key: cord-0723323-bs14oh7t
authors: Chen, Jin-Jin; Zhang, Xiao-Ai; Fan, Hang; Jiang, Fa-Chun; Jin, Mu-Zi; Dai, Ke; Wang, Ning; Zhang, Pan-He; Li, Xiao-Kun; Li, Hao; Shi, Wen-Qiang; Yang, Zhi-Cong; Fang, Li-Qun; Zhou, Hai-Sheng; Wei, Yue-Hong; Liu, Wei
title: Distribution and characteristics of Beilong virus among wild rodents and shrews in China
date: 2020-07-04
journal: Infect Genet Evol
DOI: 10.1016/j.meegid.2020.104454
sha: 0eca3f655763f8842de5632dc69aacab22221f59
doc_id: 723323
cord_uid: bs14oh7t

Beilong virus (BeiV), a member of the newly recognized genus Jeilongvirus of family Paramyxoviridae, has been reported with limited geographic and host scopes, only in Hongkong, China and from two rat species. Here, by next-generation sequencing (NGS) on dominant wild small animal species in 4 provinces in China, we obtained a complete sequence of BeiV strain from Rattus norvegicus in Guangdong, neighboring HongKong, China. We then made an expanded epidemiological investigation in 11 provinces to obtain the geographic distribution and genetic features of this virus. Altogether 7168 samples from 2005 animals (1903 rodents, 100 shrews, 2 mustelidaes) that belonged to 33 species of Cricetidae, Muridae, Sciuridae and Dipodidae family of Rodentia, 3 species of Soricidae family of Soricomorpha, 2 species of Mustelidae family of Carnivora were examined by RT-PCR and sequencing. A positive rate of 3.7% (266/7168) was obtained that was detected from 22 animal species, including 5 species of Cricetidae family, 12 species of Muridae family, 2 species of Sciuridae family and 3 species of Soricidae family. Phylogenetic analyses based on 154 partial Large gene sequences grouped the current BeiV into two lineages, that were related to their geographic regions and animal hosts. Our study showed the wide distribution of BeiV in common species of wild rodents and shrews in China, highlighting the necessity of epidemiological study in wider regions.

The family Paramyxoviridae currently contains 78 species, organized in 17 genera (Metaavulavirus, Orthoavulavirus, Paraavulavirus, Synodonvirus, Aquaparamyxovirus, Ferlavirus, Henipavirus, Jeilongvirus, Morbillivirus, Narmovirus, Respirovirus, Salemvirus, Orthorubulavirus, Pararubulavirus， Cynoglossusvirus, Hoplichthysvirus, and Scoliodonvirus) (https://talk.ictvonline.org/). During the last decade, the discovery of many new viruses has revealed a much greater genetic diversity within family Paramyxoviridae than was previously recognized. Of special interest is the newly recognized genus Jeilongvirus, which contains 7 recognized species, namely Jun jeilongvirus (J-virus, JV) (Jack et al., 2005) , Beilong jeilongvirus (Beilong virus, BeiV) (Li et al., 2006) , Tailam jeilongvirus (Tailam virus, TaiV) (Woo et al., 2011) (Vanmechelen et al., 2018) , which were all identified from rodents.

Among this variety of Jeilongvirus members, BeiV was postulated to be originated from rodent, due to the previous amplification of BeiV from a rat kidney mesangial cell line (Li et al., 2006) and its close relationship to J virus and Tailam virus, both were of rodent sources. However, the epidemiological distribution and molecular evolution of BeiV remained to be rarely investigated, with epidemiology evidence only obtained from Hong Kong, China (Woo et al., 2012) . In a territory-wide molecular survey that was performed in 2009 (Woo et al., 2012) , BeiV was detected in 40 kidney and 9 spleen samples from 40 brown rats (Rattus norvegicus) and 3 black rats (R. rattus). In 2016, the same study group obtained the complete genome of the

