Experimental SARS-CoV-2 Infection of Elk and Mule Deer.

To assess the susceptibility of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) to SARS-CoV-2, we performed experimental infections in both species. Elk did not shed infectious virus but mounted low-level serologic responses. Mule deer shed and transmitted virus and mounted pronounced serologic responses and thus could play a role in SARS-CoV-2 epidemiology.

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.

Susceptibility of Wild Canids to SARS-CoV-2.

We assessed 2 wild canid species, red foxes (Vulpes vulpes) and coyotes (Canis latrans), for susceptibility to SARS-CoV-2. After experimental inoculation, red foxes became infected and shed infectious virus. Conversely, experimentally challenged coyotes did not become infected; therefore, coyotes are unlikely to be competent hosts for SARS-CoV-2.

Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Wild animals have been implicated as the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. We show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our results expand the knowledge base of susceptible species and provide evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.

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.

Infection, Replication, and Transmission of Middle East Respiratory Syndrome Coronavirus in Alpacas.

Middle East respiratory syndrome coronavirus is a recently emerged pathogen associated with severe human disease. Zoonotic spillover from camels appears to play a major role in transmission. Because of logistic difficulties in working with dromedaries in containment, a more manageable animal model would be desirable. We report shedding and transmission of this virus in experimentally infected alpacas (n = 3) or those infected by contact (n = 3). Infectious virus was detected in all infected animals and in 2 of 3 in-contact animals. All alpacas seroconverted and were rechallenged 70 days after the original infection. Experimentally infected animals were protected against reinfection, and those infected by contact were partially protected. Necropsy specimens from immunologically naive animals (n = 3) obtained on day 5 postinfection showed virus in the upper respiratory tract. These data demonstrate efficient virus replication and animal-to-animal transmission and indicate that alpacas might be useful surrogates for camels in laboratory studies.

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.

Oral Administration of Universal Bacterium-Vectored Nucleocapsid-Expressing COVID-19 Vaccine is Efficacious in Hamsters.

Oral delivery of an inexpensive COVID-19 (coronavirus disease 2019) vaccine could dramatically improve immunization rates, especially in low- and middle-income countries. Previously, we described a potential universal COVID-19 vaccine, rLVS ΔcapB/MN, comprising a replicating bacterial vector, LVS (live vaccine strain) ΔcapB, expressing the highly conserved SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) membrane and nucleocapsid (N) proteins, which, when administered intradermally or intranasally, protects hamsters from severe COVID-19-like disease after high-dose SARS-CoV-2 respiratory challenge. Here, we show that oral administration of the vaccine also protects against high-dose SARS-CoV-2 respiratory challenge; its protection is comparable to that of intradermal, intranasal, or subcutaneous administration. Hamsters were protected against severe weight loss and lung pathology and had reduced oropharyngeal and lung virus titers. Protection against weight loss and histopathology by the vaccine, which in mice induces splenic and lung cell interferon gamma in response to N protein stimulation, was correlated in hamsters with pre-challenge serum anti-N TH1-biased IgG (IgG2/3). Thus, rLVS ΔcapB/MN has potential as an oral universal COVID-19 vaccine. IMPORTANCE The COVID-19 pandemic continues to rage into its fourth year worldwide. To protect the world's population most effectively from severe disease, hospitalization, and death, a vaccine is needed that is resistant to rapidly emerging viral variants of the causative agent SARS-CoV-2, inexpensive to manufacture, store, and transport, and easy to administer. Ideally, such a vaccine would be capable of oral administration, especially in resource-poor countries of the world where there are shortages of needles, syringes and trained personnel to administer injectable vaccines. Here, we show that oral administration of a bacterium-vectored vaccine meeting all these criteria protects naturally susceptible Syrian hamsters from severe COVID-19-like disease, including severe weight loss and lung pathology, after high-dose SARS-CoV-2 respiratory challenge. As the vaccine is based upon inducing immunity to highly conserved SARS-CoV-2 membrane and nucleocapsid proteins, as opposed to the rapidly mutating Spike protein, it should remain resistant to newly emerging SARS-CoV-2 variants.

Disease Progression and Serological Assay Performance in Heritage Breed Pigs following Brucella suis Experimental Challenge as a Model for Naturally Infected Feral Swine.

