SARS-CoV and SARS-CoV-2 display limited neuronal infection and lack the ability to transmit within synaptically connected axons in stem cell-derived human neurons.

Sarbecoviruses such as SARS and SARS-CoV-2 have been responsible for two major outbreaks in humans, the latter resulting in a global pandemic. While sarbecoviruses primarily cause an acute respiratory infection, they have been shown to infect the nervous system. However, mechanisms of sarbecovirus neuroinvasion and neuropathogenesis remain unclear. In this study, we examined the infectivity and trans-synaptic transmission potential of the sarbecoviruses SARS and SARS-CoV-2 in human stem cell-derived neural model systems. We demonstrated limited ability of sarbecoviruses to infect and replicate in human stem cell-derived neurons. Furthermore, we demonstrated an inability of sarbecoviruses to transmit between synaptically connected human stem cell-derived neurons. Finally, we determined an absence of SARS-CoV-2 infection in olfactory neurons in experimentally infected ferrets. Collectively, this study indicates that sarbecoviruses exhibit low potential to infect human stem cell-derived neurons, lack an ability to infect ferret olfactory neurons, and lack an inbuilt molecular mechanism to utilise retrograde axonal trafficking and trans-synaptic transmission to spread within the human nervous system.

A chimeric vaccine protects farmed saltwater crocodiles from West Nile virus-induced skin lesions.

West Nile virus (WNV) causes skin lesions in farmed crocodiles leading to the depreciation of the value of their hides and significant economic losses. However, there is no commercially available vaccine designed for use in crocodilians against WNV. We tested chimeric virus vaccines composed of the non-structural genes of the insect-specific flavivirus Binjari virus (BinJV) and genes encoding the structural proteins of WNV. The BinJV/WNV chimera, is antigenically similar to wild-type WNV but replication-defective in vertebrates. Intramuscular injection of two doses of BinJV/WNV in hatchling saltwater crocodiles (Crocodylus porosus) elicited a robust neutralising antibody response and conferred protection against viremia and skin lesions after challenge with WNV. In contrast, mock-vaccinated crocodiles became viraemic and 22.2% exhibited WNV-induced lesions. This suggests that the BinJV/WNV chimera is a safe and efficacious vaccine for preventing WNV-induced skin lesions in farmed crocodilians.

A novel and highly divergent Canine Distemper Virus lineage causing distemper in ferrets in Australia.

Canine distemper virus (CDV) causes a highly contagious systemic infection in an array of animal species. In this study we report an outbreak of distemper in ferrets in two research facilities in Australia, caused by a novel lineage of CDV. While the CDV strain caused mainly mild symptoms in ferrets, histopathology results presented a typical profile of distemper pathology, with multi-system virus replication. Through the development of a discriminatory PCR, paired with full genome sequencing, we revealed that the outbreak was caused by a novel lineage of CDV. The novel CDV lineage was highly divergent, with less than 93% similarity across the H gene to other described lineages, including the vaccine strain, and diverged approximately 140-400 years ago. Enhanced surveillance to determine the prevalence of CDV in ferrets, dogs and other at-risk species is critical to better understand the presence and diversity of CDV in Australia currently.

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway.

Plasmodium falciparum, the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the host's main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here, we use both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that loss of PfA-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage. In contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.

Malaria is a disease spread by mosquitoes. When infected insects bite the skin, they inject parasites called Plasmodium into the host. The symptoms of the disease then develop when Plasmodium infect host red blood cells. These parasites cannot make the raw materials to build their own proteins, so instead, they digest haemoglobin ­ the protein used by red blood cells to carry oxygen ­ and use its building blocks to produce proteins. Blocking the digestion of haemoglobin can stop malaria infections in their tracks, but it is unclear how exactly Plasmodium parasites break down the protein. Researchers think that a group of four enzymes called aminopeptidases are responsible for the final stage in this digestion, releasing the amino acids that make up haemoglobin. However, the individual roles of each of these aminopeptidases are not yet known. To start filling this gap, Edgar et al. set out to study one of these aminopeptidases, called PfA-M17. First, they genetically modified Plasmodium falciparum parasites so that the levels of this aminopeptidase were reduced during infection. Without the enzyme, the parasites were unable to grow. The next step was to confirm that this was because PfA-M17 breaks down haemoglobin, and not for another reason. To test this, Edgar et al. designed a new molecule that could stop PfA-M17 from releasing amino acids. This molecule, which they called 'compound 3', had the same effect as reducing the levels of PfA-M17. Further analysis showed that the amino acids that PfA- M17 releases match the amino acids found in haemoglobin. Malaria causes hundreds of thousands of deaths per year. Although there are treatments available, the Plasmodium parasites are starting to develop resistance. Confirming the role of PfA-M17 provides a starting point for new studies by parasitologists, biologists, and drug developers. This could lead to the development of chemicals that block this enzyme, forming the basis for new treatments.