From September 2013 to May 2019, wild small mammals, including rodents, shrews and mustelidaes were captured in 11 provinces (Beijing, Guangdong, Henan, Heilongjiang, Jilin, Liaoning, Inner Mongolia, Shandong, Xinjiang, Yunnan and Zhejiang) in China. The small animals were captured using snap traps and then identified by morphological features to the species level, which were further confirmed by sequencing of mitochondrial cytochrome b (mt-cyt b) gene (Nicolas et al., 2006) . For each animal, at least one of the following 6 sample types was collected: heart, liver, spleen, lung, kidney and intestine content. All samples were stored at -80°C until use.

The tissue samples from 5-10 animals of the same species and same sampling sites were pooled for metagenomic analysis by NGS. Briefly, the total RNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen, Germany), from which rRNA was removed using MGIEasy rRNA Depletion Kit (BGI, China). A high-throughput sequencing library was constructed using an MGIEasy RNA Library Prep Kit (BGI). Viral gene libraries were then sequenced using the MGI2000 platform (BGI), sequencer with pair-end (150-bp) reads. After processing the original data by filtering adapter contamination, cutting low-quality and complexity reads, we mapped the clean reads to a host genome sequence using BWA (Version: 0.7.15). Then the remaining reads were aligned to the non-redundant bacterial, virus, fungal, and parasite databases using BWA. The MEGAHIT (v1.1.2) software was used to assemble the reads to obtain the scaffold sequence. Blast (version 2.5.0+) software was used to compare the scaffold sequence obtained after assembly to the non-redundant nucleotide database (NT) and non-redundant protein database (NR) database sequence of NCBI and extract the valid J o u r n a l P r e -p r o o f Journal Pre-proof virus sequence. Specific primers were designed based on partial viral genomic sequences obtained by metagenomic analyses for PCR confirmation and whole genome sequencing of BeiV.

2.3. Reverse transcription-PCR (RT-PCR) sequencing for BeiV Total nucleic acid was extracted using AllPrep DNA/RNA Mini Kit (Qiagen) from tissue samples and using the QIAamp viral RNA minikit (Qiagen) from intestine contents following manufacturer's instructions. The BeiV screening was performed by PCR amplification of a 440-bp fragment of the large (L) gene, located at the 3′ end of the BeiV genome and used modified primers LPW9739-F: 5′-GGAGGATTCCCTCATAGRGAA-3′ and LPW9741-R: 5′-CTCATATGTATTTACATTTAAACCA-3′ (Woo et al., 2012) . PrimeScript™ One

Step RT-PCR Kit was used according to the manufacturer's instructions for BeiV detection. Briefly, the PCR mix was in a volume of 25 µl containing 12.5 µl of one Step Buffer (2×), 1 µl of PrimeScript one Step Enzyme Mix, 1 µl of PCR primer mix (10 µM of sense and antisense each), total RNA 2 µl and RNase free dH 2 O (8.5 µl). RT-PCR was carried with one cycle of 50ºC for 30 min and 94ºC for 2 min, followed by 40 cycles of 94ºC for 30 sec, 55ºC for 30 sec and 72 °C for 30 s in PCR System 9700 (Applied Biosystems, USA). Modified primers (5′-CTTACGARATTCGRGACCT-3′ and 5′-CCCRCTGTCAKWACTCACTA-3′) (Woo et al., 2016) targeting the 521-bp fragment of the attachment glycoprotein (G) gene were used to reconfirm the positive results. The specific PCR products were all sequenced by Sanger method.

