Discovery and Genetic Characterization of Novel Paramyxoviruses Related to the Genus Henipavirus in Crocidura Species in the Republic of Korea

Abstract

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a critical threat to human public health. Currently, 78 species, 17 genera, and 4 subfamilies of paramyxoviruses are harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Henipaviruses are critical zoonotic pathogens that cause severe acute respiratory distress and neurological diseases in humans. Using reverse transcription-polymerase chain reaction, 115 Crocidura species individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was observed in 26 (22.6%) shrews collected at five trapping sites, Republic of Korea. Herein, we report two genetically distinct novel paramyxoviruses (genus: Henipavirus): Gamak virus (GAKV) and Daeryong virus (DARV) isolated from C. lasiura and C. shantungensis, respectively. Two GAKVs and one DARV were nearly completely sequenced using next-generation sequencing. GAKV and DARV contain six genes (3'-N-P-M-F-G-L-5') with genome sizes of 18,460 nucleotides and 19,471 nucleotides, respectively. The phylogenetic inference demonstrated that GAKV and DARV form independent genetic lineages of Henipavirus in Crocidura species. GAKV-infected human lung epithelial cells elicited the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. In conclusion, this study contributes further understandings of the molecular prevalence, genetic characteristics and diversity, and zoonotic potential of novel paramyxoviruses in shrews.

Keywords: Crocidura paramyxovirus; genetic characterization and diversity; next-generation sequencing; novel virus discovery; potential zoonosis.

Figure 1

Geographic locations of the Republic of Korea, showing the shrew trapping sites described in this study. The trapping sites include Cheorwon, Chuncheon, Hwacheon, Hongcheon, and Pyeongchang in Gangwon Province; Osan, Paju, Pocheon, Suwon, and Yeoncheon in Gyeonggi Province; and Changnyeong in Gyeongsangnam Province. Red and blue dots represent trapping sites for shrews positive for the presence of Gamak virus (GAKV) and Daeryong virus (DARV), respectively. Black dots indicate that trapping sites where paramyxoviral RNA was not detected in the shrews. This figure was constructed by using Adobe Illustrator CS6 (http://www.adobe.com/products/illustrator.html, 3 March 2021).

Figure 2

Transmission electron micrograph and quantitation of Gamak virus (GAKV). (A) The appearance of GAKV isolate was obtained by using the transmission electron microscopy. Red arrows indicate virus particles. (B) The plaque assay shows infectious GAKV inoculated onto Vero E6 cells. Each plate well represents the quantitation of infectious particles at serial dilutions from 1:10 to 1:10⁵ of the virus stock.

Figure 3

Organization of the genomes of the paramyxoviruses Gamak and Daeryong viruses, in individuals of Crocidura. The genomic configurations of related paramyxoviruses are shown. The genomes of paramyxoviruses comprise 6 to 8 coding regions: 3′ N-P-M-F-SH-TM-G, HN, or H-L 5′. The colored boxes represent coding regions for each gene: N, yellow-green; P, purple; M, sky blue; F, yellow; SH, orange; TM, blue; G, light yellow; HN, red; H, Chilean pink; L, viridian. A scale bar of the length is shown under the genome structure. This figure was constructed by using Adobe Illustrator 2021 (http://www.adobe.com/products/illustrator.html, 3 March 2021).

Figure 4

A phylogenetic tree of Gamak virus, Daeryong virus, and multiple paramyxoviruses using nearly whole-genome sequences. Phylogenetic inferences were conducted by BEAST (v1.10.4) with default priors and assuming homochromous tips. The Markov chain Monte Carlo analysis was run until sufficient sample sizes (ESS > 200) were acquired. The maximum clade credibility tree from the posterior tree distribution was summarized byTreeAnnotator (v2.5.4), using a 10% burn-in. Paramyxoviridae strains were utilized as reference sequences for this phylogenetic analysis. Red and blue bold texts indicate GAKV and DARV, respectively.

Figure 5

Tanglegram comparing the phylogenies between paramyxoviruses and their reservoir hosts (insectivores and rodents). The tanglegram was generated using the R package, using Bayesian consensus tree, based on the nucleotide sequences of the paramyxoviral L gene (right panel) and cytochrome b gene of mitochondrial DNA sequences of the host species (left panel). Letters for taxa are indicated in red for Crocidura lasiura in the left panel and Gamak virus in the right panel, respectively. Letters for taxa are shown in blue for Crocidura shantungensis in the left panel and Daeryong virus in the right panel, respectively. The left panel shows shrew species in the red box, rodent species in the blue box, and mole species in the black box. The right panel shows genus Henipavirus in the red box, genus Jeilongvirus in the blue box, genus Narmovirus in the purple box, and outgroup in the grey box.

Figure 6

Enhanced expression of viral and innate antiviral genes in Gamak virus (GAKV)-infected A549 cells. A549 cells were infected with a GAKV at multiplicity of infection of 0.02. Total RNA was extracted and analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the induction of (A) GAKV RdRp, (B) interferon-β (Ifn-β), (C) Ifnl1/Il-29, (D) interferon-stimulated gene product-15 (ISG-15), (E) Ifit2/Isg54, (F) Ifit1/Isg56, (G) radical S-adenosyl methionine domain containing 2 (Rsad2/Viperin), (H) 2′, 5′-oligoadenylate synthetase 1 (OAS1), (I) Ddx58/Rig-I, (J) Ifih1/Mda5, and (K) interleukin-6 (Il-6) at 2, 6, 12, 18, and 24 h postinfection. Error bars show the standard deviation of triplicate measurements in a representative experiment. *** p < 0.001, one-way analysis of variance (ANOVA) test; ns: non-significant.