Serological characterization of lineage II insect-specific flaviviruses compared with pathogenic mosquito-borne flaviviruses.

The genus Flavivirus includes pathogenic tick- and mosquito-borne flaviviruses as well as non-pathogenic insect-specific flaviviruses (ISFVs). Phylogenetic analysis based on whole amino acid sequences has indicated that lineage II ISFVs have similarities to pathogenic flaviviruses. In this study, we used reactive analysis with immune serum against Psorophora flavivirus (PSFV) as a lineage IIa ISFV, and Barkeji virus (BJV) as a lineage IIb ISFV, to evaluate the antigenic similarity among lineage IIa and IIb ISFVs, and pathogenic mosquito-borne flaviviruses (MBFVs). Binding and antibody-dependent enhancement assays showed that anti-PSFV sera had broad cross-reactivity with MBFV antigens, while anti-BJV sera had low cross-reactivity. Both of the lineage II ISFV antisera were rarely observed to neutralize MBFVs. These results suggest that lineage IIa ISFV PSFV has more antigenic similarity to MBFVs than lineage IIb ISFV BJV.

Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle.

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

Diverse mosquito-specific flaviviruses in the Bolivian Amazon basin.

The genus Flavivirus includes a range of mosquito-specific viruses in addition to well-known medically important arboviruses. Isolation and comprehensive genomic analyses of viruses in mosquitoes collected in Bolivia resulted in the identification of three novel flavivirus species. Psorophora flavivirus (PSFV) was isolated from Psorophora albigenu. The coding sequence of the PSFV polyprotein shares 60 % identity with that of the Aedes-associated lineage II insect-specific flavivirus (ISF), Marisma virus. Isolated PSFV replicates in both Aedes albopictus- and Aedes aegypti-derived cells, but not in mammalian Vero or BHK-21 cell lines. Two other flaviviruses, Ochlerotatus scapularis flavivirus (OSFV) and Mansonia flavivirus (MAFV), which were identified from Ochlerotatus scapularis and Mansonia titillans, respectively, group with the classical lineage I ISFs. The protein coding sequences of these viruses share only 60 and 40 % identity with the most closely related of known lineage I ISFs, including Xishuangbanna aedes flavivirus and Sabethes flavivirus, respectively. Phylogenetic analysis suggests that MAFV is clearly distinct from the groups of the current known Culicinae-associated lineage I ISFs. Interestingly, the predicted amino acid sequence of the MAFV capsid protein is approximately two times longer than that of any of the other known flaviviruses. Our results indicate that flaviviruses with distinct features can be found at the edge of the Bolivian Amazon basin at sites that are also home to dense populations of human-biting mosquitoes.

Novel monoclonal antibodies against Australian strains of negeviruses and insights into virus structure, replication and host -restriction.

We report the isolation of Australian strains of Bustos virus and Ngewotan virus, two insect-specific viruses in the newly identified taxon Negevirus, originally isolated from Southeast Asian mosquitoes. Consistent with the expected insect-specific tropism of negeviruses, these isolates of Ngewotan and Bustos viruses, alongside the Australian negevirus Castlerea virus, replicated exclusively in mosquito cells but not in vertebrate cells, even when their temperature was reduced to 34 °C. Our data confirmed the existence of two structural proteins, putatively one membrane protein forming the majority of the virus particle, and one glycoprotein forming a projection on the apex of the virions. We generated and characterized 71 monoclonal antibodies to both structural proteins of the two viruses, most of which were neutralizing. Overall, these data increase our knowledge of negevirus mechanisms of infection and replication in vitro.

Inactivation of Japanese encephalitis virus in plasma by methylene blue combined with visible light and in platelet concentrates by ultraviolet C light.

Japanese encephalitis virus (JEV) is endemic to tropical areas in Asia and the Western Pacific. It can cause fatal encephalitis, although most infected individuals are asymptomatic. JEV is mainly transmitted to humans through the bite of an infected mosquito, but can also be transmitted through blood transfusion. To manage the potential risk of transfusion transmission, pathogen inactivation (PI) technologies, such as THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems, have been developed. We examined the efficacy of these two PI systems to inactivate JEV. STUDY DESIGN AND METHODS: Japanese encephalitis virus-spiked plasma units were treated using the THERAFLEX MB-Plasma system (visible light doses, 20, 40, 60, and 120 [standard] J/cm2) in the presence of methylene blue at approximately 0.8 µmol/L and spiked platelet concentrates (PCs) were treated using the THERAFLEX UV-Platelets system (UVC doses, 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2). Samples were taken before the first and after each illumination dose and tested for infectivity using an immunoplaque assay. RESULTS: Treatment of plasma with the THERAFLEX MB-Plasma system resulted in an average of 6.59 log reduction in JEV infectivity at one-sixth of the standard visible light dose (20 J/cm2). For PCs, treatment with the THERAFLEX UV-Platelet system resulted in an average of 7.02 log reduction in JEV infectivity at the standard UVC dose (0.20 J/cm2). CONCLUSIONS: The THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems effectively inactivated JEV in plasma or PCs, and thus these PI technologies could be an effective option to reduce the risk of JEV transfusion transmission.

