key: cord-0309716-1gocdcne
authors: Lee, Seung-Ho; No, Jin Sun; Kim, Kijin; Budhathoki, Shailesh; Park, Kyungmin; Lee, Geum Young; Cho, Seungchan; Choi, Hyeok Sun; Kim, Bong-Hyun; Cho, Seunghee; Kim, Jong Woo; Lee, Jin Gyeong; Cho, Seung Hye; Kim, Heung-Chul; Klein, Terry A.; Uhm, Chang-Sub; Kim, Won-Keun; Song, Jin-Won
title: Novel Paju Apodemus Paramyxovirus 1 and 2, Harbored by Apodemus agrarius in The Republic of Korea
date: 2021-03-04
journal: bioRxiv
DOI: 10.1101/2021.03.03.433816
sha: 65f3fe90e464b3a01d1ba99302f9eb87947179d8
doc_id: 309716
cord_uid: 1gocdcne

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a potential threat to public health. Currently, 78 species and 17 genera of paramyxoviruses are classified and harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Jeilongvirus has been proposed as a novel paramyxovirus genus containing J-, Beilong, and Tailam viruses, found in wild rodents. Using RT-PCR, 824 Apodemus agrarius individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was detected in 108 (13.1%) rodents captured at 14 trapping sites in Korea. We first present two genetically distinct novel paramyxoviruses (genus Jeilongvirus), Paju Apodemus paramyxoviruses 1 (PAPV-1) and 2 (PAPV-2), from A. agrarius. Six PAPV strains were completely sequenced using next-generation and Sanger sequencing. PAPV-1 genome comprised 19,716 nucleotides, with eight genes (3′-N-P/V/C-M-F-SH-TM-G-L-5′), whereas PAPV-2 genome contained 17,475 nucleotides, with seven genes (3′-N-P/V/C-M-F-TM-G-L-5′). The disparity between PAPV-1 and -2 revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogenies of PAPV-1 and -2 belong to distinct genetic lineages of Jeilongvirus despite being from the same natural host. PAPV-1 clustered with Beilong and Tailam viruses, while PAPV-2 formed a genetic lineage with Mount Mabu Lophuromys virus-1. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. Therefore, this study provides profound insights into the molecular epidemiology, virus-host interactions, and zoonotic potential of novel rodent-borne paramyxoviruses. Importance Paramyxoviruses are a critical public health and socio-economic burden to humans. Rodents play a crucial role in transmitting pathogens to humans. In the last decade, novel paramyxoviruses have been discovered in different rodents. Here, we found that Apodemus agrarius harbored two distinct genotypes of the novel paramyxoviruses, Paju Apodemus paramyxovirues 1 (PAPV-1) and 2 (PAPV-2), possessing unique genome structures that are responsible for encoding TM and G proteins of different sizes. In addition, PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III IFNs, ISGs, and proinflammatory cytokines. Thus, this study provides significant insights into molecular prevalence, virus-host interactions of paramyxoviruses. These observations raise the awareness of physicians and scientists about the emergence of new rodent-borne paramyxoviruses.

Zoonotic diseases, transmitted from reservoir hosts to humans, comprise the majority of 71 emerging and re-emerging infectious diseases and are public health and socio-economic threats 72

(1-3). Emerging outbreaks of zoonotic viruses, such as severe acute respiratory syndrome 73 coronavirus 2, have increased recently because of expanding human activities that have 74 enabled virus spillover, particularly in situations that facilitate close contact among diverse 75 wildlife species, domesticated animals, and humans (4). Rodents serve as potential mammalian 76 hosts and pose the highest risk of harboring zoonotic viruses to date (3). These animals cause 77 significant economic losses in agriculture and transmit infectious agents including viruses, 78 bacteria, and parasites that cause hemorrhagic fever, tsutsugamushi disease, and leptospirosis 79 (5, 6). Among the rodents in Asia and Europe, Apodemus species is a natural reservoir host 80 carrying pathogens that are detrimental to humans, and A. agrarius is widely distributed in 81 various natural environments (e.g., rural areas, agricultural fields, and forests). Metagenomic 82 studies and continuous surveillance of potential viruses in small mammals provided clues for 83 preventive and mitigative strategies against new emerging and re-emerging infectious diseases 84 (7-12). 85

Paramyxoviruses are non-segmented, negative-sense single-stranded RNA viruses. 86

Paramyxoviridae is divided into four subfamilies: Avulavirinae, Rubularvirinae host range, including vertebrates (mammals, birds, reptiles, and fish) (14). Some 91 paramyxoviruses, for example, human parainfluenza, Hendra, Nipah (NiV), mumps, and 92 measles viruses, pose critical public health and socio-economic burdens owing to their 93 pathogenicity in humans. 94 of PAPV-1 was confirmed by passaging two times for 14 days post-inoculation. The particles 144 of PAPV-1 were observed using a transmission electron microscope ( Figure 1A ). In addition, 145 the number of infectious PAPV particles was 3×10 5 PFU/mL, quantified using the plaque assay 146 ( Figure 1B) . 147

Whole-genome sequencing of PAPVs using next generation sequencing (NGS) and rapid 148 amplification of cDNA ends (RACE) PCR 149

