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.

We present an optimised metagenomics method for detection and characterisation of all virus types including single and double stranded DNA/RNA and enveloped and non-enveloped viruses. Initial evaluation included both spiked and non-spiked bird faecal samples as well as non-spiked human faecal samples. From the non-spiked bird samples (Australian Muscovy duck and Pacific black ducks) we detected 21 viruses, and we also present a summary of a few viruses detected in human faecal samples. We then present a detailed analysis of selected virus sequences in the avian samples that were somewhat similar to known viruses, and had good quality (Q20 or higher) and quantity of next-generation sequencing reads, and was of interest from a virological point of view, for example, avian coronavirus and avian paramyxovirus 6. Some of these viruses were closely related to known viruses while others were more distantly related with 70% or less identity to currently known/sequenced viruses. Besides detecting viruses, the technique also allowed the characterisation of host mitochondrial DNA present and thus identifying host species, while ribosomal RNA sequences provided insight into the “ribosomal activity microbiome”; of gut parasites; and of food eaten such as plants or insects, which we correlated to non-avian host associated viruses.

We evaluated the presence of coronaviruses by PCR in 918 Australian wild bird samples collected during 2016–17. Coronaviruses were detected in 141 samples (15.3%) from species of ducks, shorebirds and herons and from multiple sampling locations. Sequencing of selected positive samples found mainly gammacoronaviruses, but also some deltacoronaviruses. The detection rate of coronaviruses was improved by using multiple PCR assays, as no single assay could detect all coronavirus positive samples. Sequencing of the relatively conserved Orf1 PCR amplicons found that Australian duck gammacoronaviruses were similar to duck gammacoronaviruses around the world. Some sequenced shorebird gammacoronaviruses belonged to Charadriiformes lineages, but others were more closely related to duck gammacoronaviruses. Australian duck and heron deltacoronaviruses belonged to lineages with other duck and heron deltacoronaviruses, but were almost 20% different in nucleotide sequence to other deltacoronavirus sequences available. Deltacoronavirus sequences from shorebirds formed a lineage with a deltacoronavirus from a ruddy turnstone detected in the United States. Given that Australian duck gammacoronaviruses are highly similar to those found in other regions, and Australian ducks rarely come into contact with migratory Palearctic duck species, we hypothesise that migratory shorebirds are the important vector for moving wild bird coronaviruses into and out of Australia.

Human parechovirus type 3 (HPeV3) can cause severe sepsis-like illness in young infants and may be associated with long term neurodevelopmental delay later in childhood. We investigated the molecular epidemiology of HPeV infection in thirty three infants requiring hospitalization before, during and after the peak of the 2017/18 HPeV epidemic wave in Australia. During the peak of the epidemic, all cases were infected with an HPeV3, while before and after the peak, HPeV1 was the predominant type detected. The predominant HPeV3 was the recombinant HPeV3 also detected in the 2013/14 and 2015/16 Australian epidemics. Sepsis-like or meningitis-like symptoms were only reported in cases infected with the recombinant HPeV3. Phylogenetic analysis of the recombinant HPeV3 revealed that the virus continued to evolve, also between the Australian outbreaks, thus indicating continued circulation, despite not being detected and reported in Australia or elsewhere in between epidemic waves. The recombinant HPeV3 continued to show a remarkable stability in its capsid amino acid sequence, further strengthening our previous argument for development of a vaccine or immunotherapeutics to reduce the severity of HPeV3 outbreaks due to this virus.

Chlamydia gallinacea is a recently described bacterial species in a genus known to infect and cause disease in animals and humans. Our report describes the identification of C. gallinacea infection in free‐range laying chickens (Gallus gallus) in Australia, and the identification of C. gallinacea infection in a parrot, a wild Australian galah (Eolophus roseicapillus). There is currently little knowledge of the effects of C. gallinacea infection on avian hosts, but it has been linked to respiratory disease in humans and could potentially cause similar disease in other species. Our report highlights the need for further study and surveillance of Chlamydia species in both wild and domestic hosts in Australia.

Bacterial chondronecrosis and osteomyelitis (BCO) is increasingly recognized as a major cause of lameness in commercial broilers chickens worldwide, but the pathogenesis of the condition is incompletely understood. This was a longitudinal study of 20 commercial broiler farms in Victoria, Australia, to investigate the aetiology and pathology of BCO. Thorough postmortem examination was performed on culled and dead birds (n = 325) from 20 different flocks at either 1 week, 4 weeks or 5 weeks of age and samples were analysed by conventional bacteriology, molecular identification of infectious organisms detected, serology and histopathology. BCO occurs throughout the life of broiler flocks at a very high rate, with lesions detected in 28% (95% CI 23-34%) of the mortalities and culls. The condition occurs with similar prevalence in both the femur and tibiotarsus. BCO is an infectious process that appears to result from bacteraemia and haematological spread of bacterial pathogens, especially Escherichia coli, to the bones, with 65.3% bacterial isolates from histologically confirmed BCO identified as E. coli, 11.5% as Staphylococcus and the remainder composed of mixed infections or a range of other minor isolates. We observed that almost all E. coli isolated from cases of BCO are avian pathogenic E. coli, suggesting that preventative measures should be directed at this organism.

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.