Epidemiological survey on FB-associated human rabies cases
A retrospective survey on rabies epidemics was carried out in Zhejiang, Jiangxi and Anhui provinces in southeast China. The data were collected and summarized from the provincial CDC surveillance system and epidemiological records. Some information was obtained verbally after interviewing animal traders or hunters in the endemic areas. In human rabies cases and its potential association with FB transmission, we conducted a preliminary investigation of FB population density, exposure frequency of sick FB to humans, and management of rabies PEP after a FB bite.
FB and dog brain specimens and Fluorescent Antibody Test (FAT)
Dead or live FB collected in fields and houses, and dogs that had bitten people were sent by selected farmers to the designated laboratories for diagnoses. The surveyed mountainous areas were Huzhou, Hangzhou, Jinhua, Quzhou, Lishui in Zhejiang province; Nanchang, Jingdezhen, Shangrao, Wuyuan in Jiangxi province; and Huangshan, Xuancheng, Anqing, Jingxian in Anhui province. The animal heads were packed appropriately and shipped to our laboratory under cold conditions. The brains were removed by opening the skull under sterile conditions.
Animal experiments related to this study were approved by the Committee of Animal Welfare and Ethics of Veterinary Research Institute, Academy of Military Medical Sciences. Humane endpoints were used throughout this study in accordance with the ethical principles for in vivo studies. All animals including ferret badgers, dogs and mice that showed clinical signs of infection were killed humanely. This study did not refer to any issue of human ethics as only the epidemiological data were collected and analyzed, and no sample was collected from any healthy and contracted humans.
The FAT on brain specimens was performed according to methods described elsewhere [14]. Briefly, the brain tissue impressions were made on slides. After air drying, the slides were fixed with 80% acetone for 20 min. The FITC-conjugated anti-rabies virus nucleoprotein monoclonal antibodies (made in Laboratory of Epidemiology, Veterinary Institute, Changchun, for routine rabies diagnosis) was added and incubated for 40 min at 37ºC. After three-times' wash using PBS buffer (0.01 M, pH 7.4) -Tween-20 (0.1%), the slides were left to dry in the air at ambient temperature, and observed under a UV fluorescent microscope (Zeiss Corporation, Germany) for typical rabies virus staining.
Mouse inoculation test (MIT)
For FAT positive specimens, the suspension of the sample was injected intracerebrally to 1-day-old suckling mice (Kunming mice, Animal Core Laboratory, Changchun Institute of Biological Products) according to protocols described elsewhere [15]. The mice were observed for ~28 days. The brain smear was made to re-check for the presence of rabies virus antigen using the FAT if the inoculated mice died from day 6 to 28.
Sequences of the nucleoprotein and glycoprotein genes and phylogenetic analysis
Total RNA of the infected FB brains were extracted using Trizol reagent (Invitrogen, Carlsbad, CA). The nucleoprotein (N) and glycoprotein (G) genes were amplified by RT-PCR following the protocol described by Nadin-Davis et al [16] using the total RNA with the following primers. The positions of the primers were referred against strain FJ009 (GenBank Acc. No. FJ866836.1).
RV-N-F (1-25): 5'-ACGCTTAACAACAAAACCATAGAAG-3';
RV-N-R (1515-1538): 5'-CGGATTGACGAAGATCTTGCTCAT-3';
RV-G-F (3291-3315): 5'-CATCCCTCAAAAGACTTAAGGAAAG-3';
RV-G-R (4918-4941): 5'-CCGAGGAGATGAGGTCTTCGGGAC-3',
The TaKaRa (TaKaRa Corp Ltd, Dalian, China) was the contracted company responsible for sequencing the amplicons. We generated a phylogenetic tree using the Neighbor-joining (NJ) method in MEGA 4 (MegAlign, DNASTAR Software Suite, Version 7.1.0, Copyright 1989-2006, DNASTAR Inc.). The Bootstrap values were calculated from 1,000 repeats using 70% as the cut-off.
Rabies virus (RV) neutralizing antibodies in dog and FB serum samples
Serum samples were collected from the captured FBs and dogs in our expanded surveyed areas. All the FBs were alive and appeared healthy when the serum was collected. Dog sera were from watchdogs belonging to residents of different villages in Zhejiang, Jiangxi and Anhui provinces. The method of titration of virus neutralizing antibodies (VNA) by Fluorescent Antibody Virus Neutralization (FAVN) test was described elsewhere [17]. Briefly, 3-fold serial dilutions of standard serum (0.5 International Units, IU/ml) and test serum samples were prepared in microplates in quadruplicate. Fifty _μl of challenge rabies virus (CVS-11) containing 100 TCID50 was also added to each well. After 60 min incubation at 37°C in a humidified 5% CO2 incubator, 50 μl cell suspension containing 2 × 104 cells was added to each well and the plates were incubated for 48 h at 37°C. After fixation at room temperature for 30 min in 80% acetone, the cell monolayers were stained by addition of FITC-conjugated anti-rabies nucleoprotein monoclonal antibodies (Laboratory of Epidemiology, Veterinary Institute, Changchun, China) to each well. Staining was carried out in an incubator at 37°C for 30 min and fluorescence was observed by UV microscope (Zeiss, Germany). Wells exhibiting no specific fluorescence were considered antibody positive. Neutralizing antibody titers were calculated using the Spearman-Kärber formula and expressed in IU/ml by comparison with a reference serum (13.5 IU/ml, product of AFSSA, France), 0.1 IU/ml is considered as the cut-off of seroconversion after rabies virus infection in both ferret badgers and dogs.
