Diagnosis and molecular characterization of rabies virus from a buffalo in China: a case report

Background Rabies virus (RABV) can infect many different species of warm-blooded animals. Glycoprotein G plays a key role in viral pathogenicity and neurotropism, and includes antigenic domains that are responsible for membrane fusion and host cell receptor recognition. Case presentation A case of buffalo rabies in China was diagnosed by direct fluorescent antibody test, G gene reverse-transcriptase polymerase chain reaction, and RABV mouse inoculation test. Molecular characterization of the RABV was performed using DNA sequencing, phylogenetic analysis and amino acid sequence comparison based on the G gene from different species of animals. Conclusion The results confirmed that the buffalo with suspected rabies was infected by RABV, which was genetically closely related to HNC (FJ602451) that was isolated from cattle in China in 2007. Comparison of the G gene among different species of animal showed that there were almost no amino acid changes among RABVs isolated from the same species of animals that distributed in a near region. However, there were many changes among RABVs that were isolated from different species of animal, or the same species from different geographic regions. This is believed to be the first case report of buffalo rabies in China, and the results may provide further information to understand the mechanism by which RABV breaks through the species barrier.


Background
Rabies virus (RABV) is one of the seven species in the genus Lyssavirus in the Rhabdoviridae family [1]. All warm-blooded animals, including raccoons, skunks, bats and foxes, are susceptible to RABV, and domestic dogs act as the main reservoir and transmitter [2]. The annual number of human deaths caused by rabies is estimated to be 55,000 worldwide [3], with about 32,000 in Asia [4]. The total number of human deaths was 108,412 between 1950 and 2004 in China [5]. The average number was 1,524 from 1996 to 2008, and 50% of cases were reported in Guangxi, Hunan and Guizhou provinces [6]. Therefore, the disease continues to be a serious public and animal health problem in China.
The aims of the present study were to diagnose a case of buffalo rabies that occurred in Wuhan City, Hubei Province, using three different methods, and to compare the sequences with different RABVs that were isolated from different species, based on the G gene. This is believed to be the first report of the phylogenetic analysis of buffalo RABV in China compared with other isolates from different animals.

Case presentation
Specimens were collected from the gyrus hippocampi of the buffalo with suspected rabies in Wuhan City (114.3°E, 30 antibody test (dFAT) of the specimen was performed as previously described [21][22][23]. Normal buffalo brain samples were used as a negative control. RABV isolation by mouse inoculation test was performed as described previously [24,25]. The total RNA from buffalo brain was extracted with Trizol reagent (Invitrogen) according to the manufacturer's instructions. Primer design and reverse-transcriptase polymerase chain reaction (RT-PCR) of the G gene were performed as described previously [17]. RT-PCR products were visualized under UV light after electrophoresis on 1% agarose gels containing ethidium bromide. The amplified products were purified with a QIAquick PCR gel extraction kit (QIAGEN) according to the manufacturer's protocol. The sequencing was carried out in an Applied Biosystems 3730 DNA automated sequencer. After the raw sequences were edited by Clus-talX Version 1.82 [26], 1575 nt sequences of the G gene were obtained and submitted to GenBank.
The phylogenetic tree based on the deduced amino acid sequences was constructed by using the neighbor-joining method with 1,000 bootstrap replicates using MEGA version 4.0 software [27], based on the complete sequence of the RABV G gene from 10 different species of animal (Table 1). Bootstrap values >70% were considered significant [28]. Genetic distance analysis for the G gene was conducted with PHYLIP version 3.63 software [29]. Glycoprotein nucleotide sequences from different animals were identified, translated into amino acid sequences, edited, and pair aligned using BioEdit software [27]. Multiple alignments were performed by ClustalX software [26].

Conclusion
dFAT indicated the presence of RABV antigen in the brain specimens from the buffalo with suspected rabies (Figure 1A), whereas normal buffalo brain did not ( Figure 1B). The expected size of the G gene fragment was obtained from the suspected buffalo brain by RT-PCR (data not shown). RABV from buffalo was isolated successfully in suckling mice (data not shown), and the RABV was named Hubei070308 strain. The positive result was supported by G gene sequencing, and the sequences were submitted to GenBank under the accession number EF643518.
The G gene sequence, together with reference sequences from seven countries and 10 species of animals were aligned (Table 1). We showed that the G gene of RABV had relative territorial specificity but not species specificity ( Figure 2). The genetic relationship of the RABV in this study was differed greatly from PG(AY009097) that was used as a vaccine strain in China in 1931. Compared with 18 other RABV strains, Hubei070308 shared 85.0-99.8% sequence identity at the amino acid level (data not shown). We demonstrated that Hubei070308 strain was close to the cattle strain FJ602451 and the human strain DQ849063, which belong to Chinese group I [17], but it was far from coyote strain U52946, which was isolated in 1996 in the United States ( Figure 2). Additional File 1, Figure S1 shows that the amino acid changes were mainly focused on the transmembrane areas (aa 440-461), inner-membrane zone (aa 462-505) and signal peptide range of mature RABV glycoprotein. Glycoprotein sequences of Hubei070308 were identical to HNC (FJ602451) that was isolated from cattle in 2007. However, there were many amino acid substitutions when it was compared with RABVs from other animals such as skunk, dog, human, bat and deer. The linear epitope (aa 14-19) at antigenic site II and the minor site between aa 342 and 343 were highly conserved, which was consistent with the findings of Meng et al. [17]. Among all the RABVs, SHBRV strain that was isolated from bats was the most variable at the amino acid level. Many different animals can be infected by RABV [2], and cases of transmission from bats to humans [30], dogs to humans [17] and even dogs to pigs [31] have been reported. For a virus shed by one host to infect another, it must break through entry barriers (e.g., epithelium, mucus, and alveolar macrophages) and find its way to tissues in which it can replicate [32].
A B Figure 1 Results of dFAT of specimens from brain of buffalo with suspected rabies. (A) suspected buffalo brain sample; (B) normal buffalo brain sample.  It has been reported that several amino acids in the RABV glycoprotein are responsible for pathogenicity [33,34]. Therefore, RABV glycoprotein is the best target protein to study virus-host interaction, or it may be the main protein that is responsible for breakthrough of the species barrier. In the present study, many amino acid substitutions in G protein were found among RABVs that were isolated from different animal species, or from the same species distributed in different geographic regions. These substituted amino acids may explain why RABV can break through the host barrier to infect one species of animal from another. This hypothesis needs to be confirmed by further experiments.

Additional material
Additional file 1: Figure S1. Comparative analysis of G gene amino acid with other RABVs isolated from different animals. Dots represent identity among all sequences. Arrows mark the range of the signal peptide, antigenic site, linear epitope and endo-domain. Transmembrane (TM) domain was framed. SP: signal peptides; ENDO: endodomain; AS2: antigenic site II; AS3: antigenic site III; LE: linear epitope.