Phylogenetic analysis involving all the species from family Paramyxoviridae revealed genetic diversity, which was correlated with the phylogenetic relationship between the BeiV sequences and other Paramyxoviridae species members (Table S1 ). The amino J o u r n a l P r e -p r o o f Journal Pre-proof acid sequences of L protein from the currently detected BeiV and other representative species from family Paramyxoviridae that were downloaded from GenBank were aligned by ClustalW method using MEGA 7 (Kumar et al., 2016) with a gap opening penalty of 5 and a gap extension penalty of 1. Phylogenetic analyses based on L protein were performed using RAxML v8.2.12 (Stamatakis, 2014) with JTT as the amino acid substitution model and 1,000 bootstrap replicates. Scale bars indicate protein substitutions per site, vertical bars represent the genera, and GenBank accession numbers are shown for the reference virus sequences.

To investigate the genetic feature of BeiV in relation with its host species and geographical regions, a phylogenetic tree was constructed using partial sequences 

The multivariate logistic regression analysis was performed to compare the detection rate of BeiV regarding the animal family, sample type, and geographic region, with 95% confidence intervals (CIs) and odds ratio (OR) estimated. The chi-square or Fisher's J o u r n a l P r e -p r o o f Journal Pre-proof exact test were used for comparison of categorical variables. Statistical analyses were performed using R (version 3.5.3). All statistical tests were 2-tailed, and a significance level (P) of 0.05 was used.

The BeiV sequences generated in this study were submitted to GenBank under the accession numbers MT085491 (full-length genome sequence), MT123352-MT123505 (partial L gene, Table S2 ) and MT649415-MT649456 (partial G gene, Table S3 ).

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Four pooled samples that were prepared from R. norvegicus in Guangdong, R. norvegicus in Henan, Apodemus agrarius in Beijing and A. agrarius in Shandong, were subjected to metagenomic analysis by NGS. BeiV specific sequences were determined from pools in Guangdong (1043 reads), Beijing (115 reads) and Shandong (18 reads). A nearly complete sequence of BeiV was composed based on 1043 reads from the R. norvegicus samples in Guangdong. A set of 7 specific primers (Table S4) were designed based on parital viral genomic sequences obtained by NSG to fill in the gaps.

Finally, we obtained the full-length genome sequence of BeiV genome (named Rodent Beilong virus) from R. norvegicus captured in Guangdong. To confirm the genome sequence of Rodent Beilong virus, we desinged 28 primer pairs (Table S5) for Sanger sequencing. The obtained consensus gene sequences were consistent with those from NGS, which were deposited in GenBank (accession number MT085491). The genome of the our rodents BeiV has 19,212 nucleotides with a G+C% content of 42.61%.

Sequence comparison revealed that the current Rodent Beilong virus was highly similar to previously described BeiV that was isolated from the MHC line (GenBank accession number NC007803, with 95.21% similarity) (Li et al., 2006) and other four rodents BeiV (GenBank accession number KX940961, KX940962, KX940963 and KX940964, 97.66-98.14% similarity) that were described in HongKong, China (Woo et al., 2016) . According to the phylogenetic trees based on L protein, the current rodents BeiV was clustered with previously desribed BeiV, and had a close relationship with other 6 species in genus Jeilongvirus while distinct from other members of family Paramyxoviridae ( Figure 1 ; Table S1 ). The multiple alignment showed 74.09% Table S6 ). Six sample types were collected, including heart (n = 914), liver (n = 1356), spleen (n = 1303), lung (n = 1989), 1236 kidney (n = 1236) and small intestine content (n = 370), with two sample types tested from763 animals and three sample collected from 732 animals . An overall positive rate of 3.71% (266/7168) was obtained from the detected samples. The highest detection rate was determined from kidney (6.15%,76/1236), followed by that in spleen (5.76%, 75/1303), and much lower detection rates from liver (2.95%, 40/1356), lung (2.82%, 56/1989), heart (2.08%,19/914). No positive detection was obtained from the 370 intestinal content. (Table S7 ). Four species of rodents and two sepcies of shrew had detection rate of higher than 15%, including 33.33% (1/3) in Myodes rufocanus, 28.57% (2/7) in Crocidura shantungensis, 20.83% (10/48) in Apodemus peninsulae, 17.57% (13/74) in Suncus murinus, 16.67% (1/6) in Allocricetulus eversmanni, and 15.52% (52/335) in R.

norvegicus (Table S7 ). Both of 2 tested species of Mustelidae family were negative for (1.08-56.63), respectively. The Cricetidae species was associated with significantly higher detection rate than the other species, with OR (95 % CIs) estimated to be 2.64

(1.08-6.46). The spleen and kidney had significantly higher detection rate than the other sample types (OR = 3.08, 95 % CIs: 1.83-5.18, and OR = 2.73, 95 % CIs:

1.62-4.60, respectively) ( Table 1) .

