Serpentoviruses: More than Respiratory Pathogens.

In recent years, nidoviruses have emerged as important respiratory pathogens of reptiles, affecting captive python populations. In pythons, nidovirus (recently reclassified as serpentovirus) infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe, often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full postmortem examinations on 30 serpentovirus reverse transcription-PCR (RT-PCR)-positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether this not yet reported wider distribution of lesions is associated with previously unknown serpentoviruses or changes in the serpentovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and obtain virus isolates. Immunohistochemistry and immunofluorescence staining against serpentovirus nucleoprotein demonstrated that the virus infects not only a broad spectrum of epithelia (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts, etc.), but also intravascular monocytes, intralesional macrophages, and endothelial cells. With next-generation sequencing we obtained a full-length genome for a novel serpentovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing serpentovirus infection-associated respiratory or systemic disease did not reveal sequence association to phenotypes; however, functional studies with different strains are needed to confirm this observation. The results indicate that serpentoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated viral systemic spread.IMPORTANCE During the last years, python nidoviruses (now reclassified as serpentoviruses) have become a primary cause of fatal disease in pythons. Serpentoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previous evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that comprises important zoonotic pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. Serpentoviruses belong to the same order as the above-mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order Nidovirales.

Detection of nidoviruses in live pythons and boas. / Nachweis von Nidoviren bei lebenden Pythons und Boas.

OBJECTIVES: Nidoviruses have recently been described as a putative cause of severe respiratory disease in pythons in the USA and Europe. The objective of this study was to establish the use of a conventional PCR for the detection of nidoviruses in samples from live animals and to extend the list of susceptible species. MATERIALS AND METHODS: A PCR targeting a portion of ORF1a of python nidoviruses was used to detect nidoviruses in diagnostic samples from live boas and pythons. A total of 95 pythons, 84 boas and 22 snakes of unknown species were included in the study. Samples tested included oral swabs and whole blood. RESULTS: Nidoviruses were detected in 27.4% of the pythons and 2.4% of the boas tested. They were most commonly detected in ball pythons (Python [P.] regius) and Indian rock pythons (P. molurus), but were also detected for the first time in other python species, including Morelia spp. and Boa constrictor. Oral swabs were most commonly tested positive. CONCLUSION: The PCR described here can be used for the detection of nidoviruses in oral swabs from live snakes. These viruses appear to be relatively common among snakes in captivity in Europe and screening for these viruses should be considered in the clinical work-up. CLINICAL RELEVANCE: Nidoviruses are believed to be an important cause of respiratory disease in pythons, but can also infect boas. Detection of these viruses in live animals is now possible and can be of interest both in diseased animals as well as in quarantine situations.

Discovery and Partial Genomic Characterisation of a Novel Nidovirus Associated with Respiratory Disease in Wild Shingleback Lizards (Tiliqua rugosa).

A respiratory disease syndrome has been observed in large numbers of wild shingleback lizards (Tiliqua rugosa) admitted to wildlife care facilities in the Perth metropolitan region of Western Australia. Mortality rates are reportedly high without supportive treatment and care. Here we used next generation sequencing techniques to screen affected and unaffected individuals admitted to Kanyana Wildlife Rehabilitation Centre in Perth between April and December 2015, with the resultant discovery of a novel nidovirus significantly associated with cases of respiratory disease according to a case definition based on clinical signs. Interestingly this virus was also found in 12% of apparently healthy individuals, which may reflect testing during the incubation period or a carrier status, or it may be that this agent is not causative in the disease process. This is the first report of a nidovirus in lizards globally. In addition to detection of this virus, characterisation of a 23,832 nt segment of the viral genome revealed the presence of characteristic nidoviral genomic elements providing phylogenetic support for the inclusion of this virus in a novel genus alongside Ball Python nidovirus, within the Torovirinae sub-family of the Coronaviridae. This study highlights the importance of next generation sequencing technologies to detect and describe emerging infectious diseases in wildlife species, as well as the importance of rehabilitation centres to enhance early detection mechanisms through passive and targeted health surveillance. Further development of diagnostic tools from these findings will aid in detection and control of this agent across Australia, and potentially in wild lizard populations globally.

Development of a real-time reverse transcription PCR assay for detection of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula).

AIMS: To develop a quantitative reverse transcription PCR (RT-qPCR) assay for detection of the putative wobbly possum disease (WPD) virus and to apply this test to investigate the viral load in archival tissues from past WPD transmission studies. METHODS: The real-time assay was developed as a two-step RT-qPCR in a SYBR green format and validated using serial dilutions of a linearised plasmid containing target DNA. The copy number values were normalised to the amount of RNA in each reverse transcription reaction and presented as the number of viral copies per µg of total [corrected] RNA. The viral load was determined in archival samples from animals that had received inoculations of infectious WPD tissue suspensions. Thirty samples originating from 22 possums, comprising five samples from three healthy possums and 25 samples from 19 possums that had received inoculations of infectious WPD tissue suspensions were tested. RESULTS: The assay was linear (R(2) > 0.99) within the tested range from 1 to 10(7) target copies/µL, with an efficiency of >90%. The intra-assay variability CV values ranged from 0.8 to 4.5% for different standards, with the inter-assay variability CV values ranging from 0.4 to 2.5%, indicating good precision and reproducibility of the assay. The novel nidovirus was detected in all 25 samples from WPD-affected possums. Tissues from three control possums and from one experimentally infected rabbit were negative for WPD RNA. The viral load in WPD-positive tissues differed between individual possums and between tissue types, ranging from 2.2 to 359,980 copies/pg RNA. The highest viral load was detected in liver, followed by brain, spleen, kidney and urine. There was a more than four log difference in the viral load between pools of tissues originating from two outbreaks of WPD in different geographical regions. CONCLUSIONS: Detection of viral RNA in a variety of tissues from WPD affected possums, including brain, is consistent with the multi-organ distribution of histopathological lesions observed in WPD. Our data suggest that liver may constitute the sample of choice for diagnostic testing. Differences in the viral load in tissues from possums inoculated with infectious WPD tissue suspensions from Rotorua or Invermay origin suggest that WPD viruses with different biological properties may exist. CLINICAL RELEVANCE: We have developed a RT-qPCR assay for detection of the putative WPD virus. The test showed good sensitivity and reproducibility over the wide dynamic range of template concentrations. It provides a tool for future diagnostic and research purposes.

RT-PCR detection of yellow head virus (YHV) infection in Penaeus monodon using dried haemolymph spots.

Sample collection and RNA isolation from shrimp haemolymph for RT-PCR diagnosis of yellow head virus (YHV) infections is crucial for disease control programs for cultivated shrimp in Thailand. Problems with RNA degradation arise when field samples must be collected far from the laboratory by relatively inexperienced personnel who do not have ready access to sophisticated reagents. In an attempt to solve this problem, haemolymph samples from shrimp were collected either by mixing with 10% (w/v) sodium citrate or by spotting on ISOCODE filter paper. RNA was extracted subsequently either by a rapid boiling method or by using TRI reagent and the extracts were used in a semi-quantitative, non-stop, semi-nested RT-PCR assay for YHV. Dried haemolymph spots on ISOCODE filter paper extracted with TRI reagent gave the most reliable and reproducible results. It also allowed longer periods of storage at room temperature.