Sample collection
In 2014, several snakes in a collection of Green Tree pythons suffered from severe stomatitis and pneumonia (up to 25 animals during several weeks). Bacteriological investigations of oral swabs yielded different results in the diseased snakes. Mycological and virological investigations (paramyxovirus, arenavirus, reovirus, adenovirus) were negative. Direct microscopic evaluation of mucus showed no parasites; parasitological investigations of feces were also negative. The stomatitis was treated locally (cleansing, antibiotic ointments or fluids). For the pneumonia, a systemic treatment with antibiotics was carried out according to an antibiogram following bacteriological investigations of oral swabs. As a supportive treatment, infusions (Ringer’s solution, 10 ml/kg) and ZylexisR were applied (twice in a week interval). MetacamR was used to calm down the inflammation. Despite this therapy, most of the infected snakes died or had to be euthanized (20 out of a collection of 60 snakes).
A total of 1554 captive boid snakes were screened for snake nidoviruses. The available samples included organ tissue samples (mostly lung, in some cases liver, kidney, small intestine, brain and pancreas) from deceased animals (230) as well as oral swabs or tracheal washes from living snakes (1324). Some animals were tested at several time points.
The oral swabs and tracheal washes were sent to the institute between 2015 and 2018 from different parts of Germany (all 16 federal states) and neighboring countries. This includes oral swabs from Denmark (78), 15 samples from a collection in Italy, 20 samples from five different collections in France and 117 samples from seven collections in Austria. Some of the animals showed typical symptoms (stomatitis and / or pneumonia), others without any symptoms were investigated to obtain an overview of the infection status of the collection.
Pathological examinations
Nine green tree snakes were sent for necropsy. Diagnostic workup in the chemical and veterinary investigation office included a gross pathology, histopathology, virology and bacteriology. Necropsy and following investigations have been carried out using standard techniques. In short, after pathological examination of the carcasses tissues were fixed in formalin and stained with Haematoxylin-Eosin. The tissues were evaluated microscopically. All snakes were investigated virologically. Suspensions of liver, lung, kidney and intestine were inoculated bacteria-free into Viper Heart Cells at 29 °C (ATCC CCL 140) and incubated for 1 week. Supernatant was transferred to a new cell culture and incubated for another week. Cells were inspected daily for the presence of cytopathic effects. PCRs for paramyxovirus [18], for reovirus [19], for reptarenavirus [20], ranavirus [21] and for adenovirus [22] were performed with organ tissues and cell culture supernatant. Bacteriological investigations from liver, kidney and lung were carried out in five snakes onto blood agar, incubated at 30 °C. Isolated bacteria were typed using MALDI TOF.
RNA extraction for NGS and RT-qPCR
Small pieces of tissue samples were homogenized in 1 mL PBS with a 5 mm steel bead in a TissueLyserII (Qiagen, Hilden, Germany). Dry swab samples taken from the trachea of the snakes were resuspended in 2 ml cell culture medium by shaking for half an hour at room temperature. For metagenomics analysis 250 μl of homogenized lung tissue was mixed with 750 μl Trizol Reagent (life technologies, Darmstadt, Germany). Afterwards chloroform was added and RNA from the aqueous phase was precipitated with 75% ethanol. RNA was further purified with RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and eluted in 50 μl RNase free water. For screening purposes nucleic acids from tissue or swab samples were extracted using the King Fisher 96 Flex (Thermo Fisher Scientific, Braunschweig, Germany) in combination with the NucleoMagVet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions and eluted in 100 μl.
Next-generation sequencing
Sample material from three deceased snakes was analyzed by a metagenomics workflow as described elsewhere [23, 24].
Conventional RT-PCR
In order to find specific primers for one published ball python nidovirus (KJ541759) [4] the primer designed tool Primer-BLAST was used [25]. Three different primer pairs were tested. The RT-PCR was done with the One step RT-PCR kit (Qiagen, Hilden, Germany) and uses a forward primer (5’CAA CTC TGC ACA AAC GCG AA 3′) and a reverse primer (5’CGG CGA TCT TGA TGT TGC TG 3′) amplifying a PCR product of approximately 300 bp. The temperature profile consists of a reverse transcription step at 50 °C for 45 min, an activation step at 94 °C for 15 min, followed by 40 cycles of 94 °C for 30 s, 57 °C for 45 s and 72 °C for 45 s as well as a final extension step at 72 °C for 5 min. The PCR products were visualized by electrophoreses on an agarose gel.
