Molecular markers for malaria genetic epidemiology: progress and pitfalls.

Over recent years, progress in molecular markers for genotyping malaria parasites has enabled informative studies of epidemiology and transmission dynamics. Results have highlighted the value of these tools for surveillance to support malaria control and elimination strategies. There are many different types and panels of markers available for malaria parasite genotyping, and for end users, the nuances of these markers with respect to 'use case', resolution, and accuracy, are not well defined. This review clarifies issues surrounding different molecular markers and their application to malaria control and elimination. We describe available marker panels, use cases, implications for different transmission settings, limitations, access, cost, and data accuracy. The information provided can be used as a guide for molecular epidemiology and surveillance of malaria.

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.

Metagenomic characterisation of avian parvoviruses and picornaviruses from Australian wild ducks.

Ducks can shed and disseminate viruses and thus play a role in cross-species transmission. In the current study, we detected and characterised various avian parvoviruses and picornaviruses from wild Pacific black ducks, Chestnut teals, Grey teals and Wood ducks sampled at multiple time points from a single location using metagenomics. We characterised 46 different avian parvoviruses belonging to three different genera Dependoparvovirus, Aveparvovirus and Chaphamaparvovirus, and 11 different avian picornaviruses tentatively belonging to four different genera Sicinivirus, Anativirus, Megrivirus and Aalivirus. Most of these viruses were genetically different from other currently known viruses from the NCBI dataset. The study showed that the abundance and number of avian picornaviruses and parvoviruses varied considerably throughout the year, with the high number of virus reads in some of the duck samples highly suggestive of an active infection at the time of sampling. The detection and characterisation of several parvoviruses and picornaviruses from the individual duck samples also suggests co-infection, which may lead to the emergence of novel viruses through possible recombination. Therefore, as new and emerging diseases evolve, it is relevant to explore and monitor potential animal reservoirs in their natural habitat.

Detection of a Reassortant H9N2 Avian Influenza Virus with Intercontinental Gene Segments in a Resident Australian Chestnut Teal.

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.

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.

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.

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

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.