key: cord-0327726-alhfsdu0 authors: Dervas, Eva; Hepojoki, Jussi; Smura, Teemu; Prähauser, Barbara; Windbichler, Katharina; Blümich, Sandra; Ramis, Antonio; Hetzel, Udo; Kipar, Anja title: Python nidoviruses, more than respiratory pathogens date: 2020-04-11 journal: bioRxiv DOI: 10.1101/2020.04.10.036640 sha: 7149b8b4c8a338d95a56a676b3916da200eddc7c doc_id: 327726 cord_uid: alhfsdu0 In recent years nidoviruses have emerged as an important respiratory pathogen of reptiles, affecting especially captive python populations. In pythons, nidovirus infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe and often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full post mortem examinations on 30 nidovirus RT-PCR positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether the atypical tissue tropism associates with previously unknown nidoviruses or changes in the nidovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and to obtain virus isolates. Immunohistochemistry and immunofluorescence staining against nidovirus nucleoprotein demonstrated that the virus not only infects a broad spectrum of epithelial (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts etc.), but also intravascular monocytes, intralesional macrophages and endothelial cells. By next-generation sequencing we obtained full length genome for a novel nidovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing nidovirus infection-associated respiratory or systemic disease did not explain the observed phenotypes. The results indicate that python nidoviruses 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 systemic spread of the virus. IMPORTANCE During the last years, python nidoviruses have become a primary cause of fatal disease in pythons. Nidoviruses 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 previously 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 compromise important zoonotic pathogens such as MERS-CoV and SARS-CoV, as well as the recently emerged SARS-CoV-2. Python nidoviruses belong to the same subfamily as the 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. ABSTRACT 28 In recent years nidoviruses have emerged as an important respiratory pathogen of reptiles, (from a single snake up to a collection of 50 breeding animals) in Switzerland and Spain (Table 128 1). Sample collection and screening for infectious agents. All animals were subjected to a full 130 post mortem examination and samples from brain, lung, liver and kidney were collected and 131 stored at -80 °C for further analysis. In addition, samples from all major organs and tissues 132 (brain, respiratory tract, liver, kidney, spleen, gastrointestinal tract, reproductive tract, pancreas) 133 were collected and fixed in 4% buffered formalin for histological and immunohistological 134 examinations. The lungs of some snakes were submitted to routine bacteriological examination, and from one 136 animal, a virological examination for common respiratory snake viruses was undertaken ( unrelated to any of the others, and from tissue culture supernatants (Table 1 ) as described (6). Library preparation, data analysis, and genome assembly was done as described (6). To study the frequencies of single nucleotide polymorphisms (SNPs) within samples, the NGS 170 reads were quality filtered using Trimmomatic and the reads with Q-score over 30 were 171 assembled against the consensus sequence of a given sample using the BWA-MEM algorithm 172 (15) followed by removal of potential PCR duplicates using SAMTools version 1.8 (16). The Gross presentation of nidovirus-associated disease in pythons. 197 Full post mortem examinations were performed on all cases. Most snakes that had died 198 spontaneously exhibited the typical, previously described respiratory changes, represented by a 199 varying amount of mucoid material in the airways and particularly in the lungs (Fig. 1A) . In Table 1 . found to be infected, without evidence of correlating brain lesions ( Fig 5D) . Table 1 ) and three positive liver specimens (CH-A7, -A8 and CH-B6, for four cell culture supernatants NGS failed due to severe bacterial contamination. For three snakes (CH-A7-8, CH-C3, Table 1 The initial NeighborNet analysis of snake-associated nidoviruses suggested strong evidence of 318 recombination (PHI test p<0.001) (Fig 7A) . Similarly, the tree order scan suggested a high amount of phylogeny violations across the genome. Therefore, we conducted a more detailed 320 recombination analysis. This analysis suggested at least five highly supported recombination 321 events. Morelia viridis nidovirus isolates BH171/14-7 and BH128/14-12 (33) cluster together with whereas between nucleotides 25205-37482 it clusters with CH-A5 (MK722363) (Fig 7D) . In One Swiss breeder (CH-B, Table 1 ) lost one of three juvenile carpet pythons from the same 361 clutch that had been housed separate from each other, but in the same room (distance less than infection. In addition to the above snakes, we studied choanal and/or cloacal swabs from seven additional 369 animals from different collections for the presence of nidovirus RNA by RT-PCR. All of the 370 tested animals were found positive for nidovirus infection (Table 1) Table 1 ), and a necrotzing splenitis (CH-A1 and CH-C4, Table 1 ) and fibrinonecrotic enteritis 380 with intralesional coccoid bacterial colonies (E-B1, Table 1 ). Both conditions are likely a 381 consequence of debilation/dehydration due to chronic pulmonary disease. In our study, no differences were noted between the python species in the degree and distribution which has an impact on the blood gas exchange (40). In some snakes, we additionally observed 435 an esophagitis; this is interpreted as a consequence of overspill and swallowing of virus-laden 436 mucus, a theory also supported by previous studies (6, 7, 40) . It is possible that the esophagus is Table 1 . Animals, clinical signs, lesions and viral target cells. All animals were from breeding collections in Switzerland (CH) or Spain (E). Breeders CH A-C and CH-F were working closely together, trading/exchanging snakes. 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Antigen Distribution and Inflammatory Response Cytokine responses in severe acute respiratory 729 syndrome coronavirus-infected macrophages in vitro. Possible relevance to pathogenesis Mediators in the Mesenteric Lymph Nodes, Site of a Possible Intermediate Phase in the 733 Immune Response to Feline Coronavirus and the Pathogenesis of Feline Infectious 734 Peritonitis? Feline infectious peritonitis. Still an enigma? Veterinary 736 Acquisition of 738 macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by 739 mutations in the feline coronavirus spike protein Murine coronavirus mouse hepatitis virus is 741 recognized by MDA5 and induces type I interferon in brain macrophages/microglia Feline coronavirus: Insights into viral pathogenesis based 744 on the spike protein structure and function 582 The authors are grateful to the technical staff of the Histology Laboratory for excellent technical 583 assistance. The study was supported by the Academy of Finland (1308613) and the Finnish 584 Foundation of Veterinary Research. We also wish to thank the snake owners for submitting the 585 cases.