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.

Inactivation of yellow fever virus in plasma after treatment with methylene blue and visible light and in platelet concentrates following treatment with ultraviolet C light.

BACKGROUND: Yellow fever virus (YFV) is endemic to tropical and subtropical areas in South America and Africa, and is currently a major public health threat in Brazil. Transfusion transmission of the yellow fever vaccine virus has been demonstrated, which is indicative of the potential for viral transfusion transmission. An approach to manage the potential YFV transfusion transmission risk is the use of pathogen inactivation (PI) technology systems, such as THERAFLEX MB-Plasma and THERAFLEX UV-Platelets (Macopharma). We aimed to investigate the efficacy of these PI technology systems to inactivate YFV in plasma or platelet concentrates (PCs). STUDY DESIGN AND METHODS: YFV spiked plasma units were treated using THERAFLEX MB-Plasma system (visible light doses: 20, 40, 60, and 120 [standard] J/cm2 ) in the presence of methylene blue (approx. 0.8 µmol/L) and spiked PCs were treated using THERAFLEX UV-Platelets system (ultraviolet C doses: 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2 ). Samples were taken before the first and after each illumination dose and tested for residual virus using a modified plaque assay. RESULTS: YFV infectivity was reduced by an average of 4.77 log or greater in plasma treated with the THERAFLEX MB-Plasma system and by 4.8 log or greater in PCs treated with THERAFLEX UV-Platelets system. CONCLUSIONS: Our study suggests the THERAFLEX MB-Plasma and the THERAFLEX UV-Platelets systems can efficiently inactivate YFV in plasma or PCs to a similar degree as that for other arboviruses. Given the reduction levels observed in this study, these PI technology systems could be an effective option for managing YFV transfusion-transmission risk in plasma and PCs.

Taxonomy of the order Bunyavirales: second update 2018.

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Bunyavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).

New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

Ross River virus in Australian blood donors: possible implications for blood transfusion safety.

BACKGROUND: Emerging transfusion-transmissible pathogens, including arboviruses such as West Nile, Zika, dengue, and Ross River viruses, are potential threats to transfusion safety. The most prevalent arbovirus in humans in Australia is Ross River virus (RRV); however, prevalence varies substantially around the country. Modeling estimated a yearly risk of 8 to 11 potentially RRV-viremic fresh blood components nationwide. This study aimed to measure the occurrence of RRV viremia among donors who donated at Australian collection centers located in areas with significant RRV transmission during one peak season. STUDY DESIGN AND METHODS: Plasma samples were collected from donors (n = 7500) who donated at the selected collection centers during one peak season. Viral RNA was extracted from individual samples, and quantitative reverse transcription-polymerase chain reaction was performed. RESULTS: Regions with the highest rates of RRV transmission were not areas where donor centers were located. We did not detect RRV RNA among 7500 donations collected at the selected centers, resulting in a zero risk estimate with a one-sided 95% confidence interval of 0 to 1 in 2019 donations. CONCLUSION: Our results suggest that the yearly risk of collecting a RRV-infected blood donation in Australia is low and is at the lower range of previous risk modeling. The majority of Australian donor centers were not in areas known to be at the highest risk for RRV transmission, which was not taken into account in previous models based on notification data. Therefore, we believe that the risk of RRV transfusion transmission in Australia is acceptably low and appropriately managed through existing risk management, including donation restrictions and recall policies.

Discovery of a novel iflavirus sequence in the eastern paralysis tick Ixodes holocyclus.

Ixodes holocyclus, the eastern paralysis tick, is a significant parasite in Australia in terms of animal and human health. However, very little is known about its virome. In this study, next-generation sequencing of I. holocyclus salivary glands yielded a full-length genome sequence which phylogenetically groups with viruses classified in the Iflaviridae family and shares 45% amino acid similarity with its closest relative Bole hyalomma asiaticum virus 1. The sequence of this virus, provisionally named Ixodes holocyclus iflavirus (IhIV) has been identified in tick populations from northern New South Wales and Queensland, Australia and represents the first virus sequence reported from I. holocyclus.

Characterization of Fitzroy River Virus and Serologic Evidence of Human and Animal Infection.

In northern Western Australia in 2011 and 2012, surveillance detected a novel arbovirus in mosquitoes. Genetic and phenotypic analyses confirmed that the new flavivirus, named Fitzroy River virus, is related to Sepik virus and Wesselsbron virus, in the yellow fever virus group. Most (81%) isolates came from Aedes normanensis mosquitoes, providing circumstantial evidence of the probable vector. In cell culture, Fitzroy River virus replicated in mosquito (C6/36), mammalian (Vero, PSEK, and BSR), and avian (DF-1) cells. It also infected intraperitoneally inoculated weanling mice and caused mild clinical disease in 3 intracranially inoculated mice. Specific neutralizing antibodies were detected in sentinel horses (12.6%), cattle (6.6%), and chickens (0.5%) in the Northern Territory of Australia and in a subset of humans (0.8%) from northern Western Australia.