Snake nidovirus specific RT-qPCR
To confirm the results of the metagenomic workflow and to screen for further infected animals, a real-time RT-PCR for the detection of snake nidoviruses was developed. Primers and probes targeting the replicase open reading frame 1B (ORF1B) were selected based on an alignment of published sequence information (NCBI GenBank) together with the two newly generated complete coding sequences from this study. The PCR was performed with the AgPath-IDTM One-Step RT-PCR kit (Thermo Fisher Scientific, Braunschweig, Germany) and a snake nidovirus specific FAM-labelled primer-probe mix consisting of 800 nM Nido-Snake-20,528-F (5′ ACA TCT CGA GAC SAT YAT CCA 3′), 800 nM Nido-Snake-20,616-R (5′ CTG TAC TWG AAC AGA AYT CGT G 3′) and 200 nM Nido-Snake-20,579-FAM-as (5′ FAM-TTC CCA MGC YTT GTT CTS GTC GAC-BHQ1 3′) was used for broad-range nidovirus detection producing a 89 bp long fragment. For the RT-qPCR reaction, 10 μl master mix and 2.5 μl RNA were combined in a total reaction volume of 12.5 μl. The PCR was carried out using a Bio-Rad CFX 96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA) and the following temperature profile: 10 min at 45 °C for reverse transcription, 10 min at 95 °C for denaturation and 45 cycles of 15 s at 95 °C, 30 s at 56 °C and 30 s at 72 °C. In addition, a HEX-labelled internal control assay was used to monitor for efficient nucleic acid extraction [26].
Snake retrovirus specific SYBR green PCR
The metagenomics workflow provided some reads of snake nidoviruses. To see how frequent these viruses occur, a SYBR green based screening RT-qPCR was developed. The RT-qPCR reaction was prepared using a SensiFAST SYBR No-Rox Kit (Bioline, London, United Kingdom) in a volume of 10 μl including 400 nM Snake_RV_2624-F (5′ ACA GTG CCT GAC CCA TAC AC 3′), 400 nM Snake_RV_2716-R (5′ AAG ACC AAA ATG CAT CTT TCA GAT C 3′), 400 nM Snake_RV_2773-R (5′ TGT ATC TGG GTC AGT CCA TTC AA 3′) and 2 μl of extracted RNA. The reaction was performed for 10 min at 45 °C for reverse transcription, 2 min at 95 °C for activation of the polymerase, and 45 cycles of 5 s at 95 °C and 20 s at 60 °C followed by a melting curve analysis. The PCR was carried out using a Bio-Rad CFX 96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA).
Dideoxy chain termination sequencing
For phylogenetic analysis, primers were designed to generate a partial sequence of the ORF1AB RNA-dependet-RNA-polymerase gene by dideoxy chain-termination sequencing [27] from representative snake nidovirus RT-qPCR-positive samples. The amplification reaction was performed using the SuperScript III One-step RT-PCR Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) combined with the primers Nido-Snake-19,971-F (5′ ATC GGA GTC WCA AAA TTC CGA G 3′) and Nido-Snake-21,007-R (5′ CAC GTR TAG CAY TGC TGC TG 3′). For the amplification, 800 nM of each primer in 12.5 μl total reaction volume including 2.5 μl template RNA was used. The amplified PCR fragments were separated on agarose gels, visualized by staining with ethidium bromide and subsequently excised and purified using the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). Sequencing reactions of both strands were carried out with the primers used for amplification and the BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on a 3130 Genetic Analyzer (Thermo Fisher Scientific).
Phylogenetic analysis
The newly generated sequences of two full-length snake nidoviruses were aligned to each other as well as to further complete coding genome sequences from different members of the subfamily Tobaniviridae obtained from GenBank using the method MAFFT [28] as implemented in the Geneious software (version 10.2.1). Furthermore, the 36 partial ORF1AB sequences were aligned to each other and to other full-length snake nidovirus genomes. Based on these alignments, maximum-likelihood trees (PhyML) were calculated using the HKY85 model [29] with 1000 bootstrap replicates by the Geneious software [30].