ORCID Profile
0000-0002-1683-3555
Current Organisation
University of Queensland
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Virology | Invertebrate Biology | Microbiology |
Publisher: American Society for Microbiology
Date: 15-01-2018
DOI: 10.1128/JVI.01582-17
Abstract: The family Flaviviridae consists of four genera, Flavivirus , Pestivirus , Pegivirus , and Hepacivirus , and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo . Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against Flaviviridae viruses. IMPORTANCE The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinant Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals against Flaviviridae viruses.
Publisher: Wiley
Date: 18-01-2014
DOI: 10.1111/AJD.12012
Abstract: Trichodysplasia spinulosa is a rare polyomavirus-associated cutaneous eruption occurring in the setting of immunosuppression. Clinically it is characterised by multiple centrofacial folliculocentric papules with spinous protuberances. The histopathology is distinct and treatment with antiviral agents appears to be the most effective.
Publisher: Microbiology Society
Date: 07-2013
Abstract: 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 erse 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.
Publisher: Springer Science and Business Media LLC
Date: 20-03-2017
DOI: 10.1038/SREP44642
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 07-06-2017
DOI: 10.1038/S41598-017-03120-1
Abstract: 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.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 11-12-2019
DOI: 10.1126/SCITRANSLMED.AAX7888
Abstract: Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs). Chimeric BinJ/VIF-prME viruses remained replication defective in vertebrate cells but replicated with high efficiency in mosquito cells. Cryo-electron microscopy and monoclonal antibody binding studies illustrated that the chimeric BinJ/VIF-prME virus particles were structurally and immunologically similar to their parental VIFs. Pilot manufacturing in C6/36 cells suggests that high yields can be reached up to 10
Publisher: Springer Science and Business Media LLC
Date: 22-03-2015
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-12-2022
Abstract: All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.
Publisher: Microbiology Society
Date: 05-2015
DOI: 10.1099/VIR.0.000053
Abstract: West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1', a C-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice.
Publisher: Microbiology Society
Date: 07-2015
DOI: 10.1099/VIR.0.000097
Publisher: Springer Science and Business Media LLC
Date: 08-06-2021
DOI: 10.1038/S41467-021-23779-5
Abstract: The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical s le. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.
Publisher: Microbiology Society
Date: 09-2012
Abstract: The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNV NY99 ) and a weakly virulent Australian subtype (WNV KUN ). Five amino acid differences occur in WNV NY99 compared with WNV KUN (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNV NY99 prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNV KUN prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNV KUN prM to the same level as that of WNV NY99 , and enhanced the secretion of WNV KUN prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNV KUN infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.
Publisher: MDPI AG
Date: 29-05-2020
Abstract: Virulent strains of West Nile virus (WNV) are highly neuro-invasive and human infection is potentially lethal. However, no vaccine is currently available for human use. Here, we report the immunogenicity and protective efficacy of a vaccine derived from a chimeric virus, which was constructed using the structural proteins (prM and E) of the Kunjin strain of WNV (WNVKUN) and the genome backbone of the insect-specific flavivirus Binjari virus (BinJV). This chimeric virus (BinJ/WNVKUN-prME) exhibits an insect-specific phenotype and does not replicate in vertebrate cells. Importantly, it authentically presents the prM-E proteins of WNVKUN, which is antigenically very similar to other WNV strains and lineages. Therefore BinJ/WNVKUN-prME represents an excellent candidate to assess as a vaccine against virulent WNV strains, including the highly pathogenic WNVNY99. When CD1 mice were immunized with purified BinJ/WNVKUN-prME, they developed robust neutralizing antibody responses after a single unadjuvanted dose of 1 to 5 μg. We further demonstrated complete protection against viremia and mortality after lethal challenge with WNVNY99, with no clinical or subclinical pathology observed in vaccinated animals. These data suggest that BinJ/WNVKUN-prME represents a safe and effective WNV vaccine candidate that warrants further investigation for use in humans or in veterinary applications.
Publisher: Cold Spring Harbor Laboratory
Date: 17-09-2022
DOI: 10.1101/2022.09.15.22280012
Abstract: We conducted a prospective environmental surveillance study to investigate the air, surface, dust and water contamination of a room occupied by a patient infected with Monkeypox virus (MPXV) at various stages of his illness. The patient tested positive for MPXV from a throat swab and skin lesions. Environmental s ling was conducted in a negative pressure room with 12 unidirectional HEPA air changes per hour and daily cleaning of the surfaces. A total of 179 environmental s les were collected on days 7, 8, 13, and 21 of his illness. Air, surface, and dust contamination was highest during the first eight days of the illness, with a gradual decline to the lowest contamination level by day 21. Viable MPXV was isolated from surfaces and dust s les and no viable virus was isolated from the air and water s les. Inadequate research prevail on the mode of transmission for monkeypox virus. This study reports the findings of viable monkeypox virus from the patient environment, highlighting its implications for human health and impact on infection and prevention control measures.
