Genetic Diversity and Expanded Host Range of J Paramyxovirus Detected in Wild Small Mammals in China

J paramyxovirus (JPV) is a rodent-borne Jeilongvirus isolated from moribund mice (Mus musculus) with hemorrhagic lung lesions trapped in the 1972 in northern Queensland, Australia. The JPV antibodies have been detected in wild mice, wild rats, pigs, and human populations in Australia. Here, by next-generation sequencing (NGS), we detected JPV from M. musculus in Shandong Province of China. Molecular detection of JPV was performed to survey to survey the infection among 66 species of wild small mammals collected from six eco-climate regions in China by applying JPV specific RT-PCR and sequencing. Altogether, 21 out of 3070 (0.68%) wild small mammals of four species were positive for JPV, including 5.26% (1/19) of Microtus fortis, 3.76% (17/452) of M. musculus, 1.67% (1/60) of Apodemus peninsulae, and 0.48% (2/421) of Apodemus agrarius, which captured three eco-climate regions of China (northeastern China, northern China, and Inner Mongolia-Xinjiang). Sequence analysis revealed the currently identified JPV was clustered with other 14 Jeilongvirus members, and shared 80.2% and 89.2% identity with Australia’s JPV partial RNA polymerase (L) and glycoprotein (G) genes, respectively. Phylogenetic analysis demonstrated the separation of three lineages of the current JPV sequences. Our results show three new hosts (A. agrarius, A. peninsulae, and M. fortis) for JPV, most of which were widely distributed in China, and highlight the potential zoonotic transmission of JPV in humans. The detection of JPV in wild small mammals in China broaden the viral diversity, geographical distribution, and reservoir types of JPV. Future studies should prioritize determining the epidemiological characteristics of JPV, so that potential risks can be mitigated.


Introduction
The Paramyxoviridae family currently comprises RNA viruses of four subfamilies, 17 genera, and 86 species (https://talk.ictvonline.org/, accessed on 15 November 2022), many with high zoonotic potential. The members of the Paramyxoviridae family are negativesense single-stranded RNA viruses, and some of them are capable of infecting a wide range of natural hosts, from reptiles, birds, and fish to a variety of mammals, including humans [1]. Of special interest is the newly proposed genus Jeilongvirus, in the subfamily Orthoparamyxovirinae, to which the Henipavirus, Morbillivirus, and Respirovirus genera belong [2,3]. Several unclassified rodent-borne and bat-borne viruses that have emerged in the past decade have been classified to this newly established genus, attaining a total of 15 paramyxoviruses species, including eight rodent-borne viruses (J paramyxovirus, JPV; Beilong virus, BeiV; rodent paramyxovirus, RoPV; ruloma virus, RulV; Mount Mabu Lophuromys virus 1, MMLV-1; Mount Mabu Lophuromys virus 2, MMLV-2; Pohorje Myodes paramyxovirus 1, PMPV-1; Tailam virus, TaiV), five bat-borne viruses (Miniopterus schreibersii paramyxovirus, MisPV; bat Paramyxovirus 16797, BatPV-1; bat paramyxovirus 17770, BatPV-2; bat paramyxovirus, BatPV-3; Shaan virus, ShaV), belerina virus (BeV) from erinaceus, and feline paramyxovirus (FPaV) from felis catus, demonstrating the vast host range of Jeilongviruses [4][5][6][7][8][9]. Although the Paramyxoviridae family of viruses has been previously recognized as biomedically and veterinarily important, the recently emerging Jeilongviruses have been rarely investigated, particularly for JPV. JPV was first isolated in 1972 from moribund mice (Mus musculus) trapped in northern Queensland, Australia, by using kidney autoculture [10]. Extensive hemorrhagic lung lesions were shown in the mice from which the virus was isolated, indicating pathogenicityin rodent hosts [10]. In the subsequent study performed in Australia, JPV-LW (named after Lin-Fa Wang, who determined its sequence) that did not cause any disease in JPV-LW-infected mice was determined [11]. JPV antibodies have been detected in wild mice, wild rats, pigs, and human populations in Australia [10], suggesting a wide host range of JPV. Our previous study had confirmed the existence of BeiV in a high variety of wild animals across six ecoclimate regions in China [12]. The current study was designed to determine the molecular evidence of JPV in a great variety of wild small mammals in a wide region of China.

Sample Collection
Wild small mammals were captured with snap traps from 2013 to 2021 in 11 provinces, autonomous regions, and municipalities in China, i.e., Shandong, Guangdong, Zhejiang, Liaoning, Henan, Jilin, Heilongjiang, Yunnan, Xinjiang, Inner Mongolia, and Beijing. The spleen samples were collected and kept at −80 • C. The captured animals were morphologically identified, and confirmation of species identification was performed by amplifying the mitochondrial cytochrome b (mt-cyt b) gene region and determining the DNA sequence [13].

