Cindy S. E. Tan

Safety and immunogenicity of a delta inulin-adjuvanted inactivated Japanese encephalitis virus vaccine in pregnant mares and foals

In 2011, following severe flooding in Eastern Australia, an unprecedented epidemic of equine encephalitis occurred in South-Eastern Australia, caused by Murray Valley encephalitis virus (MVEV) and a new variant strain of Kunjin virus, a subtype of West Nile virus (WNVKUN). This prompted us to assess whether a delta inulin-adjuvanted, inactivated cell culture-derived Japanese encephalitis virus (JEV) vaccine (JE-ADVAX™) could be used in horses, including pregnant mares and foals, to not only induce immunity to JEV, but also elicit cross-protective antibodies against MVEV and WNVKUN. Foals, 74–152 days old, received two injections of JE-ADVAX™. The vaccine was safe and well-tolerated and induced a strong JEV-neutralizing antibody response in all foals. MVEV and WNVKUN antibody cross-reactivity was seen in 33% and 42% of the immunized foals, respectively. JE-ADVAX™ was also safe and well-tolerated in pregnant mares and induced high JEV-neutralizing titers. The neutralizing activity was passively transferred to their foals via colostrum. Foals that acquired passive immunity to JEV via maternal antibodies then were immunized with JE-ADVAX™ at 36–83 days of age, showed evidence of maternal antibody interference with low peak antibody titers post-immunization when compared to immunized foals of JEV-naïve dams. Nevertheless, when given a single JE-ADVAX™ booster immunization as yearlings, these animals developed a rapid and robust JEV-neutralizing antibody response, indicating that they were successfully primed to JEV when immunized as foals, despite the presence of maternal antibodies. Overall, JE-ADVAX™ appears safe and well-tolerated in pregnant mares and young foals and induces protective levels of JEV neutralizing antibodies with partial cross-neutralization of MVEV and WNVKUN.

Herpesvirus delivery to the murine respiratory tract

Herpesvirus transmission is sporadic, and infection may be asymptomatic or present only with secondary lesions after dissemination. Consequently host entry remains ill-understood. Experimental infections can be informative, but depend on inoculations that are inherently artificial and so need validation. Mice are a widely used experimental host. Alert mice inhale readily small (5 μl) liquid volumes, and Indian ink, luciferase or radiolabel delivered thus distributed to the nasopharynx and oropharynx. Murid Herpesvirus-4 or Herpes simplex virus type 1 delivered thus infected only the nose, arguing that host entry is nasal rather than oral. Marker or virus delivery to the lung depended on general anesthesia and a large inoculum volume (30 μl), and so needs further validation of physiological relevance. While lungs could be infected at lower doses than the upper respiratory tract, tracking experiments showed that nasal inocula pass mostly into the oropharynx, even when restricted to 1 μl. Thus, the relative inefficiency of experimental upper respiratory tract infection was attributable to limited liquid retention in this site. Nonetheless low volume intranasal delivery to alert mice provides a convenient way to model experimentally an apparently natural mode of herpesvirus host entry.

Natural exposure of horses to mosquito-borne flaviviruses in South-East Queensland, Australia

In 2011 an unprecedented epidemic of equine encephalitis occurred in south-eastern (SE) Australia following heavy rainfall and severe flooding in the preceding 2–4 months. Less than 6% of the documented cases occurred in Queensland, prompting the question of pre-existing immunity in Queensland horses. A small-scale serological survey was conducted on horses residing in one of the severely flood-affected areas of SE-Queensland. Using a flavivirus-specific blocking-ELISA we found that 63% (39/62) of horses older than 3 years were positive for flavivirus antibodies, and of these 18% (7/38) had neutralizing antibodies to Murray Valley encephalitis virus (MVEV), Kunjin virus (WNVKUN) and/or Alfuy virus (ALFV). The remainder had serum-neutralizing antibodies to viruses in the Kokobera virus (KOKV) complex or antibodies to unknown/untested flaviviruses. Amongst eight yearlings one presented with clinical MVEV-encephalomyelitis, while another, clinically normal, had MVEV-neutralizing antibodies. The remaining six yearlings were flavivirus antibody negative. Of 19 foals born between August and November 2011 all were flavivirus antibody negative in January 2012. This suggests that horses in the area acquire over time active immunity to a range of flaviviruses. Nevertheless, the relatively infrequent seropositivity to MVEV, WNVKUN and ALFV (15%) suggests that factors other than pre-existing immunity may have contributed to the low incidence of arboviral disease in SE-Queensland horses during the 2011 epidemic.

Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains

A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven individual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed a predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent.

An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein.

The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46-49 in the αA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modelling of these domains in West Nile virus (WNV) NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the αA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in human embryonic kidney cells with simian virus 40 large T antigen (HEK293T cells) and performed pulldown assays using an antibody to the flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically co-immunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the αA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the αA3 mutations had a significant effect on the MTase-POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase-POL interactions.

Role of enhanced vector transmission of a new West Nile virus strain in an outbreak of equine disease in Australia in 2011

BackgroundIn 2011, a variant of West Nile virus Kunjin strain (WNVKUN) caused an unprecedented epidemic of neurological disease in horses in southeast Australia, resulting in almost 1,000 cases and a 9% fatality rate. We investigated whether increased fitness of the virus in the primary vector, Culex annulirostris, and another potential vector, Culex australicus, contributed to the widespread nature of the outbreak.MethodsMosquitoes were exposed to infectious blood meals containing either the virus strain responsible for the outbreak, designated WNVKUN2011, or WNVKUN2009, a strain of low virulence that is typical of historical strains of this virus. WNVKUN infection in mosquito samples was detected using a fixed cell culture enzyme immunoassay and a WNVKUN- specific monoclonal antibody. Probit analysis was used to determine mosquito susceptibility to infection. Infection, dissemination and transmission rates for selected days post-exposure were compared using Fisher’s exact test. Virus titers in bodies and saliva expectorates were compared using t-tests.ResultsThere were few significant differences between the two virus strains in the susceptibility of Cx. annulirostris to infection, the kinetics of virus replication and the ability of this mosquito species to transmit either strain. Both strains were transmitted by Cx. annulirostris for the first time on day 5 post-exposure. The highest transmission rates (proportion of mosquitoes with virus detected in saliva) observed were 68% for WNVKUN2011 on day 12 and 72% for WNVKUN2009 on day 14. On days 12 and 14 post-exposure, significantly more WNVKUN2011 than WNVKUN2009 was expectorated by infected mosquitoes. Infection, dissemination and transmission rates of the two strains were not significantly different in Culex australicus. However, transmission rates and the amount of virus expectorated were significantly lower in Cx. australicus than Cx. annulirostris.ConclusionsThe higher amount of WNVKUN2011 expectorated by infected mosquitoes may be an indication that this virus strain is transmitted more efficiently by Cx. annulirostris compared to other WNVKUN strains. Combined with other factors, such as a convergence of abundant mosquito and wading bird populations, and mammalian and avian feeding behaviour by Cx. annulirostris, this may have contributed to the scale of the 2011 equine epidemic.

Detection of Antibodies to West Nile Virus in Horses, Costa Rica, 2004

We conducted a serosurvey for West Nile virus (WNV) infection in equines in Costa Rica in 2004. Antibodies to WNV were detected in 28% of the horses using an epitope blocking ELISA that is specific for WNV. WNV infection was confirmed for a subset of these sera by plaque reduction neutralization tests and Western blot. This is the first evidence of WNV activity in Costa Rica.

