flow cytometry. The sG1 effectively blocked infection of permissive cell lines with all of the California serogroup viruses, but did not block infection of two other bunyaviruses. These results indicate that the California serogroup bunyaviruses share a common receptor on vertebrate cells which may differ from the receptor used by other Bunyaviridae and demonstrate that the G1 glycoprotein is the virus attachment protein. sG1 will be a useful reagent in the search for a putative receptor molecule.

Requirement for the G1 protein of California encephalitis virus in infection in vitro and in vivo.

A role for the large glycoprotein (G1) of California encephalitis (CE) virus was examined in the infection of baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cell lines and the mosquito Ae. dorsalis using G1 monoclonal antibodies (MAbs) and selective protein cleavage. Five MAbs neutralized CE viral infectivity in both cell lines. One MAb, 7D4.5, efficiently neutralized the peroral infection of Ae. dorsalis females fed CE virus in artificial bloodmeals. To determine if MAbs to G1 neutralized CE virus by sterically hindering the small glycoprotein (G2), portions of G1 were trypsinized, and viral infectivity was assayed in vivo and in vitro. Cleavage of G1 resulted in a complete loss of infectivity both in mosquitoes and in culture, even though a significant amount of G2 remained intact. The loss of infectivity by both neutralization with G1 MAbs and trypsinization indicates that the G1 protein of CE virus is required for infection of mosquito and mammalian cells in vitro and of mosquitoes by the peroral route.

Vector competence of alpine, Central Valley, and coastal mosquitoes (Diptera: Culicidae) from California for Jamestown Canyon virus.

Mosquitoes collected from alpine, Central Valley, and coastal habitats in California were evaluated for their vector competence for four strains of Jamestown Canyon (JC) virus. Three of the viral strains examined were isolated from alpine Aedes species collected in California, and one, the prototype JC virus, was isolated from Culiseta inornata (Williston) collected in Colorado. Alpine Aedes tahoensis Dyar, Ae. cataphylla Dyar, Ae. hexodontus Dyar, Ae. increpitus Dyar, Ae. clivis Lanzaro and Eldridge, and coastal Aedes washinoi Lanzaro and Eldridge were variably susceptible to alpine strains of JC virus. Infection rates ranged from 22 to 77%, and peroral transmission rates of the infected females ranged from 0 to 26%. The differences were related to both mosquito species and viral strain. Coastal populations of Cs. inornata, Ae. washinoi, and Ae. sierrensis (Ludlow) were incompetent vectors when fed an alpine strain of JC virus, whereas Ae. squamiger (Coquillet) and Ae. dorsalis (Meigen) were competent vectors. Peroral transmission rates following parenteral infection of females of most species were about twofold higher than those for perorally infected females. A population of Cs. inornata from the Central Valley was highly susceptible when fed an alpine strain of JCV and transmitted virus both horizontally and vertically. Alpine strains of JC virus also were transmitted vertically by Ae. tahoensis, Ae. washinoi, and Ae. squamiger following parenteral infection of females.

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.

Genomic and biologic analyses of snowshoe hare virus field and laboratory strains.

Low-passage field strains of snowshoe hare (SSH) virus (Bunyaviridae), the prototype SSH virus (originally isolated in Montana), and La Crosse (LAC) virus were compared serologically by plaque-reduction neutralization (PRNT) and molecularly by oligonucleotide fingerprinting (ONF). The PRNT and ONF results confirmed the identity of the field strains, although some differences in the fingerprints were observed. We have examined the RNA genome variability in the two field and three laboratory strains of SSH virus, using direct sequence analysis of selected RNase T1 oligonucleotides. Few changes were observed among three Montana prototype-derived laboratory isolates, although they have different passage histories. In contrast, the field isolates differed greatly from the laboratory strains. In addition, we have located several of the larger T1 oligonucleotides within the known sequence of the small and large RNA genome segments. We then compared the viruses for their ability to replicate in and be transmitted by Aedes triseriatus mosquitoes. The oral infection rates for LAC, the field isolates, and the SSH prototype, as determined by immunofluorescent examination of midgut tissues, were 100%, 82%, and 47%, respectively. All viruses were also transmissible from mosquitoes to mice.

Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment.

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.

Vertical and venereal transmission of California group viruses by Aedes triseriatus and Culiseta inornata mosquitoes.

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.

Effect of body size on the vector competence of field and laboratory populations of Aedes triseriatus for La Crosse virus.

Aedes triseriatus females collected as pupae from 2 tireyards and one woodlot were tested for susceptibility to La Crosse virus infection and ability to transmit the virus to suckling mice. Rates of disseminated infection and oral transmission were inversely correlated with mean body size. The smallest population developed disseminated infections and transmitted virus at significantly higher rates than the 2 larger populations. Adults derived from F1 eggs of the largest and smallest populations showed no significant differences in rates of disseminated infection and transmission, suggesting that the differences between the field populations were primarily caused by larval rearing conditions. When pupae of 2 strains of Ae. triseriatus were arranged into discrete size groups and tested for susceptibility to virus, an inverse relationship was observed between rate of disseminated infections and mean pupal wet weight. In contrast, Ae. hendersoni exhibited low transmission rates (7-10%) regardless of body size.

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.

Isolation of Jamestown Canyon virus (California serogroup) from Aedes mosquitoes in an enzootic focus in Michigan.

Twenty isolates of Jamestown Canyon virus were obtained from adult females of 5 Aedes species collected at the Houghton Lake Wildlife Research Area, Missaukee County, in north-central Michigan between 1985 and 1989. Fourteen were from Aedes provocans, and 6 were from 4 other snowmelt Aedes species. One isolate of trivittatus virus and one Cache Valley-like virus were also obtained. Seasonal succession patterns for numerous mosquito species were recorded over 4 years. The temporal association of adult mosquito emergence, virus isolations, and infection and seroconversion of sentinel deer suggest that Ae. provocans is a primary enzootic vector of Jamestown Canyon virus in that focus. We hypothesize that Ae. provocans provides an overwintering reservoir for Jamestown Canyon virus at the study site. A large dry ice-baited "tent trap" was the most productive method for collecting numerous aedine and other mosquito species.