To obtain whole-genome sequences of PAPV, sequence-independent, single-primer 150 amplification-based MiSeq of the Aa17-179 and Aa17-297 isolates generated eight contigs 151 (520-976 nt in length) with significant similarities to the genomic sequence of 152 paramyxoviruses. The NGS of Aa17-179 and Aa17-297 generated 1,623,052 and 1,479,714 153 viral reads, respectively, and the depth of the viral genome sequence was 144,317 and 79,344, 154 respectively (Table S3 ). The nearly complete genome sequences of four PAPV strains (Aa17-155 255, Aa17-260, Aa17-154, and Aa17-166) were acquired via Illumina sequencing. Both the 3´ 156 and 5´ end sequences of the viral genomes revealed incomplete complementary sequences with 157 differences at nucleotide residues 4, 5, and 12. The genomic sequences of PAPV-1 and -2 have 158 been deposited in GenBank (Accession number: MT823459-MT823464). 159

The whole genomes of PAPV-1 and -2 were 19,716 and 17,475 nt in length, with GC contents 161 of 39.96-40.09% and 37.34%, respectively. PAPV-1 contained a genome structure composed 162 of eight genes in the order of 3´-N-P/V/C-M-F-SH-TM-G-L-5´, while the genome structure of 163 PAPV-2 comprised seven genes in the order of 3´-N-P/V/C-M-F-TM-G-L-5´ (Figure 2) . The 164 N, M, F, G, and L genes encode one protein, while the P gene, in addition to the viral 165 phosphoprotein, encodes some accessory proteins that arise through leaky scanning (C protein) 166 or RNA editing (V/W protein). This RNA editing occurs through the addition of one or more 167 guanine residues during transcription, following the recognition of a conserved RNA editing 168 within their P gene. This sequence matched a conserved motif sequence (YTAAAARRGGCA) 170 found in all members of the genera Henipavirus and Morbillivirus, as well as in JV, TaiV,  171 BeiV, and other rodent paramyxoviruses. PAPV-1 showed additional open reading frames 172 between the F and G genes, encoding an SH and/or TM protein. In contrast, PAPV-2 showed 173 the TM gene but not the SH gene. The 3´ leader and 5´ trailer sequences were 55 and 28 nt in 174 length, respectively. The gene start, stop, and intergenic region sequences of PAPVs are shown 175 in the Table S4 . 176

Phylogenetic inference of the whole-genome sequences of PAPVs demonstrated two distinct 178 genotypes within Jeilongviruses (Figure 3) . The genetic cluster of PAPV-1 showed a high 179 similarity (63.7-63.8%) with TaiV, while the PAPV-2 group shared a common ancestor with 180 MMLV-1, with a genomic similarity of 71.6% (Table S5 ). In addition, the amino acid to antibody neutralization and/or alternative reduction in membrane fusion and viral entry (43). 296

Based on NLG prediction, the G protein of PAPV-1 was found to contain more potential 297 glycosylation sites than that of PAPV-2. Although its precise function is still unclear, the 298 potential glycosylation sites of this protein are thought to aid in shielding the protein from 299 recognition by the host immune system. Thus, the biological consequences of the SH gene and 300 molecular characteristics of G protein in PAPV-1 and -2 remain unexplored. 301

Infectivity and expression of innate antiviral genes significantly influence the pathological 302 effects of viral infection in humans and mice (44-46). Due to the isolation of infectious particles, 303 PAPV-1 was examined for infectivity and induction of innate antiviral genes using human 304 epithelial and endothelial cells. We found that the replication of PAPV-1 increased at 1, 3, 5, 305 and 7 dpi in A549 and HUVEC, respectively. The expression of type I/III IFNs, ISGs, and 306 proinflammatory cytokines were also upregulated in response to PAPV-1. These observations 307

suggest that PAPV-1 may infect and elicit proinflammatory responses in humans. In this study, 308 PAPV-2 was not evaluated owing to the lack of infectious particles. The absence of the PAPV-309 2 SH gene might be involved in the robust induction of antiviral genes including type I IFNs 310

and cytokines, and this might be responsible for the failure to isolate infectious PAPV-2 311 particles. The comparisons of infectivity, immunogenicity, and pathogenesis between PAPV-312 1 and -2 remain to be investigated. 313

In conclusion, we presented two novel paramyxoviruses, PAPV-1 and -2, found in A. agrarius 

Kidney tissues were ground in DMEM containing 5% fetal bovine serum. After centrifugation, 354 the supernatant was inoculated into Vero E6 cells. After one and a half hours of adsorption, the 355 excess inoculum was discarded, and the mixture was replaced with 5.5 mL of DMEM. The 356

cultures were incubated at 37°C in a 5% CO2 incubator and inspected daily for cytopathic 357 effects using inverted microscopy. 358

A total of 1×10 6 cells per well were prepared in a 6-well plate. After 24 h, the cells were infected pipeline. The reads were trimmed with Trimmomatic (v0.36) to remove adapter sequences 442 (48). To exclude the reads from the host genome, they were aligned against the host sequences 443 using Bowtie2 (v2.2.6), and only unaligned reads were used for the subsequent steps (49). 444

