Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first detected in late December 2019 and has spread worldwide. Coronaviruses are enveloped, positive sense, single-stranded RNA viruses and employ a complicated pattern of virus genome length RNA replication as well as transcription of genome length and leader containing subgenomic RNAs. Although not fully understood, both replication and transcription are thought to take place in so-called double-membrane vesicles in the cytoplasm of infected cells. Here we show detection of SARS-CoV-2 subgenomic RNAs in diagnostic samples up to 17 days after initial detection of infection and provide evidence for their nuclease resistance and protection by cellular membranes suggesting that detection of subgenomic RNAs in such samples may not be a suitable indicator of active coronavirus replication/infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in China in late December 2019 and has spread worldwide. Coronaviruses are enveloped, positive sense, single-stranded RNA viruses and employ a complicated pattern of virus genome length RNA replication as well as transcription of genome length and leader containing subgenomic RNAs. Although not fully understood, both replication and transcription are thought to take place in so-called double-membrane vesicles in the cytoplasm of infected cells. We here describe detection of SARS-CoV-2 subgenomic RNAs in diagnostic samples up to 17 days after initial detection of infection, and provide a likely explanation not only for extended PCR positivity of such samples, but also for discrepancies in results of different PCR methods described by others. Overall, we present evidence that subgenomic RNAs may not be an indicator of active coronavirus replication/infection, but that these RNAs, similar to the virus genome RNA, may be rather stable, and thus detectable for an extended period, most likely due to their close association with cellular membranes.

This study reports the sequence analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from infected individuals within the Greater Geelong region, Victoria, Australia. All but one individual had recently returned from travelling abroad, and all had clinical signs consistent with SARS-CoV-2 infection. SARS-CoV-2 belonging to three lineages were detected and represent separate introductions of the virus into the region. Sequence data were consistent with the recent travel history of each case. Full virus genome sequencing can play an important role in supporting local epidemiological tracing and monitoring for community transmission. Quality of the SARS-CoV-2 sequences obtained was highly dependent on appropriate sample collection and handling.

The present study reports the genetic characterization of a low-pathogenicity H9N2 avian influenza virus, initially from a pool and subsequently from individual faecal samples collected from Chestnut teals (Anas castanea) in southeastern Australia. Phylogenetic analyses of six full gene segments and two partial gene segments obtained from next-generation sequencing showed that this avian influenza virus, A/Chestnut teal/Australia/CT08.18/12952/2018 (H9N2), was a typical, low-pathogenicity, Eurasian aquatic bird lineage H9N2 virus, albeit containing the North American lineage nucleoprotein (NP) gene segment detected previously in Australian wild birds. This is the first report of a H9N2 avian influenza virus in resident wild birds in Australia, and although not in itself a cause of concern, is a clear indication of spillover and likely reassortment of influenza viruses between migratory and resident birds, and an indication that any lineage could potentially be introduced in this way.

Gastroenteritis in young animals is a clinical presentation with many infectious and non- infectious aetiologies. We used next generation sequencing (NGS) to investigate the possible infectious causes of gastroenteritis in puppies from a dog kennel in Victoria, Australia. The near complete genome of a canine astrovirus was obtained from pooled faecal samples, and was found to be 94.7% identical with a canine astrovirus detected in the United Kingdom in 2012. The phylogenetic analysis of the capsid gene found similarities to those of canine astroviruses identified in Italy in 2005 and in UK and Hungary in 2012, but distant from that of a canine astrovirus previously identified in Australia in 2012. Thus, different serotypes of canine astrovirus are likely circulating in Australia. The close relationship to European astroviruses also suggested that there had been recent movements of ancestor canine astroviruses between Australia and Europe. NGS also detected other infections in the puppies including several canine papillomaviruses and a canine parvovirus (vaccine strain) as well as a very low level of campylobacter. Canine astrovirus was the probable cause of diarrhoea in these puppies, with the possible involvement of campylobacter bacteria. NGS was effective as a non-targeted method to determine the likely infectious cause of gastroenteritis.

