bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440555; this version posted April 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Genetic diversity, evolutionary dynamics and pathogenicity of 2 ferret badger rabies virus variants in mainland China, 2008-2018 3 4 Faming Miaoa, Jinghui Zhaoa, Nan Lia, Ye Liua, Teng Chena, Lijuan Mia, Jinjin 5 Yanga, Qi Chena, Fei Zhanga, Jie Fengb, Shunfei Lic,*, Shoufeng Zhanga,*, 6 Rongliang Hua,* 7 a 8 Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of 9 Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun, 10 China b 11 Suzhou Shangfangshan Forest Zoo, Suzhou, China c 12 Institute of Hospital Management, Medical Innovation Research Department, 13 Chinese People's Liberation Army General Hospital, Beijing, China 14 15 Faming Miao, Jinghui Zhao and Nan Li contributed equally to this work. 16 17 *Address correspondence to Shunfei Li, [email protected], or Shoufeng Zhang, 18 [email protected], or Rongliang Hu, [email protected] 19 20 ABSTRACT In contrast to dog associated human rabies cases decline year by year 21 due to the rabies vaccination coverage rates increase in China, ferret badger (FB, 22 Melogale moschata)–associated human rabies cases emerged in the 1990s, and are 23 now an increasingly recognized problem in southeast China. To investigate 24 epidemiology, temporal evolution dynamics, transmission characterization and 25 pathogenicity of FB-associated rabies viruses (RABVs), from 2008 to 2018, we 26 collected 3,622 FB brain samples in Jiangxi and Zhejiang Province, and detected 112 27 RABV isolates. Four FB-related lineages were identified by phylogenetic analysis 28 (lineages A–D), the estimated Times to Most Recent Common Ancestor were 1941, 29 1990, 1937 and 1997 for lineages A–D respectively. Furthermore, although no 30 FB-associated human rabies case has been reported there apart from Wuyuan area, bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440555; this version posted April 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 31 FB-RABV isolates are mainly distributed in Jiangxi Province. Pathogenicity of 32 FB-RABVs was assessed using peripheral inoculation in mice and in beagles with 33 masseter muscles, mortality-rates ranging from 20% to 100% in mice and 0 to 20% in 34 beagles in the groups infected with the various isolates. Screening of sera from 35 humans with FB bites and no postexposure prophylaxis to rabies, revealed that 5 of 9 36 were positive for neutralizing antibodies of RABV. All the results above indicated that 37 FB-RABV variants caused a lesser pathogenicity in mice, beagles and even humans. 38 Vaccination in mice suggests that inactivated vaccine or recombinant subunit vaccine 39 products can be used to control FB- associated rabies, however, oral vaccines for stray 40 dogs and wildlife need to be developed and licensed in China urgently. 41 IMPORTANCE In recent years FB-associated rabies virus has been identified as a 42 major life-threatening pathogen in some districts in China. To understand the risk to 43 public health and the contemporary dynamics, the present study conducted extensive 44 investigations on FB rabies, distribution, virus isolation, phylogeny analysis, 45 pathogenicity determination of various FB rabies virus strains, besides, serologic 46 epidemiology survey to those whom bit by FB was also collected. The results show 47 that the majority of FBs dwell in southeast China, like Jiangxi and Zhejiang Province, 48 Phylogenetic analysis indicates that all isolated FB RABVs evolved from dogs, and 49 the FB RABV can separate into 4 distinct lineages distributed relatively independent 50 in different areas. The isolate strains differ in pathogenicity, although they have 51 relatively lower pathogenicity compared to dog rabies virus according to our study, 52 the need for further study to licensed oral vaccines and FB RABV pathogenesis is 53 emphasized in order to control rabies. 54 KEYWORDS ferret badger, rabies virus, epidemiology and evolution, pathogenicity 55 56 Rabies is an acute, progressive and encephalitic disease. Once clinical signs 57 manifests, the disease usually is considered to be invariably fatal. It continues to 58 present public health problems worldwide and causes more than 60,000 human deaths 59 annually (1). The majority of these deaths are in developing countries, primarily in 60 Africa and Asia, more than 95% of human rabies cases are attributed to dog bites bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440555; this version posted April 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 61 worldwide and the ration is higher in developing countries (2). However, in southeast 62 China, the percentage dog-associated human rabies is relatively low, up to 80% of the 63 reported human rabies cases were inferred to be caused by ferret badger (FB) bites in 64 some districts in Zhejiang and Jiangxi province (3, 4). 