Chimeric viruses of the insect-specific flavivirus Palm Creek with structural proteins of vertebrate-infecting flaviviruses identify barriers to replication of insect-specific flaviviruses in vertebrate cells.

Here we report the generation of novel chimeric flaviviruses, which express the prM and E proteins of either dengue or Zika viruses on the genomic backbone of Palm Creek virus (PCV), an insect-specific flavivirus. The chimeric virus particles were antigenically indistinguishable from their parental prM-E donors, but were unable to infect vertebrate cells. An additional chimera (PCV structural genes in the backbone of West Nile virus - WNV/PCV-prME) was also unable to infect vertebrate cells, but transfection with RNA from this virus resulted in detectable RNA replication and translation but no infectious virion production. These data suggest multiple blocks at the entry, RNA replication and assembly/release stages of insect-specific flavivirus (ISF) infection in vertebrate cells. Serial passaging of these chimeric viruses in mosquito cells identified amino acid substitutions that may lead to increased replication efficiency. These chimeric viruses provide unique tools to further dissect the mechanisms of the host restriction of ISFs.

Author Correction: Infectious DNAs derived from insect-specific flavivirus genomes enable identification of pre- and post-entry host restrictions in vertebrate cells.

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Differential Diagnosis of Flavivirus Infections in Horses Using Viral Envelope Protein Domain III Antigens in Enzyme-Linked Immunosorbent Assay.

In Australia, infection of horses with the West Nile virus (WNV) or Murray Valley encephalitis virus (MVEV) occasionally results in severe neurological disease that cannot be clinically differentiated. Confirmatory serological tests to detect antibody specific for MVEV or WNV in horses are often hampered by cross-reactive antibodies induced to conserved epitopes on the envelope (E) protein. This study utilized bacterially expressed recombinant antigens derived from domain III of the E protein (rE-DIII) of MVEV and WNV, respectively, to determine whether these subunit antigens provided specific diagnostic markers of infection with these two viruses. When a panel of 130 serum samples, from horses with known flavivirus infection status, was tested in enzyme-linked immunosorbent assay (ELISA) using rE-DIII antigens, a differential diagnosis of MVEV or WNV was achieved for most samples. Time-point samples from horses exposed to flavivirus infection during the 2011 outbreak of equine encephalitis in south-eastern Australia also indicated that the rE-DIII antigens were capable of detecting and differentiating MVEV and WNV infection in convalescent sera with similar sensitivity and specificity to virus neutralization tests and blocking ELISAs. Overall, these results indicate that the rE-DIII is a suitable antigen for use in rapid immunoassays for confirming MVEV and WNV infections in horses in the Australian context and warrant further assessment on sensitive, high-throughput serological platforms such as multiplex immune assays.

Infectious DNAs derived from insect-specific flavivirus genomes enable identification of pre- and post-entry host restrictions in vertebrate cells.

Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are mosquito-borne pathogens that cause significant human diseases. A novel group of insect-specific flaviviruses (ISFs), which only replicate in mosquitoes, have also been identified. However, little is known about the mechanisms of ISF host restriction. We report the generation of infectious cDNA from two Australian ISFs, Parramatta River virus (PaRV) and Palm Creek virus (PCV). Using circular polymerase extension cloning (CPEC) with a modified OpIE2 insect promoter, infectious cDNA was generated and transfected directly into mosquito cells to produce infectious virus indistinguishable from wild-type virus. When infectious PaRV cDNA under transcriptional control of a mammalian promoter was used to transfect mouse embryo fibroblasts, the virus failed to initiate replication even when cell entry steps were by-passed and the type I interferon response was lacking. We also used CPEC to generate viable chimeric viruses between PCV and WNV. Analysis of these hybrid viruses revealed that ISFs are also restricted from replication in vertebrate cells at the point of entry. The approaches described here to generate infectious ISF DNAs and chimeric viruses provide unique tools to further dissect the mechanisms of their host restriction.

Chimeric viruses between Rocio and West Nile: the role for Rocio prM-E proteins in virulence and inhibition of interferon-α/ß signaling.

Mosquito-transmitted flavivirus Rocio (ROCV) was responsible for an outbreak of encephalitis in the Ribeira Valley, located in the south coast of Sao Paulo State, Brazil, in 1975-1976. ROCV also causes fatal encephalitis in adult mice. Seroprevalence studies in humans, horses and water buffaloes in different regions of Brazil have suggested that ROCV is still circulating in the country, indicating the risk of re-emergence of this virus. West Nile virus (WNV) is also a mosquito-transmitted encephalitic flavivirus, however, WNV strains circulating in Australia have not been associated with outbreaks of disease in humans and exhibit low virulence in adult mice. To identify viral determinants of ROCV virulence, we have generated reciprocal chimeric viruses between ROCV and the Australian strain of WNV by swapping structural prM and E genes. Chimeric WNV containing ROCV prM-E genes replicated more efficiently than WNV or chimeric ROCV containing WNV prM-E genes in mammalian cells, was as virulent as ROCV in adult mice, and inhibited type I IFN signaling as efficiently as ROCV. The results show that ROCV prM and E proteins are major virulence determinants and identify unexpected function of these proteins in inhibition of type I interferon response.