For each of the 154 BeiV-positive animals, partial sequences of L gene (440-bp) were obtained for the phylogenetic analyses. The current BeiVs were clustered in two major lineages, with mean(± standard deviation) of the amino acid sequence identity between two lineages as 95.78 ± 0.1%, lower than the identity within each lineage ( 98.28 ± 0.1% for Lineage 1 and 99.02 ± 0.1% for Lineage 2) (Figure 3a, Table S8 (Table   S9) .

From the 154 BeiV-positive animals, 42 partial sequences of G gene (521-bp) were obtained for the phylogenetic analyses, which was grouped into four distinct clusters that were related to geographic origins ( Figure S1 ). Sequences obtained from Northeast, North and South region were identical to those from the BeiV strain ERN081008_1S and 

Wild animals are extremely important natural hosts for the natural circulation of viral zoonosis (Bengis et al., 2004) , posing increased global public health concerns, with the very recent example of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) that were hypothesized to be originated from bats or from Pangolin (Zhou et al., 2020) . During the past few decades, many new viruses from the Paramyxoviridae family have been isolated either from disease outbreaks or surveillance programs or simply by chance (Wang et al., 2008) . Of special interest are the viruses originating from a wide range of animal hosts, such as bats, snakes, tree shrews, and rodents (Li et al., 2006) . In recent years, the continuous discovery and characterization of Paramyxoviruses from rodents further expanded the genome diversity of these viruses, such as Mossman virus (Miller et al., 2003) , Nariva virus (Lambeth et al., 2009) , Mojiang paramyxovirus (Wu et al., 2014) and seven recognized species of genus Jeilongvirus (Jack et al., 2005; Li et al., 2006; Vanmechelen et al., 2018; Woo et al., 2011; Wu et al., 2018) . In many of these reservoir host species, emerging viruses appear to be well adapted, with little or no evidence of clinical disease. However, the effects can sometimes be devastating when these viruses spill over into humans. This possibility underscored the need for persistent surveillance of these viral communities and the evolutionary processes that drive the emergence and adaptation of zoonotic viruses.

Here we provided evidence that BeiV, a rarely investigated Paramyxovirus, was widely distributed in a great variety of wide rodents and shrews in wide-range regions J o u r n a l P r e -p r o o f analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases. Microbiome 6, 178-178. Wu, Z., Yang, L., Yang, F., Ren, X., Jiang, J., Dong, J., Sun, L., Zhu, Y., Zhou, H., Jin, Q., 2014 . Novel Henipa-like virus, Mojiang Paramyxovirus, in rats, China, 2012 . Emerging infectious diseases 20, 1064 -1066 . Xing, Y., Zhou, L., Zhang, Y., Wang, X., 2008 

The role of wildlife in emerging and re-emerging zoonoses

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MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets

Complete genome sequence of Nariva virus, a rodent paramyxovirus

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Full-length genome sequence of Mossman virus, a novel paramyxovirus isolated from rodents in Australia

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RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies

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Disease outbreaks caused by emerging paramyxoviruses of bat origin

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Complete genome sequence of a novel paramyxovirus, Tailam virus, discovered in Sikkim rats

Comparative genome and evolutionary analysis of naturally occurring Beilong virus in brown and black rats

analyzed the data and wrote the manuscript

All of the authors reviewed the manuscript and approved the

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