65 The Chinese ferret badger, which dwells mainly in southeastern China. These 66 mustelids have several names like crab-eating mongoose, rice field dog, 67 viviparid-eating dog, loach-eating dog, and white face weasel—mainly because of 68 their omnivorous behavior and external appearance. FB-associated human rabies 69 cases in China were emerged in 1994 (5), forty-six human rabies cases exposed to FB 70 bites were reported in Zhejiang and Jiangxi province from 1994-2007 (6). 71 In 2008, we isolated the first FB rabies virus strain, since then, we continued our 72 surveillance, and collected additional positive samples mainly distributed in Jiangxi 73 and some in Zhejiang province that were isolated from FB. We have conducted the 74 laboratory-based surveillance, virus isolation, nucleotide sequencing and molecular 75 characterization analysis (7-9). Recently, we investigated the temporal dynamics of 76 FB-RABVs in China based on N sequences from samples collected from 2014 to 77 2016 (10), and demonstrated that the increasing genetic diversity of RABVs in FBs is 78 a result of the selective pressure from coexisting dog rabies virus, and FB rabies is 79 likely occurring as an independent enzootic that became established in the FB 80 populations from a dog RABV variant distributed in southeast China (11), which 81 served as a reservoir for human infections and a source of new variants capable of 82 emerging or reemerging in the future; however, little is known about the genetic 83 diversity of FB-associated RABVs, their evolutionary dynamics and the role of FBs in 84 maintaining RABV transmission in the epizootic regions, especially the pathogenicity 85 of FB RABV remains to be unclear. 86 In this study, we collected 3,622 FB brain samples from 2008 through 2018 in 87 these regions and detected 112 FB-RABV isolates from the collected samples, and 88 sequenced the full-length of the nucleoprotein (N) gene from each isolate, constructed 89 the phylogenetic and temporal evolution dynamics analysis. Furthmore, we 90 determined the pathogenic characteristics of FB RABV in mice and beagles, tested bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440555; this version posted April 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 91 the serum antibody titers for RABV who bit by FBs. Moreover, we reported that using 92 inactivated vaccines or adenovirus recombinant vaccine can protect mice from lethal 93 challenge with FB RABV infection. 94 95 Results 96 FB RABVs were constantly detected over the years in Poyang and Qiandao 97 lake regions 98 As depicted in Table 1, from 2008 to 2018, FB-associated RABV nucleic acids 99 were regularly detected in our collected samples from the Poyang lake region, with a 100 positive rate ranging from 1.7%–6.3%. In the Qiandao lake region, we detected 5 101 RABV positive among 138 samples from 2008 through 2013 (Table 1). A total of 112 102 FB RABV strains were detected and 70% infectious viruses were successfully 103 isolated from all positive tissues and cultured in suckling mouse brain. The full-length 104 of the N gene from each positive sample was amplified by RT-PCR and sequenced for 105 phylogenetic and evolutionary studies. 106 Segregation of FB RABVs into geographic clades and evidence of RABV 107 cross species transmission between FBs and dogs 108 The 112 FB-associated RABVs were classified into 4 lineages (A–D) using data 109 from 132 Chinese RABV N gene sequences, and each lineage was grouped 110 geographically by the location of the virus isolate (Fig 1). Lineage A, with 41 111 FB-associated RABV isolates, 40 distributed in east of Poyang lake and 1 distributed 112 in Zhejiang province, was embedded in China canine RABV Group II (China II). The 113 FB RABVs in lineage A were closely related to Chinese vaccine strains CTN181 and 114 CTN7, which were derived from a dog street virus in the 1950s (12). 115 Lineage B, C and D were grouped with China canine RABV Group I (China I) 116 (13). Lineage B had 17 isolates from the Fuzhou district, south of Poyang lake (7). 117 Lineage D had 7 FB isolates, 4 FB isolates (ZJ12-03, ZJ13-66, ZJ13-130, ZJ13-431), 118 located in Taizhou district, Zhejiang province, other 3 isolates located in east Poyang 119 lake. Of note, a canine RABV isolate denoted as LH from Zhejiang province, where bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440555; this version posted April 20, 2021.