Invasive feral swine (Sus scrofa) are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of concern for the health of humans and domestic animals. Brucella suis, the causative agent of swine brucellosis, is one such pathogen carried and transmitted by feral swine. Serology assays are the preferred field diagnostic for B. suis infection, as whole blood can be readily collected and antibodies are highly stable. However, serological assays frequently have lower sensitivity and specificity, and few studies have validated serological assays for B. suis in feral swine. We conducted an experimental infection of Ossabaw Island Hogs (a breed re-domesticated from feral animals) as a disease-free proxy for feral swine to (1) improve understanding of bacterial dissemination and antibody response following B. suis infection and (2) evaluate potential changes in the performance of serological diagnostic assays over the course of infection. Animals were inoculated with B. suis and serially euthanized across a 16-week period, with samples collected at the time of euthanasia. The 8% card agglutination test performed best, whereas the fluorescence polarization assay demonstrated no capacity to differentiate true positive from true negative animals. From a disease surveillance perspective, using the 8% card agglutination test in parallel with either the buffered acidified plate antigen test or the Brucella abortus/suis complement fixation test provided the best performance with the highest probability of a positive assay result. Application of these combinations of diagnostic assays for B. suis surveillance among feral swine would improve understanding of spillover risks at the national level.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

Immunogenicity, Safety, and Anti-Viral Efficacy of a Subunit SARS-CoV-2 Vaccine Candidate in Captive Black-Footed Ferrets (Mustela nigripes) and Their Susceptibility to Viral Challenge.

A preliminary vaccination trial against the emergent pathogen, SARS-CoV-2, was completed in captive black-footed ferrets (Mustela nigripes; BFF) to assess safety, immunogenicity, and anti-viral efficacy. Vaccination and boosting of 15 BFF with purified SARS-CoV-2 S1 subunit protein produced a nearly 150-fold increase in mean antibody titers compared to pre-vaccination titers. Serum antibody responses were highest in young animals, but in all vaccinees, antibody response declined rapidly. Anti-viral activity from vaccinated and unvaccinated BFF was determined in vitro, as well as in vivo with a passive serum transfer study in mice. Transgenic mice that received BFF serum transfers and were subsequently challenged with SARS-CoV-2 had lung viral loads that negatively correlated (p < 0.05) with the BFF serum titer received. Lastly, an experimental challenge study in a small group of BFF was completed to test susceptibility to SARS-CoV-2. Despite viral replication and shedding in the upper respiratory tract for up to 7 days post-challenge, no clinical disease was observed in either vaccinated or naive animals. The lack of morbidity or mortality observed indicates SARS-CoV-2 is unlikely to affect wild BFF populations, but infected captive animals pose a potential risk, albeit low, for humans and other animals.

American alligators are capable of West Nile virus amplification, mosquito infection and transmission.

West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle.

Susceptibility of livestock to SARS-CoV-2 infection.

We report pilot studies to evaluate the susceptibility of common domestic livestock (cattle, sheep, goat, alpaca, rabbit, and horse) to intranasal infection with SARS-CoV-2. None of the infected animals shed infectious virus via nasal, oral, or faecal routes, although viral RNA was detected in several animals. Further, neutralizing antibody titres were low or non-existent one month following infection. These results suggest that domestic livestock are unlikely to contribute to SARS-CoV-2 epidemiology.

Activity of Galidesivir in a Hamster Model of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) has claimed the lives of millions of people worldwide since it first emerged. The impact of the COVID-19 pandemic on public health and the global economy has highlighted the medical need for the development of broadly acting interventions against emerging viral threats. Galidesivir is a broad-spectrum antiviral compound with demonstrated in vitro and in vivo efficacy against several RNA viruses of public health concern, including those causing yellow fever, Ebola, Marburg, and Rift Valley fever. In vitro studies have shown that the antiviral activity of galidesivir also extends to coronaviruses. Herein, we describe the efficacy of galidesivir in the Syrian golden hamster model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Treatment with galidesivir reduced lung pathology in infected animals compared with untreated controls when treatment was initiated 24 h prior to infection. These results add to the evidence of the applicability of galidesivir as a potential medical intervention for a range of acute viral illnesses, including coronaviruses.

Biodetection of a specific odor signature in mallard feces associated with infection by low pathogenic avian influenza A virus.

Outbreaks of avian influenza virus (AIV) infection included the spread of highly pathogenic AIV in commercial poultry and backyard flocks in the spring of 2015. This resulted in estimated losses of more than $8.5 million from federal government expenditures, $1.6 billion from direct losses to produces arising from destroyed turkey and chicken egg production, and economy-wide indirect costs of $3.3 billion from impacts on retailers and the food service industries. Additionally, these outbreaks resulted in the death or depopulation of nearly 50 million domestic birds. Domesticated male ferrets (Mustela putorius furo) were trained to display a specific conditioned behavior (i.e. active scratch alert) in response to feces from AIV-infected mallards in comparison to feces from healthy ducks. In order to establish that ferrets were identifying samples based on odors associated with infection, additional experiments controlled for potentially confounding effects, such as: individual duck identity, housing and feed, inoculation concentration, and day of sample collection (post-infection). A final experiment revealed that trained ferrets could detect AIV infection status even in the presence of samples from mallards inoculated with Newcastle disease virus or infectious laryngotracheitis virus. These results indicate that mammalian biodetectors are capable of discriminating the specific odors emitted from the feces of non-infected versus AIV infected mallards, suggesting that the health status of waterfowl can be evaluated non-invasively for AIV infection via monitoring of volatile fecal metabolites. Furthermore, in situ monitoring using trained biodetectors may be an effective tool for assessing population health.