Next-Generation Sequencing (NGS)
Twelve randomly selected spleen samples from four dominant wild small animal species (M. musculus, Apodemus agrarius, Rattus norvegicus, and Tscherskia triton) in Shandong Province were subject to metagenomic analysis by NGS [14]. Briefly, total RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), from which rRNA was removed using the MGIEasy rRNA Depletion Kit (BGI, China). A highthroughput 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, trimming, and error removal, the remaining reads were mapped to NCBI viral reference genome sequences using Bowtie2 (version: 2.3.5.1) [15]. De novo assembly was performed using MEGAHIT (v1.2.9) software [16]. The assembled contigs were compared against the NCBI non-redundant nucleotide database using Blastn (version 2.12.0+) [17].

Reverse Transcription-PCR (RT-PCR) Sequencing for JPV
Total nucleic acid was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen) from spleen samples. The JPV screening was performed by PCR amplification of a 199-bp fragment of the glycoprotein (G) gene using JPV-specific primers (JPVF 5 -AAAACTTAGGAGTGAAT-GAGCGTC -3 and JPVR 5 -TGGTAAATAAGCTCAACAGTCCAG -3 ) designed based on genomic sequences obtained by metagenomic analyses and all nucleotide sequences from the currently determined JPV. RT-PCR amplification was performed using the PrimeScript™ One Step RT-PCR kit. RT-PCR conditions were identical to those described previously except for an annealing temperature of 55 • C for JPV. The 199-bp amplicon was separated on a 3% agarose gel. PCR products from positive samples were used to determine sequences with an applied biosystem (ABI 3730XL DNA sequencer). All PCR tests were conducted in parallel with the positive control (RNA from the positive sample) and the negative control (RNase-free water).

Phylogenetic Analysis
The nucleotide and amino acid sequences from the currently determined JPV and representative species belonging to the family Phenuiviridae that were downloaded from GenBank (Table S1) were aligned by the ClustalW method using MEGA-X [18]. Phylogenetic trees were constructed with MEGA-X by the maximum likelihood (ML) method with 1000 bootstrap replications. Based on the Bayesian information criterion (BIC), the best evolutionary model was selected to run the analysis and trees were generated using the general time reversible model.

Statistical Analysis
The chi-square or Fisher exact test was performed to compare the detection rate of JPV regarding the geographic region. 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.

Nucleotide Sequence Accession Numbers
The JPV partial sequences generated in this study were submitted to GenBank under the accession numbers OP795118-OP795139.

Study Site and Sample Collection
A total of 3070 wild small mammals that belonged to 66 species in six families were trapped, including 2518 from 29 species of the Muridae family, 239 from 15 species of the Cricetidae family, 148 from 12 species of the Soricidae family, 100 from 6 species of Sciuridae family, 47 from 2 species of Spalacidae family, and 18 from 2 species of the Dipodidae family ( Figure 1, Table S2). The 11 surveyed locations were grouped into six eco-climate regions [19]. Eco-climate regions were used for the geographic description.  Figure 1).

Eco-Climate Regions
Year  The nucleotide sequences of the current JPV (strain SD634) share 80.2% and 89.2% identity for partial RNA polymerase (L) and G genes, respectively, with Australian JPVs that were isolated from feral rodents in 1972 (GenBank accession numbers NC007454 and AY900001). The genome of JPV is composed of 18,954 nucleotides with a genome organization similar to that of other Jeilongviruses (Figure 2). Phylogenetic trees based on a 709-bp fragment of the L gene indicated that the current JPV strain SD634 grouped with previously described Australian JPVs, showing a close relationship with the other 14 species in the genus Jeilongvirus (Figure 1 (Table S2).

JPV Screening in Wild Small Mammals by RT-PCR
All specimens (n = 3070) were individually screened for JPV by RT-PCR using a selfdesigned primer targeting the JPV G gene. Altogether, 21 out of 3070 (0.68%) mammals of four species were tested positive for JPV (

Phylogenetic Analysis of JPV Sequences
For each of the 21 JPV-positive animals, partial sequences (199 bp) of the G gene were obtained for the phylogenetic analyses, together with sequences of 14 Jeilongvirus members retrieved from GenBank. Three major lineages were formed, which showed a close relatedness between the viral clades and their ecoclimate regions. For instance, strains from Inner Mongolia-Xinjiang were clustered into Lineage 1, strains from northeastern China were clustered into Lineage 2, and sequences from northern China were observed in all three lineages. No host specificity can be observed, since M. musculus can harbor all three lineages, and A. agrarius can harbor both Lineages 1 and 2. (Figure 4). The Australian JPV was clustered in Lineage 2, together with the current JPV from both northern and northeastern China (Figure 4).

