Molecular Epidemiology Of Ferret Badger(Melogale Moschata) Rabies Virus In China

Ferret badger (FB, Melogale moschata), mainly distributed in southern andeastern China. Rabies in FBs was first reported in the1990s. Subsequently, ferretbadger–associated human rabies cases were reported in Jiangxi, Zhejang and Anhui.Although the doctor make a definite diagnosis based on clinical symptoms and casehistory, no rabies virus was obtained. Ferret badgers are a major reservoir and vectorof rabies in China, and rabies in FBs is an increasing public health threat to humans insoutheast China (including northern Jiangxi, middle to western Zhejiang, and easternAnhui provinces). However, little is known about the genetic diversity ofFB-associated RABVs, their evolutionary dynamics and the role of FBs inmaintaining RABV transmission in the epizootic regions, mainly due to limitedanimal samples and RABV isolates. Oral immunization has been shown to be apractical method for wildlife rabies management in Europe and North America. InChina, no practical rabies vaccine has been developed for wildlife and no anti-rabiesoral vaccine has been registered.To investigate this further, we carried out the following studies:1. Epidemiological surveillance of rabies in FBs and phylogenetic analysis ofFBs RABV isolates.From2008to2014, we collected2,932FB samples in Jiangxi, Zhejiang, Anhuiand Fujian. A total of91FB RABVs were confirmed by immunofluorescence assaymethod (FAT), and all infectious viruses were successfully isolated and cultured insuckling mice (positive ratio as3.1%). The full-length of the RABV N gene and Ggene was amplified by RT-PCR and sequenced for phylogenetic studies.Four FB-related lineages were identified by phylogenetic analysis (Clades A–D).Clade A had46FB-associated RABV isolates, mainly distributed in Qiandao Lake(Zhejiang province) and east Poyang Lake (Jiangxi province) regions. It wasembedded in China RABV Group II. Clades B and D were grouped into China RABVGroup I. Clade B had8isolates from Fuzhou district, south of Poyang Lake. Clade Dhad10FB isolates, located in Taizhou district, Zhejiang province. Of note, a dogRABV isolate LH from Zhejiang province, was embedded in the FB Clade D. Clade C included27FB-associated RABVs, share approximately95%nucleotide identitywith China RABV Group I, and88%nucleotide identity with China RABV Group II,forming an independent Clade.We use Bayesian Markov Chain Monte Carlo (MCMC) method in the BEASTpackage to reconstruct the Maximum Clade Credibility (MCC) phylogenetic tree,estimate the rate of nucleotide substitution (per site, per year), and calculate the Timeto Most Recent Common Ancestor (TMRCA). The estimated Time to Most RecentCommon Ancestor was1941for clade A,1990for B,1937for C, and1997for D.Clades B and D could be recent events diverged from dog RABV spillovers to the FBpopulations, while Clades A and C could represent old historical spillovers withsubsequent establishment in the FB population.2. Genomes of three ferret badger rabies virus isolates from Jiangxi province,ChinaWe sequenced three FB rabies viruses JX09-17(fb), JX09-18and JX10-37,whichwere isolated from3different regions in Jiangxi province, in2009and2010. Thecomplete genome sequences of the three FB isolates were compared with37fullgenome sequences available in GenBank of vaccine strains and street isolates fromvarious regions of China.The complete nucleotide sequence identity between these three isolates was87-93%. Compared with the other Chinese rabies virus isolates and vaccine strains,101substitutions (53in JX10-37,23in JX09-17(fb) and25in JX09-18) in the fivestructural proteins were observed and47of these substitutions (27in JX10-37,14inJX09-17(fb) and6in JX09-18) were unique among lyssaviruses.3. Experimental oral immunization of ferret badgers with a recombinant canineadenovirus vaccine CAV-2-E3Δ-RGP and an attenuated rabies virus SRV9Two groups of20ferret badgers were given a single oral dose of a recombinantcanine adenovirus-rabies vaccine, CAV-2-E3Δ-RGP, or an experimental attenuatedrabies virus vaccine, SRV9. Blood serums were separated at14and21days postimmunization; the antibody levels were tested by FAVN. At21days, all ferret badgershad seroconverted, with serum virus-neutralizing antibodies ranging from0.1–4.5IU/mL. Titers were>0.50IU/ml (indicative of an acceptable level) in17/20and16/20animals receiving CAV-2-E3Δ-RGP or SRV9, respectively. Conclusions:1. Four clades A, B, C and D were identified and each clade was reconstructedgeographically. Clades B and D could be recent events diverged from dog RABVspillovers to the FB populations, while Clades A and C could represent old historicalspillovers with subsequent establishment in the FB population. We also foundevidence of rabies virus cross species transmission from FBs to dogs, suggesting dograbies virus variants may have been independently introduced multiple times to FBs,and subsequently adapted to FBs in southeast China. Reintroduction of rabies fromFBs to dogs is possible without intervention.2. Amino acid substitutions of S231and Q333were noted respectively in the Gprotein antigenic site I of JX10-37and site III in JX09-17(fb). Phylogenetic analysisshows that JX09-17(fb) is rooted within China I lineage, JX09-18China II, andJX10-37is independent. The substitutions may help provide an explanation for theadaptation and transmission of rabies virus in ferret badgers.3. The serologic results indicate that the recombinant CAV-2-E3Δ-RGP is at leastas effective as the attenuated rabies virus vaccine SRV9. Both may be considered foradditional research as oral rabies vaccine candidates for ferret badgers.