Powassan Virus Experimental Infections in Three Wild Mammal Species.

Powassan virus (POWV) is a tick-borne virus maintained in sylvatic cycles between mammalian wildlife hosts and ticks (primarily Ixodes spp.). There are two currently recognized lineages, POWV-lineage 1 (POWV-L1) and deer tick virus (DTV; lineage 2), both of which can cause fatal neurologic disease in humans. Increased numbers of human case reports in the northeastern and north central United States in recent years have fueled questions into POWV epidemiology. We inoculated three candidate wildlife POWV reservoir hosts, groundhogs (Marmota monax), striped skunks (Mephitis mephitis), and fox squirrels (Sciurus niger), with either POWV-L1 or DTV. Resulting viremia, tissue tropism, and pathology were minimal in most inoculated individuals of all three species, with low (peak titer range, 101.7-103.3 plaque-forming units/mL serum) or undetectable viremia titers, lack of detection in tissues except for low titers in spleen, and seroconversion in most individuals by 21 days postinoculation (DPI). Pathology was limited and most commonly consisted of mild inflammation in the brain of POWV-L1- and DTV-inoculated skunks on four and 21 DPI, respectively. These results reveal variation in virulence and host competence among wild mammalian species, and a likely limited duration of host infectiousness to ticks during enzootic transmission cycles. However, POWV can transmit rapidly from tick to host, and tick co-feeding may be an additional transmission mechanism. The rare and low-level detections of viremia in these three, common, wild mammal species suggest that vector-host dynamics should continue to be explored, along with eco-epidemiological aspects of enzootic POWV transmission in different regions and virus lineages.

Δcps1 vaccine protects dogs against experimentally induced coccidioidomycosis.

Coccidioidomycosis is a significant health problem of dogs and humans in endemic regions, especially California and Arizona in the U.S. Both species would greatly benefit from a vaccine to prevent this disease. A live avirulent vaccine candidate, Δcps1, was tested for tolerability and efficacy to prevent pulmonary coccidioidomycosis in a canine challenge model. Vaccine injection-site reactions were transient and there were no systemic effects observed. Six of seven vaccine sites tested and all draining lymph nodes were sterile post-vaccination. Following infection with Coccidioides posadasii, strain Silveira, arthroconidia into the lungs, dogs given primary and booster vaccinations had significantly reduced lung fungal burdens (P = 0.0003) and composite disease scores (P = 0.0002) compared to unvaccinated dogs. Dogs vaccinated once had fungal burdens intermediate between those given two doses or none, but disease scores were not significantly different from unvaccinated (P = 0.675). Δcps1 was well-tolerated in the dogs and it afforded a high level of protection when given as prime and boost. These results drive the Δcps1 vaccine toward a licensed veterinary vaccine and support continued development of this vaccine to prevent coccidioidomycosis in humans.

An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces protective immunity and prevents transmission of SARS-CoV-2 between cats.

Early in the SARS-CoV-2 pandemic concerns were raised regarding infection of new animal hosts and the effect on viral epidemiology. Infection of other animals could be detrimental by causing clinical disease, allowing further mutations, and bares the risk for the establishment of a non-human reservoir. Cats were the first reported animals susceptible to natural and experimental infection with SARS-CoV-2. Given the concerns these findings raised, and the close contact between humans and cats, we aimed to develop a vaccine candidate that could reduce SARS-CoV-2 infection and in addition to prevent spread among cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus, known to be safe and efficacious in a variety of animal species, could induce neutralizing antibody responses in guinea pigs and cats. The design of the SARS-CoV-2 spike immunogen was critical in developing a strong neutralizing antibody response. Vaccination of cats was able to induce high neutralizing antibody responses, effective also against the SARS-CoV-2 B.1.1.7 variant. Interestingly, in contrast to control animals, the infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after SARS-CoV-2 challenge. Correspondingly, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit the virus. The results show that the RP vaccine induces protective immunity preventing SARS-CoV-2 infection and transmission. These data suggest that this RP vaccine could be a multi-species vaccine useful to prevent infection and spread to and between animals should that approach be required.