Characterisation and natural progression of SARS-CoV-2 infection in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the infectious disease COVID-19, which has rapidly become an international pandemic with significant impact on healthcare systems and the global economy. To assist antiviral therapy and vaccine development efforts, we performed a natural history/time course study of SARS-CoV-2 infection in ferrets to characterise and assess the suitability of this animal model. Ten ferrets of each sex were challenged intranasally with 4.64 × 104 TCID50 of SARS-CoV-2 isolate Australia/VIC01/2020 and monitored for clinical disease signs, viral shedding, and tissues collected post-mortem for histopathological and virological assessment at set intervals. We found that SARS-CoV-2 replicated in the upper respiratory tract of ferrets with consistent viral shedding in nasal wash samples and oral swab samples up until day 9. Infectious SARS-CoV-2 was recovered from nasal washes, oral swabs, nasal turbinates, pharynx, and olfactory bulb samples within 3-7 days post-challenge; however, only viral RNA was detected by qRT-PCR in samples collected from the trachea, lung, and parts of the gastrointestinal tract. Viral antigen was seen exclusively in nasal epithelium and associated sloughed cells and draining lymph nodes upon immunohistochemical staining. Due to the absence of clinical signs after viral challenge, our ferret model is appropriate for studying asymptomatic SARS-CoV-2 infections and most suitable for use in vaccine efficacy studies.

In vitro characterisation of SARS-CoV-2 and susceptibility of domestic ferrets (Mustela putorius furo).

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

Evaluation of henipavirus chemical inactivation methods for the safe removal of samples from the high-containment PC4 laboratory.

Henipaviruses, Hendra (HeV) and Nipah (NiV), are highly pathogenic zoonotic agents that pose a serious health risk to human life, and as such are restricted to physical containment 4 (PC4) laboratories. For further analysis of virus-infected biological specimens, it is necessary to ensure absolute inactivation of any infectious virus present before removal from the PC4 laboratory. To evaluate the inactivation of HeV and NiV within infected samples, two chemical inactivation methods were assessed. Henipavirus-infected cell monolayers treated with 4 % paraformaldehyde (PFA) showed the complete inactivation of infectious virus, with an inactivation period of 15 min resulting in more than 8-log decrease in infectious titre. NiV-infected tissue samples treated with 10 % neutral-buffered formalin (NBF) showed a complete reduction of infectious virus in 7/8 ferret organs incubated for 24 h, with the remaining tissue demonstrating complete virus inactivation after 48 h. The chemical inactivation methods described herein evaluated two simple methods of henipavirus inactivation, resulting in the complete inactivation of infectious virus - an essential requirement for the safe removal and handling of biological samples from the PC4 laboratory.

ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets.

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

West Nile Virus: An Update on Pathobiology, Epidemiology, Diagnostics, Control and "One Health" Implications.

West Nile virus (WNV) is an important zoonotic flavivirus responsible for mild fever to severe, lethal neuroinvasive disease in humans, horses, birds, and other wildlife species. Since its discovery, WNV has caused multiple human and animal disease outbreaks in all continents, except Antarctica. Infections are associated with economic losses, mainly due to the cost of treatment of infected patients, control programmes, and loss of animals and animal products. The pathogenesis of WNV has been extensively investigated in natural hosts as well as in several animal models, including rodents, lagomorphs, birds, and reptiles. However, most of the proposed pathogenesis hypotheses remain contentious, and much remains to be elucidated. At the same time, the unavailability of specific antiviral treatment or effective and safe vaccines contribute to the perpetuation of the disease and regular occurrence of outbreaks in both endemic and non-endemic areas. Moreover, globalisation and climate change are also important drivers of the emergence and re-emergence of the virus and disease. Here, we give an update of the pathobiology, epidemiology, diagnostics, control, and "One Health" implications of WNV infection and disease.

