Identification of Reptarenaviruses, Hartmaniviruses and a Novel Chuvirus in Captive Brazilian Native Boa Constrictors with Boid Inclusion Body Disease

Abstract

Word number in abstract and importance: 221 and 143 Word number in text: 4687 ABSTRACT 3 Boid Inclusion Body Disease (BIBD) is a transmissible viral disease of captive snakes that 4 causes severe losses in snake collections worldwide. It is caused by reptarenavirus infection, 5 which can persist over several years without overt signs, but is generally associated with the 6 eventual death of the affected snakes. Thus far, reports have confirmed existence of 7 reptarenaviruses in captive snakes in North America, Europe, and Australia, but there is no 8 evidence that it also occurs in wild snakes. BIBD affects both boas and pythons, the habitats 9 of which do not naturally overlap. Herein, we studied Brazilian captive snakes with BIBD 10 using a metatranscriptomic approach, and report the identification of novel reptarenaviruses, 11 hartmaniviruses, and a new species in the family Chuviridae. The reptarenavirus L segments 12 identified represent six novel species, while we only found a single novel reptarenavirus S 13 segment. Until now, hartmaniviruses had been identified only in European captive boas with 14 BIBD, and the present results increase the number of known hartmanivirus species from four 15 to six. The newly identified chuvirus showed 38.4%, 40.9%, and 48.1% amino acid identity to 16 the nucleoprotein, glycoprotein, and RNA-dependent RNA polymerase of its closest relative, 17Guangdong red-banded snake chuvirus-like virus. Although we cannot rule out the possibility 18 that the found viruses originated from imported snakes, the results suggest that the viruses 19 would circulate in indigenous snake populations. 20Reptarenavirus NP expression was mainly restricted to the IBs (Figs. 2B and 3B, C), whereas 129 hartmanivirus NP was also detected throughout the cytoplasm (Fig. 2C). 130 Analysis of the identified reptarenavirus sequences. 131We used the PAirwise Sequence Comparison (PASC) web tool (available at 132 https://www.ncbi.nlm.nih.gov/sutils/pasc/viridty.cgi?textpage=overview), recommended by 133 the Arenaviridae study group of the International Committee on Taxonomy of Viruses 134 (ICTV) for arenavirus classification (16), to analyze the identified reptarenavirus segments. 135The PASC results showed that we had recovered CDSs for seven novel L and two novel S 136 segments ( Table 2). The PASC analysis identified one of the L segments in snake #1 as UHV-137

Arenavirus nucleoprotein localizes to mitochondria

“…mouse anti-α-tubulin (Calbiochem CP06, dilution 1:500 in immunoblot), rabbit anti-mitochondrial outer membrane translocase 20 (TOM20, Santa Cruz sc-11415, dilution 1:1000 in immunoblot), rabbit anti-cytochrome c oxidase subunit IV (COX IV, Abcam ab16056, dilution 1:2000 in immunoblot), mouse anti-voltage-dependent anion-channel (VDAC, Abcam ab14734, dilution 1:500 in immunoblot), rabbit anti-mitochondrial ribosomal protein S35 (MRPS35, Proteintech 16457-1-AP, dilution 1:1000 in immunoblot), mouse anti-mitofusin 2 (MFN2, Abcam ab56889, dilution 1:200 in immunoblot), mouse anti-mitochondria “mtCO2” (Abcam ab3298, clone mtCO2, dilution 1:1000 in IF), mouse anti-FLAG (Flarebio CSB-MA000021M0m, dilution 1:500 in immunoblot and 1:1000 in IF), rabbit anti-FLAG (Flarebio CSB-PA000337, dilution 1:1000 in IF), mouse anti-HA (Sigma, H3663, dilution 1:500 in immunoblot and 1:1000 in IF), rabbit anti-HA (Flarebio, CSB-PA275079, dilution 1:1000 in IF), affinity-purified rabbit anti-UHV-NP 49 (dilution 1:500 in immunoblot), rabbit anti-pan reptarenavirus NP 39 (anti-pan-RAVs NP, dilution 1:6000 in IF and 1:1000 in immune EM). The polyclonal rabbit antiserum against the putative MTSs of arenavirus NPs (named MTS-NP) was raised against a synthetic multiepitope protein with the following sequence: MAALQRAAVNQLALKKKLNKMLSPFQRELNNQIFGGGGGMAALQEAAVNQLALEE ELNEMLSPFQEELNNQIFGGGGGMSLSKEVKSFQWTQALRRELQGGGGGMSLSEEV ESFQWTQALEEELQGGGGGMAAFQKAAVNQLALKKKLNKMLAPYQRELNNQIFGG GGGMAAFQEAAVNQLALEEELNEMLAPYQEELNNQIFGGGGGMAHSKEVPSFRWT QSLRRGLSGGGGGMAHSEEVPSFEWTQSLEEGLSHHHHHH.…”

“…The IBs, comprising mainly reptarenavirus NP 29,31,34 , appear not to be cytopathic 31,35 . However, snakes with BIBD often die from secondary bacterial, fungal or protozoal infections, or neoplastic processes 36,37 , suggesting that reptarenaviruses cause immunosuppression 38,39 . This is supported by the fact that snakes with BIBD carry IBs in leukocytes and myelopoietic cells and often exhibit a weak antibody response against reptarenavirus NP 31,38,40 .…”

Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia

“…This Hardyhead chuvirus displayed three open reading frames (ORFs), representing the L protein (RdRp), glycoprotein and nucleoprotein. Our analysis identified Guangdong red-banded snake chuvirus [18] as the closest relative of the L protein (44% amino acid similarity), Wenling fish chu-like virus [18] as the closest relative of the glycoprotein (41%), and Herr Frank virus 1 [41] as the closest relative of the nucleoprotein (34%). Hardyhead chuvirus formed a distinct phylogenetic clade with all other vertebrate-associated chuviruses (Figure 5).…”

Experimental Reptarenavirus Infection of Boa constrictor and Python regius

“…Negative control slides ( B and D ). IHC employing a broadly cross-reactive rabbit anti-pan-reptarenavirus antiserum (25), hematoxylin counterstain. Bars = 20 µm.…”

“…Sections (3-4 µm) were prepared and stained with hematoxylin-eosin (HE). For all RT-PCR-positive animals, consecutive sections were prepared and subjected to IHC for viral NP, employing the recently described broadly cross-reactive rabbit anti-pan-reptarenavirus antiserum (25), following a previously described protocol (9, 23).…”