Vaccination demonstration zone successfully controls rabies in Guangxi Province, China.

BACKGROUND: Guangxi is the province most seriously affected by rabies virus (RABV) in China. Those most affected by RABV each year are people in rural areas, where dogs are the main cause of human infection with the virus. METHODS: In this study, we established a rabies vaccination demonstration program that included eradication, core, and peripheral areas. This program was implemented for 9 years and comprised three stages: 12 counties in the first stage (2008-2010), 21 counties in the second stage (2011-2013), and then extending to all counties of Guangxi Province in the third stage (2014-2016). The program included a dog vaccination campaign, surveillance of clinically healthy dogs who may be potential RABV carriers, monitoring anti-RABV antibody titers in vaccinated dogs, and compiling and reporting statistics of human rabies cases. RESULTS: The target effectiveness was achieved in the eradication, core, and peripheral areas in all three stages. The vaccination demonstration program successfully promoted RABV vaccination of domestic dogs throughout Guangxi Province by drawing upon the experience gained at key points. Compared with a vaccination coverage rate of 39.42-46.85% in Guangxi Province overall during 2003-2007, this rate gradually increased to 48.98-52.67% in 2008-2010, 60.24-69.67% in 2011-2013, and 70.09-71.53% in 2014-2016, thereby meeting World Health Organization requirements. The total cases of human rabies in the province decreased from 602 in 2004 to 41 cases in 2017. CONCLUSIONS: The present pilot vaccination program obviously increased the rabies vaccination and seroconversion rates, and effectively reduced the spread of rabies from dogs to humans as well as the number of human rabies cases, thus successfully controlling rabies in Guangxi.

Evolutionary analysis of rabies virus isolates from Guangxi Province of southern China.

BACKGROUND: Rabies is a severe epidemic in Guangxi province, China, with hundreds of deaths occurring each year. In the past six decades, rabies has emerged three times in Guangxi, and the province has reported the largest number of rabies cases in China. The domestic dog is the principal vector for rabies, and 95% of human cases are associated with transmission from dogs. RESULTS: To understand the genetic relationship between street rabies virus (RABV) from Guangxi, genetic diversity analysis was performed using RABV isolates collected between 1999 and 2012. The N gene of 42 RABV isolates, and the P and M genes, as well as fragments of the 3' terminus (L1-680) and the polymerase activity module of the L gene (Lpam) of 36 RABV isolates were sequenced. In addition, whole genome sequencing was performed for 5 RABV isolates. There was evidence of topological discrepancy in the phylogenetic trees based on different genes of the RABV isolates. Amino acid variation of the deduced N protein exhibited different patterns to those obtained from the P and M proteins reported here, and the previously reported G protein (Tang H. et al., PLoS Negl Trop Dis, 8(10): e3114, 2014), and L1-680 and Lpam. These RABV isolates were divided into three main branches against fixed strains. CONCLUSION: RABV is prevalent in Guangxi province and strains collected over the last two decades belong mainly to three groups (I, II, III). These RABV isolates reveal genetic diversity. Individual RABV genes from Guangxi exhibit different evolutionary characteristics. The results will have benefits for continuing comprehensive rabies surveillance, prevention and control in China.

Generation of transgenic-cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein.

Huanjiang Xiang pig is a unique native minipig breed originating in Guangxi, China, and has great utility value in agriculture and biomedicine. Reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) and SCNT-mediated genetic modification show great potential value in genetic preservation and utilization of Huanjiang Xiang pigs. Our previous work has successfully produced cloned and transgenic-cloned embryos using somatic cells from a Huanjiang Xiang pig. In this study, we firstly report the generation of transgenic-cloned Huanjiang Xiang pigs carrying an enhanced green fluorescent protein (eGFP) gene. A total of 504 SCNT-derived embryos were transferred to two surrogate recipients, one of which became pregnant and gave birth to three live piglets. Exogenous eGFP transgene had integrated in all of the three Huanjiang Xiang piglets identified by genotyping. Furthermore, expression of eGFP was also detected from in vitro cultured skin fibroblast cells and various organs or tissues from positive transgenic-cloned Huanjiang Xiang pigs. The present work provides a practical method to preserve this unique genetic resource and also lays a foundation for genetic modification of Huanjiang Xiang pigs with improved values in agriculture and biomedicine.

Characterization of the biological properties and complete genome sequence analysis of a cattle-derived rabies virus isolate from the Guangxi province of southern China.