Vaccine Candidate Brucella melitensis 16MΔvjbR Is Safe in a Pregnant Sheep Model and Confers Protection.

As a natural host species for Brucella melitensis, pregnant sheep offer an ideal model to evaluate vaccine candidates for safety. B. melitensis strain Rev. 1 has been used almost exclusively to prevent brucellosis in small ruminants, but it causes abortions when given to pregnant animals. To evaluate the comparative safety of the candidate Brucella melitensis 16MΔvjbR, pregnant sheep (n = 6) were vaccinated subcutaneously with 1 × 1010 CFU/ml of 16MΔvjbR or 1 × 109 CFU/ml Rev. 1 at a highly susceptible stage of gestation (approximately 70 days). 16MΔvjbR resulted in only 1 abortion (1 of 6) compared with 4 of 6 (66.7%) abortions in the Rev. 1 cohort. The placenta was evaluated by culture to determine if vaccination resulted in colonization. As another measure of safety, effects of B. melitensis on the fetus/offspring (vertical transmission) was evaluated by culture and histopathology of fetal tissues to determine if vaccination prevented infection of the fetus. Vaccination with 16MΔvjbR resulted in less vertical transmission than Rev. 1. To determine if vaccination was efficacious and could reduce tissue colonization in sheep, the same cohort of sheep were challenged 5 weeks postpartum by conjunctival inoculation with 1 × 107 CFU/ml B. melitensis Protection was similar between Rev. 1 and 16MΔvjbR, with no statistical difference in colonization in the target organs. Overall, the 16MΔvjbR vaccine was considered safer than Rev. 1 based on a reduced number of abortions and limited infection in the offspring. Future experiments are needed to further refine the vaccine dose to increase the safety margin and to evaluate protection in pregnant ewes.IMPORTANCE Brucellosis is one of the most commonly reported zoonotic disease with a worldwide distribution. Of the 12 Brucella species, Brucella melitensis is considered the most virulent and causes reproductive failure (abortions/stillbirths) in small ruminants, which can spread the disease to other animals or to humans. Vaccination of small ruminants is a key measure used to protect both human and animal health. However, the commercially available live-attenuated vaccine for Brucella melitensis Rev. 1 retains virulence and can cause disease in animals and humans. In order to evaluate the safety and efficacy in sheep, we vaccinated pregnant sheep with 16MΔvjbR Our results indicate that 16MΔvjbR was safer for use during pregnancy, provided a similar level of protection as Rev. 1, and could be considered an improved candidate for future vaccine trials.

NS4/5 mutations enhance flavivirus Bamaga virus infectivity and pathogenicity in vitro and in vivo.

Flaviviruses such as yellow fever, dengue or Zika viruses are responsible for significant human and veterinary diseases worldwide. These viruses contain an RNA genome, prone to mutations, which enhances their potential to emerge as pathogens. Bamaga virus (BgV) is a mosquito-borne flavivirus in the yellow fever virus group that we have previously shown to be host-restricted in vertebrates and horizontally transmissible by Culex mosquitoes. Here, we aimed to characterise BgV host-restriction and to investigate the mechanisms involved. We showed that BgV could not replicate in a wide range of vertebrate cell lines and animal species. We determined that the mechanisms involved in BgV host-restriction were independent of the type-1 interferon response and RNAse L activity. Using a BgV infectious clone and two chimeric viruses generated as hybrids between BgV and West Nile virus, we demonstrated that BgV host-restriction occurred post-cell entry. Notably, BgV host-restriction was shown to be temperature-dependent, as BgV replicated in all vertebrate cell lines at 34°C but only in a subset at 37°C. Serial passaging of BgV in Vero cells resulted in adaptive mutants capable of efficient replication at 37°C. The identified mutations resulted in amino acid substitutions in NS4A-S124F, NS4B-N244K and NS5-G2C, all occurring close to a viral protease cleavage site (NS4A/2K and NS4B/NS5). These mutations were reverse engineered into infectious clones of BgV, which revealed that NS4B-N244K and NS5-G2C were sufficient to restore BgV replication in vertebrate cells at 37°C, while NS4A-S124F further increased replication efficiency. When these mutant viruses were injected into immunocompetent mice, alongside BgV and West Nile virus chimeras, infection and neurovirulence were enhanced as determined by clinical scores, seroconversion, micro-neutralisation, viremia, histopathology and immunohistochemistry, confirming the involvement of these residues in the attenuation of BgV. Our studies identify a new mechanism of host-restriction and attenuation of a mosquito-borne flavivirus.