Phylogenetic Analysis of JPV Sequences
For each of the 21 JPV-positive animals, partial sequences (199 bp) of the G gene were obtained for the phylogenetic analyses, together with sequences of 14 Jeilongvirus members retrieved from GenBank. Three major lineages were formed, which showed a close relatedness between the viral clades and their ecoclimate regions. For instance, strains from Inner Mongolia-Xinjiang were clustered into Lineage 1, strains from northeastern China were clustered into Lineage 2, and sequences from northern China were observed in all three lineages. No host specificity can be observed, since M. musculus can harbor all three lineages, and A. agrarius can harbor both Lineages1 and 2. (Figure 4). The Australian JPV was clustered in Lineage 2, together with the current JPV from both northern and northeastern China (Figure 4).

Discussion
The Paramyxoviridae family contains several zoonotic viruses, including highly pathogenic viruses, such as Nipah virus and Hendra virus, and an increasing number of largely uncharacterized animal viruses [20][21][22]. After its first emergence in Australia in

Discussion
The Paramyxoviridae family contains several zoonotic viruses, including highly pathogenic viruses, such as Nipah virus and Hendra virus, and an increasing number of largely uncharacterized animal viruses [20][21][22]. After its first emergence in Australia in 1972, no distribution in other countries was reported. Here, we determined JPV in three other rodent species than the already known one in Australia for the first time, indicating a diverse geographical range and a over long period during which JPV can circulate in China.
Recently, new Jeilongviruses have emerged from an extensive range of small mammals, however, very little is known about the distribution and diversity of the rapidly expanding genus Jeilongvirus. Among this variety of Jeilongvirus genus, BeiV and JPV have been highlighted with the potential for zoonotic spread to humans. Serological evidence suggests JPV has previously spilled over into the human population, although only limited to Australia [10], while BeiV was capable of cross-contaminating human cell cultures from rodent cell cultures [5]. JPV and BeiV have similar genome organizations and interchange-able genome replication machines. In contrast to most other paramyxoviruses, the JPV and BeiV V proteins did not interact with or inhibit signaling by STAT1 or STAT2; however, V proteins of both viruses interacted with melanoma differentiation-associated protein 5 (MDA5) and inhibited MDA5-dependent activation of the IFN-β promoter in HEK293T cells [23].
While both belong to the Jeilongvirus genus of the Paramyxoviridae family, JPV appeared to have a lower prevalence, host diversity, and a narrower geographic range as compared to BeiV. BeiV was discovered in a human kidney cell line in 2006 in Hong Kong, China [5], and R. norvegicus and R. rattus were suggested as natural reservoirs of BeiV by phylogenetic analysis of the naturally occurring BeiV [24]. Subsequent epidemiological studies from our group have determined the presence of BeiV in a total of 22 species of wild rodents and shrews in China, with R. norvegicus and R. rattus as the most predominant species that carry the virus [12]. In this study, the same batch of wild small mammals was tested for the presence of JPV, and thus, provided comparisons for the distribution and hosts of JPV and BeiV. It was determined that the three ecoclimate regions (northeastern China, Inner Mongolia-Xinjiang, and northern China) identified both viruses, and three rodent hosts (A. agrarius, A. peninsulae, and M. musculus) harbored both viruses. No codetection in one single sample was determined. After its first emergence in Australia in 1972, little is known about the ecology of JPV in other parts of the world. In this study, in addition to M. musculus, our results show three new potential hosts, A. agrarius, A. peninsulae, and M. fortis for JPV, most of which were widely distributed in China and wide range and highlight potential zoonotic transmission of JPV in humans. A wide screen for JPV is needed in regions where these potential hosts are highly abundant. Serological tests in wildlife and humans should also be carried out to extend the knowledge on its potential importance in causing disease.
In this study, although JPV was mainly tested in M. musculus from three ecoclimate regions, M. musculus is extensively distributed in China. It is plausible to assume that both ecological climates and topographic features as well as the ecological characteristics of animals, may play a role in the natural circulation of JPV. However, these results warrant further confirmation in future studies.
In conclusion, we determined the presence of JPV in four rodent species in China, also representing its first identification outside Australia. Although with low prevalence, these results expand on the known diversity of rodent species that can host JPV and their geographical distribution in the worldwide range. Further studies that combine active surveillance, viral isolation, and a phylogenetic approach might help to gain better knowledge on this scarcely investigated virus and guide the diagnosis and management of its potential infection in human beings.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v15010049/s1, Table S1: GenBank accession numbers of viruses used for phylogenetic analysis in this study; Table S2: Nucleotide similarity (%) between our J paramyxovirus and other 14 species in genus Jeilongvirus based on a 709-bp fragment of the L gene; Table S3: Positive rate of J paramyxovirus in wild small mammals in six eco-climate regions in China.