Type I Interferons and NK Cells Restrict Gammaherpesvirus Lymph Node Infection

ABSTRACT Gammaherpesviruses establish persistent, systemic infections and cause cancers. Murid herpesvirus 4 (MuHV-4) provides a unique window into the early events of host colonization. It spreads via lymph nodes. While dendritic cells (DC) pass MuHV-4 to lymph node B cells, subcapsular sinus macrophages (SSM), which capture virions from the afferent lymph, restrict its spread. Understanding how this restriction works offers potential clues to a more comprehensive defense. Type I interferon (IFN-I) blocked SSM lytic infection and reduced lytic cycle-independent viral reporter gene expression. Plasmacytoid DC were not required, but neither were SSM the only source of IFN-I, as IFN-I blockade increased infection in both intact and SSM-depleted mice. NK cells restricted lytic SSM infection independently of IFN-I, and SSM-derived virions spread to the spleen only when both IFN-I responses and NK cells were lacking. Thus, multiple innate defenses allowed SSM to adsorb virions from the afferent lymph with relative impunity. Enhancing IFN-I and NK cell recruitment could potentially also restrict DC infection and thus improve infection control. IMPORTANCE Human gammaherpesviruses cause cancers by infecting B cells. However, vaccines designed to block virus binding to B cells have not stopped infection. Using a related gammaherpesvirus of mice, we have shown that B cells are infected not via cell-free virus but via infected myeloid cells. This suggests a different strategy to stop B cell infection: stop virus production by myeloid cells. Not all myeloid infection is productive. We show that subcapsular sinus macrophages, which do not pass infection to B cells, restrict gammaherpesvirus production by recruiting type I interferons and natural killer cells. Therefore, a vaccine that speeds the recruitment of these defenses might stop B cell infection.

Type I Interferons Direct Gammaherpesvirus Host Colonization

Gamma-herpesviruses colonise lymphocytes. Murid Herpesvirus-4 (MuHV-4) infects B cells via epithelial to myeloid to lymphoid transfer. This indirect route entails exposure to host defences, and type I interferons (IFN-I) limit infection while viral evasion promotes it. To understand how IFN-I and its evasion both control infection outcomes, we used Mx1-cre mice to tag floxed viral genomes in IFN-I responding cells. Epithelial-derived MuHV-4 showed low IFN-I exposure, and neither disrupting viral evasion nor blocking IFN-I signalling markedly affected acute viral replication in the lungs. Maximising IFN-I induction with poly(I:C) increased virus tagging in lung macrophages, but the tagged virus spread poorly. Lymphoid-derived MuHV-4 showed contrastingly high IFN-I exposure. This occurred mainly in B cells. IFN-I induction increased tagging without reducing viral loads; disrupting viral evasion caused marked attenuation; and blocking IFN-I signalling opened up new lytic spread between macrophages. Thus, the impact of IFN-I on viral replication was strongly cell type-dependent: epithelial infection induced little response; IFN-I largely suppressed macrophage infection; and viral evasion allowed passage through B cells despite IFN-I responses. As a result, IFN-I and its evasion promoted a switch in infection from acutely lytic in myeloid cells to chronically latent in B cells. Murine cytomegalovirus also showed a capacity to pass through IFN-I-responding cells, arguing that this is a core feature of herpesvirus host colonization.

CD8+ T cell evasion mandates CD4+ T cell control of chronic gamma-herpesvirus infection

Gamma-herpesvirus infections are regulated by both CD4+ and CD8+ T cells. However clinical disease occurs mainly in CD4+ T cell-deficient hosts. In CD4+ T cell-deficient mice, CD8+ T cells control acute but not chronic lung infection by Murid Herpesvirus-4 (MuHV-4). We show that acute and chronic lung infections differ in distribution: most acute infection was epithelial, whereas most chronic infection was in myeloid cells. CD8+ T cells controlled epithelial infection, but CD4+ T cells and IFNγ were required to control myeloid cell infection. Disrupting the MuHV-4 K3, which degrades MHC class I heavy chains, increased viral epitope presentation by infected lung alveolar macrophages and allowed CD8+ T cells to prevent disease. Thus, viral CD8+ T cell evasion led to niche-specific immune control, and an essential role for CD4+ T cells in limiting chronic infection.