Characterization of Teviot virus, an Australian bat-borne paramyxovirus.

Bats are the reservoir hosts for multiple viruses with zoonotic potential, including coronaviruses, paramyxoviruses and filoviruses. Urine collected from Australian pteropid bats was assessed for the presence of paramyxoviruses. One of the viruses isolated was Teviot virus (TevPV), a novel rubulavirus previously isolated from pteropid bat urine throughout the east coast of Australia. Here, we further characterize TevPV through analysis of whole-genome sequencing, growth kinetics, antigenic relatedness and the experimental infection of ferrets and mice. TevPV is phylogenetically and antigenically most closely related to Tioman virus (TioPV). Unlike many other rubulaviruses, cell receptor attachment by TevPV does not appear to be sialic acid-dependent, with the receptor for host cell entry being unknown. The infection of ferrets and mice suggested that TevPV has a low pathogenic potential in mammals. Infected ferrets seroconverted by 10 days post-infection without clinical signs of disease. Furthermore, infected ferrets did not shed virus in any respiratory secretions, suggesting a low risk of onward transmission of TevPV. No productive infection was observed in the mouse infection study.

An Australian Newcastle Disease Virus With a Virulent Fusion Protein Cleavage Site Produces Minimal Pathogenicity in Chickens.

Newcastle disease is an important disease of poultry caused by virulent strains of Newcastle disease virus (NDV). During the 1998 to 2002 outbreaks of Newcastle disease in Australia, it was observed that the mild clinical signs seen in some chickens infected with NDV did not correlate with the viruses' virulent fusion protein cleavage site motifs or standard pathogenicity indices. The pathogenicity of 2 Australian NDV isolates was evaluated in experimentally challenged chickens based on clinical evaluation, histopathology, immunohistochemistry, and molecular techniques. One of these virus isolates, Meredith/02, was shown to induce only very mild clinical signs with no mortalities in an experimental setting, in contrast to the velogenic Herts 33/56 and Texas GB isolates. This minimal pathogenicity was associated with decreased virus replication and antigen distribution in tissues. This demonstrates that the Australian Meredith/02 NDV, despite possessing a virulent fusion protein cleavage site, did not display a velogenic phenotype.

Henipavirus-induced neuropathogenesis in mice.

Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses that can cause fatal encephalitis in humans. Many animal models have been used to study henipavirus pathogenesis. In the mouse, HeV infection has previously shown that intranasal challenge can lead to neurological infection, however mice similarly challenged with NiV show no evidence of virus infecting the brain. We generated recombinant HeV (rHeV) and NiV (rNiV) where selected proteins were switched to examine their role in neuroinvasion in the mouse. These viruses displayed similar growth kinetics when compared to wildtype in vitro. In the mouse, infection outcomes with recombinant virus did not differ to infection outcomes of wildtype viruses. Virus was detected in the brain of 5/30 rHeV-challenged mice, but not rNiV-challenged mice. To confirm the permissiveness of mouse neurons to these viruses, primary mouse neurons were successfully infected in vitro, suggesting that other pathobiological factors contribute to the differences in disease outcomes in mice.

Transmission routes for nipah virus from Malaysia and Bangladesh.

Human infections with Nipah virus in Malaysia and Bangladesh are associated with markedly different patterns of transmission and pathogenicity. To compare the 2 strains, we conducted an in vivo study in which 2 groups of ferrets were oronasally exposed to either the Malaysia or Bangladesh strain of Nipah virus. Viral shedding and tissue tropism were compared between the 2 groups. Over the course of infection, significantly higher levels of viral RNA were recovered from oral secretions of ferrets infected with the Bangladesh strain. Higher levels of oral shedding of the Bangladesh strain of Nipah virus might be a key factor in onward transmission in outbreaks among humans.

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.

In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Erratum: Johnson et al. Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets. Viruses 2018, 10, 675.

The authors wish to make the following corrections to this paper [...].

Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV.

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.

Assessment of cellular immune responses of healthy and diseased Tasmanian devils (Sarcophilus harrisii).

The Tasmanian devil (TD) (Sarcophilus harrisii) is under threat from devil facial tumour disease (DFTD), a cancer that is transmitted between devils by direct cell implantation. As no devil is known to have rejected the tumour allograft, an understanding of the immune status of this species is essential to help explain the unique infectious nature of this cancer. We analysed differential white blood cell counts, the phagocytic response of neutrophils as well as mitogen-induced lymphocyte proliferation. Devils analysed included healthy TDs kept in captivity, healthy devils from disease-free and diseased areas as well as diseased devils. Neutrophils isolated from the peripheral blood of healthy devils showed competent phagocytosis and peripheral blood mononuclear cells from healthy and diseased devils proliferated in response to Con A, PHA and PWM stimulation. Although a wide range of responses was observed and relatively high doses of mitogens were required, there was no significant difference between males and females, adults and juveniles or between normal and diseased animals, suggesting that transmission of DFTD is not a consequence of a severely impaired immune system. As lymphocytes from all TDs appear to require strong stimulation for activation, this threshold may contribute to all devils being susceptible to DFTD.