Publisher: Springer Science and Business Media LLC
Date: 20-02-2017
DOI: 10.1007/S00705-017-3279-3
Abstract: Cacipacoré virus (CPCV) is a potential emerging virus classified in the genus Flavivirus, family Flaviviridae. In the present study, we present the genetic characterization of a CPCV isolated from ticks (Amblyomma cajennense) collected from a sick capybara (Hydrochoerus hydrochaeris) in São Paulo State, Brazil. The CPCV isolate shares the typical genomic organization of flaviviruses with 10,857 nucleotides in length and a single open reading frame of 10,284 nucleotides encoding a polyprotein of 3,427 amino acids. Phylogenetic analysis revealed that CPCV is unique, as a potentially tick-borne virus, in the Japanese encephalitis virus serogroup.
Publisher: Wiley
Date: 02-10-2018
DOI: 10.1002/JCB.27575
Abstract: Wealth of information gleaned from decades of high‐impact research work scientists have disentangled the complicated web of versatile regulators that underlie cancer development and progression. Use of structural biology approaches and functional genomics have helped us to gain new insights into complex nature of cancer, and it is now clear that genetic/epigenetic mutations, overexpression of oncogenes, inactivation of tumor suppressors, loss of apoptosis, and versatility of protein binding partners have contributory roles in carcinogenesis and metastatic spread. It is becoming progressively more understandable that reprogramming of gene expression during and nontranscriptional changes during cancer development and progression are initiated and controlled by deregulated signal transduction cascades, all of which collectively create an incalculable complexity. Data obtained through preclinical and clinical trials revealed that alterations in the targeted oncogenes and other downstream, and parallel pathways played a central role in the development of resistance against different therapeutics. Phytochemicals have regained limelight, and different natural products are currently being tested for efficacy in preclinical studies. Apigenin, a plant‐derived flavonoid has considerable pharmacological value and is reportedly involved in the regulation of different signaling cascades. In this review, we have attempted to summarize rapidly evolving understanding of molecular biologists and pharmacologists about the potential of apigenin in the regulation of deregulated signaling pathways in different cancers. We have emphasized on the regulation of WNT/β‐catenin and janus kinase/signal transducers and activators of transcription (JAK‐STAT) pathways. We also comprehensively discuss how apigenin restored apoptosis in tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐resistant cancers. The review also gives a snapshot of microRNAs (miRNAs) that regulate wide‐ranging biological processes, and it is now clear that each miRNA can control hundreds of gene targets. Apigenin was noted to upregulate miR‐520b and miR‐101 in different cancers to inhibit tumor growth. Moreover, apigenin‐induced apoptotic rate was significantly higher when used in combination with miR‐423‐5p inhibitors or miR‐138 mimics. Better comprehension of linear and integrated signaling pathways will be helpful in effective therapeutic targeting of deregulated signaling pathways to inhibit revent cancer.
Publisher: American Society for Microbiology
Date: 28-06-2017
DOI: 10.1128/MSPHEREDIRECT.00190-17
Abstract: The major complications of an ongoing Zika virus outbreak in the Americas and Asia are congenital defects caused by the virus’s ability to cross the placenta and infect the fetal brain. The ability to generate molecular tools to analyze viral isolates from the current outbreak is essential for furthering our understanding of how these viruses cause congenital defects. The majority of existing viral isolates and infectious cDNA clones generated from them have undergone various numbers of passages in cell culture and/or suckling mice, which is likely to result in the accumulation of adaptive mutations that may affect viral properties. The approach described herein allows rapid generation of new, fully functional Zika virus isolates directly from deep sequencing data from virus-infected tissues without the need for prior virus passaging and for the generation and propagation of full-length cDNA clones. The approach should be applicable to other medically important flaviviruses and perhaps other positive-strand RNA viruses.
Publisher: MDPI
Date: 07-2020
Publisher: Springer Science and Business Media LLC
Date: 22-09-2018
DOI: 10.1007/S00705-017-3561-4
Abstract: Rocio virus (ROCV) is an arbovirus belonging to the genus Flavivirus, family Flaviviridae. We present an updated sequence of ROCV strain SPH 34675 (GenBank: AY632542.4), the only available full genome sequence prior to this study. Using next-generation sequencing of the entire genome, we reveal substantial sequence variation from the prototype sequence, with 30 nucleotide differences amounting to 14 amino acid changes, as well as significant changes to predicted 3'UTR RNA structures. Our results present an updated and corrected sequence of a potential emerging human-virulent flavivirus uniquely indigenous to Brazil (GenBank: MF461639).
Publisher: Public Library of Science (PLoS)
Date: 27-02-2013
Publisher: Springer Science and Business Media LLC
Date: 07-05-2015
Publisher: Springer Science and Business Media LLC
Date: 26-03-2018
DOI: 10.1038/S41467-018-03662-6
Abstract: Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictu s mosquitoes. The “Sementis Copenhagen Vector” (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR −/− mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal lacental infection in female IFNAR −/− mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR −/− mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced “shot burden” for these often co-circulating arboviruses.