Characterization of non-lethal West Nile Virus (WNV) infection in horses: Subclinical pathology and innate immune response.

Most natural West Nile virus (WNV) infections in humans and horses are subclinical or sub-lethal and non-encephalitic. Yet, the main focus of WNV research remains on the pathogenesis of encephalitic disease, mainly conducted in mouse models. We characterized host responses during subclinical WNV infection in horses and compared outcomes with those obtained in a novel rabbit model of subclinical WNV infection (Suen et al. 2015. Pathogens, 4: 529). Experimental infection of 10 horses with the newly emerging WNV-strain, WNVNSW2011, did not result in neurological disease in any animal but transcriptional upregulation of both type I and II interferon (IFN) was seen in peripheral blood leukocytes prior to or at the time of viremia. Likewise, transcript upregulation for IFNs, TNFα, IL1ß, CXCL10, TLRs, and MyD88 was detected in lymphoid tissues, while IFNα, CXCL10, TLR3, ISG15 and IRF7 mRNA was upregulated in brains with histopathological evidence of mild encephalitis, but absence of detectable viral RNA or antigen. These responses were reproduced in the New Zealand White rabbits (Oryctolagus cuniculus) experimentally infected with WNVNSW2011, by intradermal footpad inoculation. Kinetics of the anti-WNV antibody response was similar in horses and rabbits, which for both species may be explained by the early IFN and cytokine responses evident in circulating leukocytes and lymphoid organs. Given the similarities to the majority of equine infection outcomes, immunocompetent rabbits appear to represent a valuable small-animal model for investigating aspects of non-lethal WNV infections, notably mechanisms involved in abrogating morbidity.

Reduction of Zika virus infectivity in platelet concentrates after treatment with ultraviolet C light and in plasma after treatment with methylene blue and visible light.

BACKGROUND: Zika virus (ZIKV) has emerged as a potential threat to transfusion safety worldwide. Pathogen inactivation is one approach to manage this risk. In this study, the efficacy of the THERAFLEX UV-Platelets system and THERAFLEX MB-Plasma system to inactivate ZIKV in platelet concentrates (PCs) and plasma was investigated. STUDY DESIGN AND METHODS: PCs spiked with ZIKV were treated with the THERAFLEX UV-Platelets system at 0.05, 0.10, 0.15, and 0.20 J/cm2 UVC. Plasma spiked with ZIKV was treated with the THERAFLEX MB-Plasma system at 20, 40, 60, and 120 J/cm2 light at 630 nm with at least 0.8 µmol/L methylene blue (MB). Samples were taken before the first and after each illumination dose and tested for residual virus. For each system the level of viral reduction was determined. RESULTS: Treatment of PCs with THERAFLEX UV-Platelets system resulted in a mean of 5 log reduction in ZIKV infectivity at the standard UVC dose (0.20 J/cm2 ), with dose dependency observed with increasing UVC dose. For plasma treated with MB and visible light, ZIKV infectivity was reduced by a mean of at least 5.68 log, with residual viral infectivity reaching the detection limit of the assay at 40 J/cm2 (one-third the standard dose). CONCLUSIONS: Our study demonstrates that the THERAFLEX UV-Platelets system and THERAFLEX MB-Plasma system can reduce ZIKV infectivity in PCs and pooled plasma to the detection limit of the assays used. These findings suggest both systems have the capacity to be an effective option to manage potential ZIKV transfusion transmission risk.

Discovery of new orbiviruses and totivirus from Anopheles mosquitoes in Eastern Australia.

Three new viruses classifiable within the Totivirus and Orbivirus genera were detected from Anopheles mosquito species collected in Eastern Australia. The viruses could not be isolated in C6/36 mosquito cell cultures but were shown to replicate in their mosquito hosts by small RNA analysis. The viruses grouped phylogenetically with other viruses recently detected in insects. These discoveries contribute to a better understanding of commensal viruses in Australian mosquitoes and the evolution of these viruses.

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.

A newly discovered flavivirus in the yellow fever virus group displays restricted replication in vertebrates.

A novel flavivirus, provisionally named Bamaga virus (BgV), was isolated from Culex annulirostris mosquitoes collected from northern Australia. Phylogenetic analysis of the complete nucleotide sequence of the BgV genome revealed it clustered with the yellow fever virus (YFV) group, and was most closely related to Edge Hill virus (EHV), another Australian flavivirus, with 61.9 % nucleotide and 63.7 % amino acid sequence identity. Antigenic analysis of the envelope and pre-membrane proteins of BgV further revealed epitopes common to EHV, dengue and other mosquito-borne flaviviruses. However, in contrast to these viruses, BgV displayed restricted growth in a range of vertebrate cell lines with no or relatively slow replication in inoculated cultures. There was also restricted BgV replication in virus-challenged mice. Our results indicate that BgV is an evolutionary divergent member of the YFV group of flaviviruses, and represents a novel system to study mechanisms of virus host-restriction and transmission.