Molecular Identication of a Novel Iavirus in Brown-Spotted Pitvipers (Protobothrops Mucrosquamatus)

Background: Iaviridae is a family of small non-enveloped viruses with monopartite, positive-stranded RNA genomes, which are identied in arthropod hosts, primarily infecting insect species. Herein, we rstly identify the sequence of an iavirus (YB-PMP20) found in brown-spotted pitvipers in China. Results: The sequence of YB-PMP20 showed high identity to the sequences of Hubei picorna-like virus (HUPV) (99.2% in nt), Vespa velutina-associated iavirus like virus (VVAIV) (58.6% in nt) and Lygus lineolaris virus (LyIV-1) (46.6% in nt) in nucleotides encoding polyproteins. It contained a single large ORF (304–9291 nt) encoding 2996 amino acids. The deduced amino acid sequences were compared with those of iavirus. Helicase, protease and the RdRp domain were found to be located at the 3´ end, and structural genes (VP1, VP2 and VP3) were found to be located at the 5´ end. Phylogenetic analysis indicated that YB-PMP20 belongs to the iavirus cluster, and is similar to HUPV, LyIV-1 and VVAIV. Conclusion: The present study described the genetic characterization of a PmIFV strain in brown-spotted pitvipers. Our genomic data extend knowledge of the diversity of viruses in snakes.

The present study rst investigated the brown-spotted pitviper (Protobothrops mucrosquamatus) i avirus (PmIFV), denoted YB-PMP20, which was identi ed from the feces of P. mucrosquamatus (Crotalinae, Viperidae) through high-throughput sequencing. Sequence and phylogenetic analyses indicated that YB-PMP20 showed high sequence identity with Hubei picorna-like virus 36 (HUPV 36) and Vespa velutina associated i a-like virus (VVIAV), which were isolated from insects and closely clustered with i avirus. This sequence analysis may contribute to understanding the evolution of i avirus in snakes.

Sample collection
From July to September 2020, we collected seven anal swabs from brown-spotted pitvipers with lassos from quebrada and bamboo forests located in the Laojun mountains, 110 kilometers from Yibin city in Sichuan province (Fig. 1). To prevent sample contamination, we placed the animals individually in sterilized tubs and cleaned their skins with 75% alcohol. All samples were collected opportunistically with sterilized swabs in areas where pitvipers were captured. The swabs were placed in RNase-free tubes and immediately transported on dry ice to Shanghai Biozeron Biothchnology Co., Ltd. (Shanghai, China.) the same day. The pitvipers were then released back into the wild.

Read quality control and mapping
The raw paired end reads were trimmed and subjected to quality control in Trimmomatic with parameters (SLIDINGWINDOW:4:15 MINLEN:75) (version 0.36 http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic). Then, clean reads that aligned to the host genome were also removed. This set of high-quality reads was then used for further analysis. A total of 10.0 gigabases (Gb) of paired-end reads was obtained for the sample.

Identi cation of the genome
Reverse transcription of 1 µg RNA from feces was conducted with random primers according to the manufacturer's protocol (TAKARA). Twelve pairs of primers were developed for PCR ampli cation, as previously described [27].

Sequence alignment and phylogenetic analysis
Sequence data were assembled and analyzed in Clustal X software and DNASTAR. To determine the relationship between the i avirus representative isolates and YB-PMP20 strain, phylogenetic trees based on the whole gene sequence were constructed in molecular evolutionary genetics analysis (MEGA) software (version 6.0) with the maximum-likelihood method. Bootstrap values were estimated for 1,000 replicates. The sequences obtained in this study were assembled and submitted to GenBank under the accession number MZ005704.

Results
The genome of YB-PMP20 was 9808 nucleotides (nt) in length, with a nucleotide composition of 2827, 2329, 2680 and 1972, A, G, T and C nucleotides, respectively. The G + C content of the YB-PMP20 genome was 43.85% higher than that of other i aviruses, including VVAILV144 (35.71%), Aedes I a-like virus However, the deduced VP4 was not found in the consensus sequences. RNA helicase domains were identi ed in the polyprotein from 1437-1583 aa, and showed 25.93% amino acid identity with 2C helicase from enterovirus 71 (EV71) and 2C ATPase of picornavirus. Three conserved helicase motifs (A, B and C) are present in the picornavirus and i avirus [30]. The highly conserved amino acids within motif A (GxxGxxGKS) and motif B (QxxxxxDD) were identi ed in the YB-PMP20 sequence, between amino acids 1449-1456 and 1495-1503. In YB-PMP20, the amino acids within motif C were KKxxxxPxxxxxATN, in contrast to the consensus motif, KGxxxxSxxxxxSTN. Proteases were identi ed from aa 2174-2379 and showed 22.29% amino acid identity with 3C protease from coxsackievirus. The putative residues H 2217 , E 2290 and C 2340 may form the catalytic triad in the protease. RdRp domains were identi ed in aa 2416-2974, and showed 20.86% amino acid identity with RdRp of sapporovirus. Eight conserved RdRp amino acid motifs are found in RNA viruses [30]. The putative RdRp conserved domains are shown in Fig. 3C. Furthermore, we constructed phylogenetic trees based on the full sequences, by using the maximum likelihood method. The i aviruses clustered in a large clade, and two subclades were present. The phylogenetic trees indicated that YB-PMP20 clustered in the same subclade with HUPV 36, LyIV-1 and VVILV.

Discussion
In present study, we rst identi ed and characterized an i avirus strain (YB-PMP20) from brown-spotted pitvipers without apparent clinical symptoms, which shows characteristics typical of the family I aviridae, including the capsid protein, helicase, protease and RdRp domains. Sequence analysis suggested that YB-PMP20 is similar to HUPV 36, LyIV-1 and VVAIV, identi ed in Diptera, Lygus lineolaris and Vespa velutina nigrithorax, respectively.
Metagenomic studies of invertebrate viruses have recently been undertaken. More than 220 invertebrate species, including 9 metazoan phyla, were identi ed at least 1,445 distinct virus genomes segments [31]. It indicated that invertebrates acted as viral vectors, could play a key role in transmit and reservoir of pathogens.
Snakes may be a common predator of monkeys [32], shell snails [33], kangaroos [33], shes [33], leeches [33], earthworms [33], frogs [33], tadpoles [33], sh eggs [34], lizards [35,36], eld voles [36] and shrews [36]. There are no reports to support the idea that snakes prey on insects. We speculated that 1) snakes may occasionally prey on insects, thus, resulting in transmission of the virus from insects to snakes; 2) generally, amphibians including frogs feed on insects, thus, resulting in pathogen transmission from insects to amphibians. Then, snakes preying on amphibians might become a reservoir of insect and amphibian pathogens. To investigate the epidemiology of i avirus in snakes, a large-scale survey is needed. Such an investigation would aid in understanding the prevalence of i avirus and across the range of all snake species.

Conclusion
We rst identi ed and characterized an i avirus strain (YB-PMP20) from brown-spotted pitvipers. Phylogenetic analysis indicated that YB-PMP20 belongs to the i avirus cluster, and is similar to HUPV, LyIV-1 and VVAIV. These ndings contribute to our understanding of the prevalence of viruses in snake species.

Declarations
Ethics approval and consent to participate The present study was approved by the Animal Ethics Committee of Yibin University, Yibin, China, according to the OIE standards for use of animals in research and education.

Consent for publication
Not applicable.

Availability of data and materials
The complete sequences obtained in this study have been submitted to the GenBank database (accession number: MZ005704).

Competing interests
The authors declare no competing interests.