Publisher: MDPI AG
Date: 02-11-2017
DOI: 10.3390/V9110326
Publisher: Wiley
Date: 08-12-2016
DOI: 10.1111/TRF.13443
Abstract: 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 s les. Plasma s les 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 lification (TMA) blood screening assay (Grifols). All WNV strains used were detectable by RT-PCR after being spiked into plasma. Additionally, all viral spiked s les were reactive by WNV TMA. 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.
Publisher: Mary Ann Liebert Inc
Date: 12-2017
Abstract: 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 h ered 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 s les, 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 s les. Time-point s les 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.
Publisher: Microbiology Society
Date: 06-2015
DOI: 10.1099/VIR.0.000069
Abstract: 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 in idual 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 in idual 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.
Publisher: American Society for Microbiology
Date: 09-2014
DOI: 10.1128/JVI.01304-14
Abstract: 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 (WNV NY99 ), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNV KUN ), the African WNV-like Koutango virus (WNV KOU ), and a WNV-like isolate from Sarawak, Malaysia (WNV Sarawak ), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro . While WNV NY99 replicated to the highest levels in vitro , in vivo mouse challenge revealed that WNV KOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNV KOU - and WNV NY99 -infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNV KOU -infected mice. Enhanced virulence of WNV KOU 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 (WNV KOU ), that was more virulent than a known virulent lineage I virus, WNV NY99 . The enhanced virulence of WNV KOU 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.
Publisher: MDPI AG
Date: 03-10-2018
DOI: 10.3390/V10100541
Abstract: The recent emergence of Zika virus (ZIKV) in Brazil was associated with an increased number of fetal brain infections that resulted in a spectrum of congenital neurological complications known as congenital Zika syndrome (CZS). Herein, we generated de novo from sequence data an early Asian lineage ZIKV isolate (ZIKV-MY Malaysia, 1966) not associated with microcephaly and compared the in vitro replication kinetics and fetal brain infection in interferon α/β receptor 1 knockout (IFNAR1−/−) dams of this isolate and of a Brazilian isolate (ZIKV-Natal Natal, 2015) unequivocally associated with microcephaly. The replication efficiencies of ZIKV-MY and ZIKV-Natal in A549 and Vero cells were similar, while ZIKV-MY replicated more efficiently in wild-type (WT) and IFNAR−/− mouse embryonic fibroblasts. Viremias in IFNAR1−/− dams were similar after infection with ZIKV-MY or ZIKV-Natal, and importantly, infection of fetal brains was also not significantly different. Thus, fetal brain infection does not appear to be a unique feature of Brazilian ZIKV isolates.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 08-01-2021
Abstract: Flaviviruses are a group of RNA viruses that include the human pathogens dengue virus, Zika virus, and West Nile virus. The envelope protein (E) on the virus surface has been the target of vaccine development, but problems have arisen with antibodies against E, leading to enhanced infection. Now, Modhiran et al. and Biering et al. describe two different antibodies that bind to the flavivirus NS1 protein and prevent it from disrupting epithelial cells, which is associated with severe disease. Both antibodies cross-react with multiple flavivirus NS1 proteins. The antibodies reduce viremia and increase survival in mouse models of flavivirus disease. Both papers include structures of NS1 bound to an antibody, which give insight into the protective mechanism. Science , this issue p. 190 , p. 194
Publisher: Springer Science and Business Media LLC
Date: 18-03-2019
Publisher: Microbiology Society
Date: 02-2016
DOI: 10.1099/JGV.0.000356
Abstract: 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.
Publisher: Centers for Disease Control and Prevention (CDC)
Date: 08-2016
Publisher: Springer Science and Business Media LLC
Date: 05-05-2020
DOI: 10.1038/S41467-020-16086-Y
Abstract: Flaviviruses, including Zika virus (ZIKV), utilise host mRNA degradation machinery to produce subgenomic flaviviral RNA (sfRNA). In mammalian hosts, this noncoding RNA facilitates replication and pathogenesis of flaviviruses by inhibiting IFN-signalling, whereas the function of sfRNA in mosquitoes remains largely elusive. Herein, we conduct a series of in vitro and in vivo experiments to define the role of ZIKV sfRNA in infected Aedes aegypti employing viruses deficient in production of sfRNA. We show that sfRNA-deficient viruses have reduced ability to disseminate and reach saliva, thus implicating the role for sfRNA in productive infection and transmission. We also demonstrate that production of sfRNA alters the expression of mosquito genes related to cell death pathways, and prevents apoptosis in mosquito tissues. Inhibition of apoptosis restored replication and transmission of sfRNA-deficient mutants. Hence, we propose anti-apoptotic activity of sfRNA as the mechanism defining its role in ZIKV transmission.
Publisher: Microbiology Society
Date: 11-2019
DOI: 10.1099/JGV.0.001326
Abstract: 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.
Start Date: 06-2021
End Date: 07-2021
Amount: $808,584.00
Funder: Australian Research Council
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