Virulence and Evolution of West Nile Virus, Australia, 1960-2012.

Worldwide, West Nile virus (WNV) causes encephalitis in humans, horses, and birds. The Kunjin strain of WNV (WNVKUN) is endemic to northern Australia, but infections are usually asymptomatic. In 2011, an unprecedented outbreak of equine encephalitis occurred in southeastern Australia; most of the ≈900 reported cases were attributed to a newly emerged WNVKUN strain. To investigate the origins of this virus, we performed genetic analysis and in vitro and in vivo studies of 13 WNVKUN isolates collected from different regions of Australia during 1960-2012. Although no disease was recorded for 1984, 2000, or 2012, isolates collected during those years (from Victoria, Queensland, and New South Wales, respectively) exhibited levels of virulence in mice similar to that of the 2011 outbreak strain. Thus, virulent strains of WNVKUN have circulated in Australia for >30 years, and the first extensive outbreak of equine disease in Australia probably resulted from a combination of specific ecologic and epidemiologic conditions.

End-point disease investigation for virus strains of intermediate virulence as illustrated by flavivirus infections.

Viruses of intermediate virulence are defined as isolates causing an intermediate morbidity/mortality rate in a specific animal model system, involving specific host and inoculation parameters (e.g. dose and route). Therefore, variable disease phenotype may exist between animals that develop severe disease or die and those that are asymptomatic or survive after infection with these isolates. There may also be variability amongst animals within each of these subsets. Such potential variability may confound the use of time-point sacrifice experiments to investigate pathogenesis of this subset of virus strains, as uniformity in disease outcome is a fundamental assumption for time-course sacrifice experiments. In the current study, we examined the disease phenotype, neuropathology, neural infection and glial cell activity in moribund/dead and surviving Swiss white (CD-1) mice after intraperitoneal infection with various Australian flaviviruses, including West Nile virus (WNV) strains of intermediate virulence (WNVNSW2011 and WNVNSW2012), and highly virulent Murray Valley encephalitis virus (MVEV) isolates. We identified notable intragroup variation in the end-point disease in mice infected with either WNVNSW strain, but to a lesser extent in mice infected with MVEV strains. The variable outcomes associated with WNVNSW infection suggest that pathogenesis investigations using time-point sacrifice of WNVNSW-infected mice may not be the best approach, as the assumption of uniformity in outcomes is violated. Our study has therefore highlighted a previously unacknowledged challenge to investigating pathogenesis of virus isolates of intermediate virulence. We have also set a precedent for routine examination of the disease phenotype in moribund/dead and surviving mice during survival challenge experiments.

Detection of emergent strains of West Nile virus with a blood screening assay.

BACKGROUND: West Nile virus (WNV) is a threat to transfusion safety. WNV Kunjin strain (WNVKUN ) is endemic across parts of Australia; however, human infection is believed to be infrequent and is often associated with relatively minor symptoms. A virulent strain, closely related to WNVKUN (termed WNVNSW2011 ) was recently identified as the etiologic agent of encephalitis in Australian horses. The aim of this project was to investigate whether a commercially available WNV blood screening assay can detect different strains of WNVKUN , including the virulent WNVNSW2011 , in human blood donor samples. STUDY DESIGN AND METHODS: Plasma samples were spiked with four different strains of WNVKUN , as well as a prototype WNV strain, at high, medium, and low viral loads. Spiking was confirmed with real-time reverse transcription-polymerase chain reaction (RT-PCR), before testing with the Procleix WNV transcription-mediated amplification (TMA) blood screening assay (Grifols). RESULTS: All WNV strains used were detectable by RT-PCR after being spiked into plasma. Additionally, all viral spiked samples were reactive by WNV TMA. CONCLUSION: We experimentally demonstrate that a commercially available WNV blood screening assay can detect different strains of WNVKUN . Given that WNV can be transfusion transmissible, it is essential to confirm that emergent strains are detectable by existing blood screening methods.

The West Nile virus-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response.

UNLABELLED: The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II. IMPORTANCE: In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango virus (WNVKOU), that was more virulent than a known virulent lineage I virus, WNVNY99. The enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralizing antibody response. These findings provide new insights into the pathogenesis of West Nile virus.

Genetic divergence among members of the Kokobera group of flaviviruses supports their separation into distinct species.

The Kokobera virus group comprises mosquito-borne flaviviruses that cluster together phylogenetically. These viruses are unique to Australia and Papua New Guinea, and have been associated with a mild polyarticular disease in humans. Recent isolation of genetically diverse viruses within this group has prompted analysis of their genetic and phenotypic relationships. Phylogenetic analysis based on complete ORF, the envelope gene or the NS5/3' untranslated region supported the separation of the group into distinct species: Kokobera virus (KOKV), Stratford virus, New Mapoon virus, MK7979 and TS5273. Virulence studies in 3-week-old mice also provided the first evidence that a member of the KOKV group (MK7979) was neuroinvasive after intraperitoneal inoculation. In this context, our recent detection of KOKV group-specific antibodies in horses in the field suggests that these viruses should be considered in the epidemiology of flavivirus encephalitis in Australia.