Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete ‘infectome’ (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing (“metatranscriptomics”) we documented the presence of contaminant viral sequences in multiple ‘blank’ negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae, Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.

The iron prawn syndrome (IPS), also described as sexual precocity, results in the reduced size of farmed prawns at harvest and significant economic losses. IPS has been frequently reported in populations of farmed Macrobrachium rosenbergii since 2010, but the cause was heretofore unknown.

The diversity of pathogens associated with acute respiratory infection (ARI) makes diagnosis challenging. Traditional pathogen screening tests have a limited detection range and provide little additional information. We used total RNA sequencing (“meta-transcriptomics”) to reveal the full spectrum of microbes associated with paediatric ARI. Throat swabs were collected from 48 paediatric ARI patients and 7 healthy controls. Samples were subjected to meta-transcriptomics to determine the presence and abundance of viral, bacterial, and eukaryotic pathogens, and to reveal mixed infections, pathogen genotypes/subtypes, evolutionary origins, epidemiological history, and antimicrobial resistance. We identified 11 RNA viruses, 4 DNA viruses, 4 species of bacteria, and 1 fungus. While most are known to cause ARIs, others, such as echovirus 6, are rarely associated with respiratory disease. Co-infection of viruses and bacteria and of multiple viruses were commonplace (9/48), with one patient harboring 5 different pathogens, and genome sequence data revealed large intra-species diversity. Expressed resistance against eight classes of antibiotic was detected, with those for MLS, Bla, Tet, Phe at relatively high abundance. In summary, we used a simple total RNA sequencing approach to reveal the complex polymicrobial infectome in ARI. This provided comprehensive and clinically informative information relevant to understanding respiratory disease.

Background: Rotavirus A (RVA) and adenovirus (Adv) are important causes of acute diarrhea in children. RVAs are classified into G and P genotypes based on viral proteins (VP)7 and VP4 gene and Adv contains over 70 genotypes based on hexon and fiber gene. This study aimed to characterize the molecular epidemiology of RVA and Adv in children with acute diarrhea during 2017–2018 in Hangzhou. Methods: The stool samples were collected and tested for RVA and Adv by reverse transcription-quantitative PCR (RT-qPCR) assay. The RVA positive samples were detected by RT-PCR for VP7(G) and VP4([P]) genotypes, and the Adv positive samples were detected by PCR for genotyping by the target to hexon gene. Results: Among 228 RVA-positive samples, G9 was detected as the most frequent genotype (195/228, 85.5%), followed by G3 (20/228, 8.8%), G2 (7/228, 3.1%) and G1 (6/228, 2.6%). G9 strains were closely related to strains from China and neighboring countries, as well as the USA. On the other hand, P[8] strains were detected in 219 (96.1%) samples with most closely related to one strain from Malawi, and P[4] in 9 (3.9%) samples. G9P[8] (84.6%, 193/228) was the most prevalent rotavirus A strains, followed by G3P[8] (8.8%, 20/228), G2P[4] (3.1%, 7/228), G1P[8] (2.6%, 6/228) and G9P[4] (0.9%, 2/228). Of 167 Adv-positive cases, 2 different genotypes were identified with 152 (91.0%) of Adv-41and 15 (9%) of Adv-40. All Adv strains were closely related to prototype strains of Adv types 40 and 41 in India. Conclusions: G9P[8] of RVA and Adv-41 were the most common genotypes that caused children’s acute diarrhea in Hangzhou, 2017–2018.

Papillomaviruses infect the skin and mucosal surfaces of diverse animal hosts with consequences ranging from asymptomatic colonization to highly malignant epithelial cancers. Increasing evidence suggests a role for papillomaviruses in the most common cutaneous malignancy of domestic cats, squamous cell carcinoma (SCC). Using total DNA sequencing we identified a novel feline papillomavirus in a nasal biopsy taken from a cat presenting with both nasal cavity lymphoma and recurrent squamous cell carcinoma affecting the nasal planum. We designate this novel virus as Felis catus papillomavirus 6 (FcaPV6). The complete FcaPV6 7453 bp genome was similar to those of other feline papillomaviruses and phylogenetic analysis revealed that it was most closely related to FcaPV3, although was distinct enough to represent a new viral type. Classification of FcaPV6 in a new genus alongside FcaPVs 3, 4 and 5 is supported. Archived excisional biopsy of the SCC, taken 20 months prior to presentation, was intensely positive on p16 immunostaining. FcaPV6, amplified using virus-specific, but not consensus, PCR, was the only papillomavirus detected in DNA extracted from the SCC. Conversely, renal lymphoma, sampled at necropsy two months after presentation, tested negative on FcaPV6-specific PCR. In sum, using metagenomics we demonstrate the presence of a novel feline papillomavirus in association with cutaneous squamous cell carcinoma.

Viral pathogens are being increasingly described in association with mass morbidity and mortality events in reptiles. However, our knowledge of reptile viruses remains limited. Herein, we describe the meta-transcriptomic investigation of a mass morbidity and mortality event in a colony of central bearded dragons (Pogona vitticeps) in 2014. Severe, extensive proliferation of the respiratory epithelium was consistently found in affected dragons. Similar proliferative lung lesions were identified in bearded dragons from the same colony in 2020 in association with increased intermittent mortality. Total RNA sequencing identified two divergent DNA viruses: a reptile-infecting circovirus, denoted bearded dragon circovirus (BDCV), and the first exogeneous reptilian chaphamaparvovirus—bearded dragon chaphamaparvovirus (BDchPV). Phylogenetic analysis revealed that BDCV was most closely related to bat-associated circoviruses, exhibiting 70% amino acid sequence identity in the Replicase (Rep) protein. In contrast, in the nonstructural (NS) protein, the newly discovered BDchPV showed approximately 31%–35% identity to parvoviruses obtained from tilapia fish and crocodiles in China. Subsequent specific PCR assays revealed BDCV and BDchPV in both diseased and apparently normal captive reptiles, although only BDCV was found in those animals with proliferative pulmonary lesions and respiratory disease. This study expands our understanding of viral diversity in captive reptiles.