Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis)

Abstract

In 2014 we observed a noticeable increase in sudden deaths of green tree pythons (). Pathological examination revealed accumulation of mucoid material within airways and lung, associated with enlargement of the entire lung. We performed full necropsy and histological examination on 12 affected green tree pythons from 7 different breeders to characterise the pathogenesis of this "mucinous" pneumonia. By histology we could show a marked hyperplasia of the airway epithelium and of faveolar type II pneumocytes. Since routine microbiological tests failed to identify a causative agent, we studied lung samples of a few diseased snakes by next-generation sequencing (NGS). From the NGS data we could assemble a piece of RNA genome <85% identical to nidoviruses previously identified in ball pythons and Indian pythons. We then employed RT-PCR to demonstrate the presence of the novel nidovirus in all diseased snakes. To attempt virus isolation, we established primary cell cultures of liver and brain, which we inoculated with lung homogenates of infected individuals. Ultrastructural examination of concentrated cell culture supernatants showed the presence of nidovirus particles, and subsequent NGS analysis yielded the full genome of the novel virus, Morelia viridis nidovirus (MVNV). We then generated an antibody against MVNV nucleoprotein, which we used alongside RNA hybridisation to demonstrate viral antigen and RNA in the affected lungs. This suggests that in natural infection MVNV damages the respiratory tract epithelium which then results in epithelial hyperplasia, most likely as an exaggerated regenerative attempt in association with increased epithelial turnover. Fairly recently novel nidoviruses associated with severe respiratory disease were identified in ball pythons and Indian pythons. Herein we report isolation and identification of a further nidovirus from green tree pythons () with fatal pneumonia. We thoroughly characterize the pathological changes in the infected individuals, and show that nidovirus infection is associated with marked epithelial proliferation in the respiratory tract. We speculate that this and the associated excess mucus production can lead to the animals' death, by inhibitingthe normal gas exchange in the lung. The virus was predominantly detected in the respiratory tract, which renders transmission via the respiratory route likely. Nidoviruses cause sudden outbreaks with high mortality in breeding collections, most affected snakes die without prior clinical signs. These findings, together with those of other groups, indicate that nidoviruses are a likely cause of severe pneumonia in pythons.

Investigations into the presence of nidoviruses in pythons

“…These sequences are a little bit shorter than the other published fulllength sequences of snakes, but belong still to the longest RNA genomes. The sequence identity to the other published genomes is rather low (< 66.9% on nucleotide sequence) with the highest similarity to the virus described in green tree pythons from Switzerland (Table 3) [7], whereas the three sequences published in 2014 are more similar to each other. Nevertheless, all reptile nidoviruses cluster together within the genus Pregotovirus (Fig.…”

“…After that, a working group from Switzerland detected a nidovirus in different captive breeding colonies of green tree pythons (Morelia viridis). Twelve deceased animals with pneumonia were investigated and this virus shared only < 85% sequence similarity to the previous published sequences [7]. Marschang and Kolesnik detected the virus not only in deceased animals, but also in swab samples from living animals.…”

“…These two sequences share a nucleotide identity of 99.7% to each other. The highest overall sequence similarity to published sequences appears to be 66.8 and 66.9%, respectively to a nidovirus sequence derived from a green tree python in Switzerland (MF351889 [7]). The overall sequence similarities to other published snake nidoviruses is between 64.8 and 65.5%.…”

“…To confirm the results from the metagenomic analysis different RT-qPCRs and RT-PCRs for the snake nidoviruses as well as a RT-qPCR for the snake retrovirus were developed. The snake nidovirus RT-qPCRs were compared to other published RT-qPCRs [7] and thereby the results further validated (Additional file 1: Table S1). For this, 60 negative and 46 positive oral swabs were investigated using two different PCRs.…”

“…We further validated the new developed real-time PCR by comparing it to the PCR used from Dervas et al [7]. In total, 60 negative and 46 positive swabs were tested using both methods.…”