Owing to the absence of the completely sequenced genome of the host species, only the 445 complete mitochondrial sequence of the species on the NCBI RefSeq was used as a host 446 reference (50). The remaining reads were filtered for quality using FaQCs (v0.11.5), and de-447 novo assembly was performed to produce contigs using SPAdes (v3.11.1) (51, 52). The 448 assembled contigs were subsequently examined in a database consisting of complete viral 449 genomes collected from the NCBI RefSeq database (updated in May 2018) using BLASTn 450 (v2.6.0). 451

To obtain the 3′ and 5′ terminal genome sequences of paramyxovirus, we performed RACE 453 PCR using a SMARTer® RACE 5'/3' Kit (Takara Bio), according to the manufacturer's 454 specifications. We purified the PCR products using the LaboPass PCR Purification Kit (Cosmo 

Full-length amino acid sequences were submitted to the NetNlyc 1.0 (Kemitorvet, Denmark) 471 to predict the NLG sites of the G gene of Jeilongviruses (55). 472

To find homology, we ran NCBI BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using 474 PAPV-1 and -2 G protein sequences against the NR database using default settings. When we 475 ran BLASTP using PAPV-1 G protein and not PAPV-2 G protein, we found the alignments 476 shown in the supplement covering both domains. indicate the standard deviation of triplicate measurements in a representative experiment. 719 (*p<0.05; ***p<0.001, unpaired student t-test; ns: non-significant). 720 

Evaluation of viral genome assembly 538 and diversity estimation in deep metagenomes

Viral Diversity of House Mice

The complete genome sequence of J 549 virus reveals a unique genome structure in the family Paramyxoviridae

Beilong virus, a novel paramyxovirus with the largest genome of non-segmented 553 negative-stranded RNA viruses

Complete genome 555 sequence of a novel paramyxovirus, Tailam virus, discovered in Sikkim rats

Discovery and genome characterization of three new Jeilongviruses, 560 a lineage of paramyxoviruses characterized by their unique membrane proteins

metapneumovirus small hydrophobic protein has properties consistent with those of a 589 viroporin and can modulate viral fusogenic activity

Navaratnarajah CK, Generous AR, Yousaf I, Cattaneo R. 2020. Receptor-mediated cell 594 entry of paramyxoviruses: Mechanisms, and consequences for tropism and 595 pathogenesis

Structural rearrangements of the central 598 region of the morbillivirus attachment protein stalk domain trigger F protein refolding 599 for membrane fusion

Unraveling a 602 three-step spatiotemporal mechanism of triggering of receptor-induced Nipah virus 603 fusion and cell entry

Sequential conformational 606 changes in the morbillivirus attachment protein initiate the membrane fusion process

Residues in the stalk domain of the hendra virus g 610 glycoprotein modulate conformational changes associated with receptor binding

Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of

Timing is everything: Fine-tuned molecular 616 machines orchestrate paramyxovirus entry

Intrinsically disordered 618 proteins of viruses: Involvement in the mechanism of cell regulation and pathogenesis

N-Glycans on the Nipah virus attachment glycoprotein modulate 624 fusion and viral entry as they protect against antibody neutralization

Human immunodeficiency virus type 1 V1-627 V2 envelope loop sequences expand and add glycosylation sites over the course of 628 infection, and these modifications affect antibody neutralization sensitivity

N-Glycans in the gp120 V1/V2 domain of the HIV-1 strain

NL4-3 are indispensable for viral infectivity and resistance against antibody 632 neutralization

Antibody neutralization and escape by HIV-1

N-glycans on Nipah virus fusion protein protect against 639 neutralization but reduce membrane fusion and viral entry

Cytokines and respiratory syncytial virus 641 infection

Hendra and Nipah viruses: 643 different and dangerous

Defective viral genomes arising in vivo provide critical danger signals for the 646 triggering of lung antiviral immunity

Sensitive and broadly 648 reactive reverse transcription-PCR assays to detect novel paramyxoviruses

Trimmomatic: a flexible trimmer for Illumina 651 sequence data

Fast gapped-read alignment with Bowtie 2

NCBI reference sequences (RefSeq): a 655 curated non-redundant sequence database of genomes, transcripts and proteins

Rapid evaluation and quality control of next generation 658 sequencing data with FaQCs

661 applications to single-cell sequencing

Molecular Evolutionary Genetics

Analysis Version 7.0 for Bigger Datasets

Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus 668 Evol 4:vey016. 669 55. Gavel Y, von Heijne G. 1990. Sequence differences between glycosylated and non-670 glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering

A Completely Reimplemented MPI Bioinformatics Toolkit 674 with a New HHpred Server at its Core

HH-676 suite3 for fast remote homology detection and deep protein annotation

PROMALS: towards accurate multiple sequence alignments 679 of distantly related proteins

* The positive rate of PAPV RNA indicates the detection of the partial L segment, targeting 724 pan-Orthoparamyxovirinae and/or the genera Respirovirus, Morbillivirus, and Henipavirus 725 using RT-PCR and Sanger sequencing

The authors declare no competing financial interests. 513