A novel group A rotavirus associated with acute illness and hepatic necrosis in pigeons (Columba livia), in Australia.

Cases of vomiting and diarrhoea were reported in racing pigeons in Western Australia in May, 2016. Morbidity and mortality rates were high. Similar clinical disease was seen in Victoria in December and by early 2017 had been reported in all states except the Northern Territory, in different classes of domestic pigeon-racing, fancy and meat bird-and in a flock of feral pigeons. Autopsy findings were frequently unremarkable; histological examination demonstrated significant hepatic necrosis as the major and consistent lesion, often with minimal inflammatory infiltration. Negative contrast tissue suspension and thin section transmission electron microscopy of liver demonstrated virus particles consistent with a member of the Reoviridae. Inoculation of trypsin-treated Vero, MDBK and MA-104 cell lines resulted in cytopathic changes at two days after infection. Next generation sequencing was undertaken using fresh liver samples and a previously undescribed group A rotavirus (genotype G18P[17]) of avian origin was identified and the virus was isolated in several cell lines. A q-RT-PCR assay was developed and used to screen a wider range of samples, including recovered birds. Episodes of disease have continued to occur and to reoccur in previously recovered lofts, with variable virulence reported. This is the first report of a rotavirus associated with hepatic necrosis in any avian species.

Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets.

Multiple viruses with zoonotic potential have been isolated from bats globally. Here we describe the isolation and characterization of a novel paramyxovirus, Alston virus (AlsPV), isolated from urine collected from an Australian pteropid bat colony in Alstonville, New South Wales. Characterization of AlsPV by whole-genome sequencing and analyzing antigenic relatedness revealed it is a rubulavirus that is closely related to parainfluenza virus 5 (PIV5). Intranasal exposure of mice to AlsPV resulted in no clinical signs of disease, although viral RNA was detected in the olfactory bulbs of two mice at 21 days post exposure. Oronasal challenge of ferrets resulted in subclinical upper respiratory tract infection, viral shedding in respiratory secretions, and detection of viral antigen in the olfactory bulb of the brain. These results imply that AlsPV may be similar to PIV5 in its ability to infect multiple mammalian host species. This isolation of a novel paramyxovirus with the potential to transmit from bats to other mammalian species reinforces the importance of continued surveillance of bats as a source of emerging viruses.

A network approach for provisional assay recognition of a Hendra virus antibody ELISA: test validation with low sample numbers from infected horses.

Obtaining statistically sound numbers of sera from Hendra virus (HeV)-infected horses is problematic because affected individuals usually die or are euthanized before developing a serum antibody response. As a consequence, test validation becomes a challenge. Our approach is an extension of OIE principles for provisional recognition and included 7 validation panels tested across multiple laboratories that provided estimates for test performance characteristics. At a 0.4 S/P cutoff, 16 of 19 sera from HeV-infected horses gave positive results in the HeV soluble G, indirect ELISA (HeVsG iELISA; DSe 84.2% [95% CI: 60.4-96.6%]); 463 of 477 non-infected horse sera tested negative (DSp 97.1% [95% CI: 95.1-98.4%]). The HeVsG iELISA eliminated almost all false-positive results from the previously used HeV iELISA, with marginally decreased relative sensitivity. Assay robustness was evaluated in inter-laboratory and proficiency testing panels. The HeVsG iELISA is considered to be fit for purpose for serosurveillance and international movement of horses when virus neutralization is used for follow-up testing of positive or inconclusive serum samples.

The diagnosis and management of a case of leishmaniosis in a dog imported to Australia.

This case study discusses in detail for the first time the diagnosis and management of a case of leishmaniosis in a dog imported to Australia. The dog presented with epistaxis and a non-regenerative anaemia five years after being imported from Europe. Protozoa were identified within macrophages in bone marrow and splenic cytology. A Leishmania indirect fluorescent antibody test was performed and was positive while an Ehrlichia canis antibody test was negative. Polymerase chain reaction of the ITS-1 and ITS-2 regions of skin, lymph node, spleen and bone marrow were all positive for Leishmania infantum. The dog was treated with amphotericin B with a strong clinical response. The importance of thorough diagnostics in non-endemic areas, particularly Australia, is discussed. Treatment with amphotericin B is discussed. Vigilance, disease reporting and response frameworks are recommended for non-endemic areas.