Metagenomics detection and characterisation of viruses in faecal samples from Australian wild birds

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

Novel Human Parechovirus 3 Diversity, Recombination, and Clinical Impact Across 7 Years: An Australian Story

BACKGROUND A novel human parechovirus 3 Australian recombinant (HPeV3-AR) strain emerged in 2013 and coincided with biennial outbreaks of sepsis-like illnesses in infants. We evaluated the molecular evolution of the HPeV3-AR strain and its association with severe HPeV infections. METHODS HPeV3-positive samples collected from hospitalized infants aged 5-252 days in 2 Australian states (2013-2020) and from a community-based birth cohort (2010-2014) were sequenced. Coding regions were used to conduct phylogenetic and evolutionary analyses. A recombinant-specific polymerase chain reaction was designed and utilized to screen all clinical and community HPeV3-positive samples. RESULTS Complete coding regions of 54 cases were obtained, which showed the HPeV3-AR strain progressively evolving, particularly in the 3' end of the nonstructural genes. The HPeV3-AR strain was not detected in the community birth cohort until the initial outbreak in late 2013. High-throughput screening showed that most (>75%) hospitalized HPeV3 cases involved the AR strain in the first 3 clinical outbreaks, with declining prevalence in the 2019-2020 season. The AR strain was not statistically associated with increased clinical severity among hospitalized infants. CONCLUSIONS HPeV3-AR was the dominant strain during the study period. Increased hospital admissions may have been from a temporary fitness advantage and/or increased virulence

Molecular epidemiology of avian nephritis virus in commercial chicken flocks

© 2016 Dr. Anthony Neal ChamingsAvian Nephritis Virus (ANV) is an astrovirus which infects chickens, turkeys and pigeons. In commercial chicken production, the virus has been associated with poor growth, increased mortality and kidney disease (Imada et al., 1979; Imada et al., 1981). In 2009, the virus was identified for the first time in a flock of commercial meat chickens in Australia suffering from an increased mortality rate with gross and histological evidence of renal disease (Hewson et al., 2010). The work described in this thesis aimed to isolate, propagate and genetically characterize the Australian ANV isolates, and develop reliable methods for detection of the virus in clinical samples, determine the distribution of the virus in Australia, and to look for any association between the presence of the virus and poor performance or disease in commercial chickens. A robust PCR targeting the 3’ Untranslated region (UTR) of the virus was developed and found to be capable of detecting the virus in a range of clinical samples. In a survey of meat chicken flocks, the virus was detected by PCR in 84% of farms and 61% of flocks. The virus was detected in both clinically normal flocks and flocks showing signs of poor performance or disease. No statistical association between the presence of the virus and poor performance or disease was detected. In contrast to some overseas ANV isolates, Australian ANVs could not be cultured in vitro either in chicken cells or fertile eggs. These viruses however replicated readily in young SPF chickens. From the attempts to culture ANV in vitro, reovirus was found to be a common co-infection with ANV. Using PCR and molecular cloning in conjunction with High Resolution Melting (HRM) curve analysis of the UTR PCR amplicon, it was found that many flocks were simultaneously infected with two genetically different ANVs. Genetic sequencing of the Australian ANVs found unique genetic groups not described anywhere else in the world. Several of the ANV’s also showed genetic evidence of recombination with other ANVs. By comparing the pattern of recombination in ANVs isolated on different farms, it was found that farms with similar isolates of ANV were the progeny of parent flocks from the same breeder company. This indicated that ANV was likely being spread to broiler farms in or on fertile eggs. Rapid typing of ANV isolates using HRM curve analysis targeting different regions of the genome was explored. Two PCR-HRMs were tested in parallel to determine if they could discriminate between the isolates collected in this study. The combination of both tests could discriminate between most genetic groups of ANV, however the high frequency of co-infection with different genotypes limited the test’s capacity for routine diagnostics. To address this limitation, a mathematical method of determining the composition of a mixed sample from the HRM curves was developed. It was found that the shape of a melt curve from a mixed sample closely resembled the predict curve generated from the weighted average of the fluorescence of the melt curve of the pure samples. Using a database of these predicted mixed curves, the exact composition of, and predominant genotype in, the mixed ANV samples could be correctly determined in 70% and 90% of the cases respectively. Results of this study have made significant contribution to our understanding of the biology of ANV, and of the epidemiology and significance of ANV infection in the poultry industry

Metagenomic characterisation of additional and novel avian viruses from Australian wild ducks

Birds, notably wild ducks, are reservoirs of pathogenic and zoonotic viruses such as influenza viruses and coronaviruses. In the current study, we used metagenomics to detect and characterise avian DNA and RNA viruses from wild Pacific black ducks, Chestnut teals and Grey teals collected at different time points from a single location. We characterised a likely new species of duck aviadenovirus and a novel duck gyrovirus. We also report what, to the best of our knowledge, is the first finding of an avian orthoreovirus from Pacific black ducks and a rotavirus F from Chestnut teals. Other viruses characterised from the samples from these wild ducks belong to the virus families Astroviridae, Caliciviridae and Coronaviridae. Some of the viruses may have potential cross-species transmissibility, while others indicated a wide genetic diversity of duck viruses within a genus. The study also showed evidence of potential transmission of viruses along the East Asian—Australasian Flyway; potentially facilitated by migrating shorebirds. The detection and characterisation of several avian viruses not previously described, and causing asymptomatic but potentially also symptomatic infections suggest the need for more virus surveillance studies for pathogenic and potential zoonotic viruses in wildlife reservoirs

Exploring the cause of diarrhoea and poor growth in 8–11-week-old pigs from an australian pig herd using metagenomic sequencing

Diarrhoea and poor growth among growing pigs is responsible for significant economic losses in pig herds globally and can have a wide range of possible aetiologies. Next generation sequencing (NGS) technologies are useful for the detection and characterisation of diverse groups of viruses and bacteria and can thereby provide a better understanding of complex interactions among microorganisms potentially causing clinical disease. Here, we used a metagenomics approach to identify and characterise the possible pathogens in colon and lung samples from pigs with diarrhoea and poor growth in an Australian pig herd. We identified and characterized a wide diversity of porcine viruses including RNA viruses, in particular several picornaviruses—porcine sapelovirus (PSV), enterovirus G (EV-G), and porcine teschovirus (PTV), and a porcine astrovirus (PAstV). Single stranded DNA viruses were also detected and included parvoviruses like porcine bocavirus (PBoV) and porcine parvovirus 2 (PPV2), porcine parvovirus 7 (PPV7), porcine bufa virus (PBuV), and porcine adeno-associated virus (AAV). We also detected single stranded circular DNA viruses such as porcine circovirus type 2 (PCV2) at very low abundance and torque teno sus viruses (TTSuVk2a and TTSuVk2b). Some of the viruses detected here may have had an evolutionary past including recombination events, which may be of importance and potential involvement in clinical disease in the pigs. In addition, our metagenomics data found evidence of the presence of the bacteria Lawsonia intracellularis, Brachyspira spp., and Campylobacter spp. that may, together with these viruses, have contributed to the development of clinical disease and poor growth.</jats:p

Detection and characterisation of canine astrovirus, canine parvovirus and canine papillomavirus in puppies using next generation sequencing

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

Detection and characterisation of coronaviruses in migratory and non-migratory Australian wild birds

Abstract 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