Tiger (Panthera tigris) populations are in danger across their entire range due to habitat loss, poaching and the demand for tiger parts. The Bengal tiger (Panthera tigris tigris) is an endangered apex predator with a population size estimated to be less than 200 in Nepal. In spite of strict wildlife protection laws, illegal trade of tiger parts is increasing; and Nepal has become one of the major sources and transit routes for poached wildlife parts. Identification of wildlife parts is often challenging for law enforcement officials due to inadequate training and lack of available tools. Here, we describe a molecular forensic approach to gain insight into illegally trafficked tiger parts seized across Nepal. We created Nepal’s first comprehensive reference genetic database of wild tigers through the Nepal Tiger Genome Project (2011–2013). This database has nuclear DNA microsatellite genotype and sex profiles, including geo-spatial information, of over 60% (n = 120) of the wild tigers of Nepal. We analyzed 15 putative cases of confiscated poached tiger parts and all were confirmed to be of tiger. Ten samples were identified as male and five were female. We determined probable geo-source location for 9 of the 14 samples with 6–8 nuclear DNA microsatellite loci using inferences from four different statistical assignment methods. Six samples were assigned to Bardia National Park and one of these was an exact match to a female tiger previously profiled in our fecal DNA reference database. Two tiger samples were assigned to Shuklaphanta Wildlife Reserve and one to Chitwan National Park. We are unable to definitively assign five tiger samples which could be offspring dispersers or might have come from tiger population outside of Nepal. Our study revealed that the western region, particularly Bardia National Park, is a poaching hotspot for illegal tiger trade in Nepal. We present feasibility of using molecular forensic based evidence to incriminate criminals in a court of law in the fight against wildlife crime.

Nepal boarders India and China and all three countries lie within the Central Asian Flyway for migratory birds. Novel influenza A H7N9 caused human fatalities in China in 2013. Subclinical infections of influenza A H7N9 in birds and the potential for virus dispersal by migratory birds prompted this study to assess avian H7N9 viral intrusion into Nepal. Surveillance of influenza A virus in migratory birds was implemented in early 2014 with assistance from the Food and Agricultural Organization (FAO). Of 1811 environmental fecal samples collected from seven wetland migratory bird roosting areas, influenza A H9N2 was found in one sample from a ruddy shelduck in Koshi Tappu Wildlife Reserve located in southern Nepal. Avian H7N9 and other highly pathogenic avian influenza viruses were not detected. This study provides baseline data on the status of avian influenza virus in migratory bird populations in Nepal.

Influenza A virus (IAV) in swine, so-called swine influenza A virus (swIAV), causes respiratory illness in pigs around the globe. In Danish pig herds, a H1N2 subtype named H1N2dk is one of the main circulating swIAV. In this cohort study, the infection dynamic of swIAV was evaluated in a Danish pig herd by sampling and PCR testing of pigs from two weeks of age until slaughter at 22 weeks of age. In addition, next generation sequencing (NGS) was used to identify and characterize the complete genome of swIAV circulating in the herd, and to examine the antigenic variability in the antigenic sites of the virus hemagglutinin (HA) and neuraminidase (NA) proteins. Overall, 76.6% of the pigs became PCR positive for swIAV during the study, with the highest prevalence at four weeks of age. Detailed analysis of the virus sequences obtained showed that the majority of mutations occurred at antigenic sites in the HA and NA proteins of the virus. At least two different H1N2 variants were found to be circulating in the herd; one H1N2 variant was circulating at the sow and nursery sites, while another H1N2 variant was circulating at the finisher site. Furthermore, it was demonstrated that individual pigs had recurrent swIAV infections with the two different H1N2 variants, but re-infection with the same H1N2 variant was also observed. Better understandings of the epidemiology, genetic and antigenic diversity of swIAV may help to design better health interventions for the prevention and control of swIAV infections in the herds.