Protective Efficacy of a Chimeric Insect-Specific Flavivirus Vaccine against West Nile Virus.

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.

Mosquito-Independent Transmission of West Nile virus in Farmed Saltwater Crocodiles (Crocodylus porosus).

West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2-17 dpi and in-contact animals 11-21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies.

Rhinosinusitis in an Australian mare caused by Flavodon flavus, a recently recognized invasive fungal pathogen of the horse.

We describe herein the clinical, endoscopic, computed tomography (CT), pathologic, and microbiologic features of an infection caused by an under-recognized fungal pathogen, Flavodon flavus, in a 25-y-old Australian Quarter Horse. The horse had a unilateral obstructive nasal mass, resulting in stertor and dyspnea. On endoscopy, the mass was tan, multinodular, and completely obstructed the nasal passage. CT analysis revealed a large, soft tissue-attenuating and partially mineralized mass in the right nasal passage and dorsal-conchofrontal sinus, expanding into adjacent paranasal sinuses with associated bone lysis and rhinosinusitis. Histopathology of the mass on 2 occasions revealed suppurative inflammation initially, and pyogranulomatous inflammation subsequently. The inflammatory reaction surrounded numerous spherical fungal structures (~60-80 µm diameter) that stained positively on periodic acid-Schiff and Grocott methenamine silver stains. PCR for the fungal internal transcribed spacer 1 and 2 regions followed by Sanger sequencing on a cultured isolate identified the agent as F. flavus, which has only been reported previously as pathogenic in one horse in the United States, to our knowledge. Previous reports described this fungus as a nonpathogenic, environmental commensal fungus associated with insects and plants.

An Acute Stress Model in New Zealand White Rabbits Exhibits Altered Immune Response to Infection with West Nile Virus.

The immune competence of an individual is a major determinant of morbidity in West Nile virus (WNV)-infection. Previously, we showed that immunocompetent New Zealand White rabbits (NZWRs; Oryctolagus cuniculus) are phenotypically resistant to WNV-induced disease, thus presenting a suitable model for study of virus-control mechanisms. The current study used corticosteroid-treated NZWRs to model acute "stress"-related immunosuppression. Maximal effects on immune parameters were observed on day 3 post dexamethasone-treatment (pdt). However, contrary to our hypothesis, intradermal WNV challenge at this time pdt produced significantly lower viremia 1 day post-infection (dpi) compared to untreated controls, suggestive of changes to antiviral control mechanisms. To examine this further, RNAseq was performed on RNA extracted from draining lymph node-the first site of virus replication and immune detection. Unaffected by dexamethasone-treatment, an early antiviral response, primarily via interferon (IFN)-I, and induction of a range of known and novel IFN-stimulated genes, was observed. However, treatment was associated with expression of a different repertoire of IFN-α-21-like and IFN-ω-1-like subtypes on 1 dpi, which may have driven the different chemokine response on 3 dpi. Ongoing expression of Toll-like receptor-3 and transmembrane protein-173/STING likely contributed to signaling of the treatment-independent IFN-I response. Two novel genes (putative HERC6 and IFIT1B genes), and the SLC16A5 gene were also highlighted as important component of the transcriptomic response. Therefore, the current study shows that rabbits are capable of restricting WNV replication and dissemination by known and novel robust antiviral mechanisms despite environmental challenges such as stress.

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.

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.

Kinetics of the West Nile virus induced transcripts of selected cytokines and Toll-like receptors in equine peripheral blood mononuclear cells.

West Nile virus (WNV) is one of the most common causes of epidemic viral encephalitis in horses worldwide. Peripheral blood mononuclear cells (PBMCs) are amongst the first to encounter the virus following a mosquito bite. This study aimed to elucidate the transcription kinetics of cytokine, Toll-like receptor (TLRs) and TLRs-associated genes following WNV challenge of equine PBMCs. PBMCs were challenged with an Australian strain of WNV (WNVNSW2011) and transcriptomes were quantified at 2, 6, 12 and 24 h post-infection (pi) using qRT-PCR. Type I and II interferons (IFNα, ß and γ) mRNA transcription increased following WNV exposure, as did the transcripts for IL1α, IL1ß, IL6, IL8, and IL22, but with slightly varying kinetics. TLR1, 3, 5, 7-9 transcripts were also upregulated in equine PBMCsin response to WNV challenge, as were those for MyD88, NF-κB, TRAF3, STAT1 and 2, IRF3 and 7, ISG15, as well as SOCS1 and 3 compared to the control cells. Expression of selected genes in the draining lymph node, spleen and brain (medulla oblongata) of experimentally infected horses was also assessed and transcription of most of these genes was also upregulated here. Although qRT-PCR detected higher viral RNA at 24 h pi compared to 6 h pi, the virus did not replicate productively in equine PBMCs. The up-regulation of gene-transcription for selected cytokines, IFNs, TLRs and TLRs-associated molecules suggests their involvement in virus recognition and control of WNV infection in the horse.

Tissue-specific transcription profile of cytokine and chemokine genes associated with flavivirus control and non-lethal neuropathogenesis in rabbits.

We previously showed that New Zealand White (NZWRs) and cottontail rabbits (CTRs) are a suitable model for studying immune mechanisms behind virus control and non-lethal neuropathogenesis associated with West Nile virus (WNV) and Murray Valley encephalitis virus (MVEV) infections. In the current study, we observed that MVEV infection induced high IFNα, TNFα, IL6, and CXCL10 transcript levels in the brains of weanling NZWRs, unlike infection with the less virulent WNVNSW2011. These transcript levels also correlated with encephalitis severity. Widespread STAT1 protein expression in brain with moderate neuropathology suggests that IFN-I signaling is crucial for limiting neural infection and mediating non-lethal neuropathogenesis. Unlike NZWRs, CTRs limit neuroinvasion without upregulation of many cytokine/chemokine transcripts, suggesting a species-dependent virus control mechanism. However, the common IFNγ, TNFα and IL6 transcript upregulation in specific lymphoid organs suggest some conserved elements in the response against flaviviruses, unique to all rabbits.

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.

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.

West Nile Virus Challenge Alters the Transcription Profiles of Innate Immune Genes in Rabbit Peripheral Blood Mononuclear Cells.

The peripheral innate immune response to West Nile virus (WNV) is crucial for control of virus spread to the central nervous system. Therefore, transcriptomes encoding the innate immune response proteins against WNV were investigated in peripheral blood mononuclear cells (PBMCs) of New Zealand White rabbits, a recently established novel rabbit model for WNV pathogenesis studies. PBMCs were challenged with an Australian WNV strain, WNVNSW2011, in vitro, and mRNA expression of selected immune response genes were quantified at 2-, 6-, 12-, and 24-h post-infection (pi) using qRT-PCR. Compared to mock-inoculated PBMCs, WNV-stimulated PBMCs expressed high levels of interferon (IFN) alpha (IFNA), gamma (IFNG), IL6, IL12, IL22, CXCL10, and pentraxin 3 (PTX3) mRNA. Likewise, TLR1, 2, 3, 4, 6, and 10 mRNA became up-regulated with the highest expression seen for TLR3, 4, and 6. TLRs-signaling downstream genes (MyD88, STAT1, TRAF3, IRF7, and IRF9) subsequently became up-regulated. The high expression of IFNs, TLR3, TLR4, TRAF3, STAT1, IRF7, and IRF9 are in accordance with antiviral activities, while expression of TNFA, HO1, iNOS, caspase 3, and caspase 9 transcripts suggests the involvement of oxidative stress and apoptosis in WNV-stimulated rabbit PBMCs, respectively. The level of WNVNSW2011 RNA increased at 24-h pi in PBMCs challenged with virus in vitro compared to input virus. The expression dynamics of selected genes were validated in PBMCs from rabbits experimentally infected with WNV in vivo. Higher expression of IFNA, IFN beta (IFNB), IFNG, TNFA, IL6, IL22, PTX3, TLR3 and TLR4, IRF7, IRF9, STST1, TRAF3, caspase 3, and caspase 9 were seen in PBMCs from WNV-infected rabbits on day 3 post-intradermal virus inoculation compared to PBMCs from uninfected control rabbits. This study highlights the array of cytokines and TLRs involved in the host innate immune response to WNV in the rabbit leukocytes and suggests that these cells may be a useful in vitro model for WNV infection study.