In this study, a street rabies virus isolate, GXHXN, was obtained from the brain of one rabid cattle in Guangxi province of southern China. To characterize the biological properties of GXHXN, we first evaluated its pathogenicity using 4-week-old adult mice. GXHXN was highly pathogenic with a short incubation period and course of disease. Its LD50 of 10(-6.86)/mL is significantly higher than the LD50 of 10(-5.19)/mL of GXN119, a dog-derived rabies virus isolate. It also displayed a higher neurotropism index than the rRC-HL strain. However, the relative neurotropism index of GXHXN was slightly lower than that of GXN119. Analyzing antigenicity using anti-N and anti-G monoclonal antibodies (MAbs), all tested anti-N MAbs reacted similarly to GXHXN, CVS, and rRC-HL, but the reaction of anti-N MAbs to GXHXN was slightly different from GXN119. Moreover, 2/11 tested anti-G mAbs showed weaker reactivity to GXHXN than rRC-HL, whereas 4/11 showed stronger reactivity to GXHXN than CVS and GXN119, indicating that the structures of G might differ. In order to understand its genetic variation and evolution, the complete GXHXN genome sequence was determined and compared with the known 12 isolates from other mammals. A total of 42 nucleotide substitutions were found in the full-length genome, including 15 non-synonymous mutations. The G gene accounts for the highest nucleotide substitution rate of 0.70 % in ORF and an amino acid substitution rate of 0.95 %. Phylogenetic trees based on the complete genome sequence as well as the N and G gene sequences from 37 known rabies isolates from various mammals demonstrated that the GXHXN is closely related to the BJ2011E isolate from a horse in Beijing, the WH11 isolate from a donkey in Hubei, and isolates from dogs in the Fujian and Zhejiang provinces. These findings will be helpful in exploring the molecular mechanisms underlying interspecies transmission and the genetic variation of the rabies virus in different mammal species.

CRISPR/Cas9-Mediated Generation of Guangxi Bama Minipigs Harboring Three Mutations in α-Synuclein Causing Parkinson's Disease.

Parkinson's disease (PD) is a common, progressive neurodegenerative disorder characterized by classical motor dysfunction and is associated with α-synuclein-immunopositive pathology and the loss of dopaminergic neurons in the substantia nigra (SN). Several missense mutations in the α-synuclein gene SCNA have been identified as cause of inherited PD, providing a practical strategy to generate genetically modified animal models for PD research. Since minipigs share many physiological and anatomical similarities to humans, we proposed that genetically modified minipigs carrying PD-causing mutations can serve as an ideal model for PD research. In the present study, we attempted to model PD by generating Guangxi Bama minipigs with three PD-causing missense mutations (E46K, H50Q and G51D) in SCNA using CRISPR/Cas9-mediated gene editing combining with somatic cell nuclear transfer (SCNT) technique. We successfully generated a total of eight SCNT-derived Guangxi Bama minipigs with the desired heterozygous SCNA mutations integrated into genome, and we also confirmed by DNA sequencing that these minipigs expressed mutant α-synuclein at the transcription level. However, immunohistochemical analysis was not able to detect PD-specific pathological changes such as α-synuclein-immunopositive pathology and loss of SN dopaminergic neurons in the gene-edited minipigs at 3 months of age. In summary, we successfully generated Guangxi Bama minipigs harboring three PD-casusing mutations (E46K, H50Q and G51D) in SCNA. As they continue to develop, these gene editing minipigs need to be regularly teseted for the presence of PD-like pathological features in order to validate the use of this large-animal model in PD research.

Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4.

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4.

Molecular epidemiology and phylogenetic analysis of diverse bovine astroviruses associated with diarrhea in cattle and water buffalo calves in China.

Astroviruses are the principal causative agents of gastroenteritis in humans and have been associated with diarrhea in other mammals as well as birds. However, astroviral infection of animals had been poorly studied. In the present study, 211 rectal swabs collected from cattle and water buffalo calves with mild to severe diarrhea were tested for bovine astrovirus (BAstV) by RT-PCR. Results: 92/211 (43.6%) samples were positive for BAstV, at a rate of 46.10% (71/154) in cattle and 36.84% (21/57) in water buffalo. Phylogenetic analysis based on the partial and full-length of 25 ORF2 amino acid sequences obtained in this study classified the Guangxi BAstVs isolates into five subgroups under the genus of Mamastrovirus, genotype MAstV33, which suggested that the water buffalo was a new host of this genogroup that previously included only cattle and roe deer. Despite the origin of the host, the Guangxi BAstV isolates were closely related to the BAstV Hong Kong isolates (B18/HK and B76-2/HK), but highly divergent from the BAstV NeuroS1 isolate previously associated with neurologic disease in cattle in the U.S.A. Nucleotide sequence-based characterization of the ORF1b/ORF2 junction and corresponding overlapping regions showed distinctive properties, which may be common to BAstVs. Our results suggested that cattle and water buffalo are prone to infection of closely related astroviruses, which probably evolved from the same ancestor. The current study described astroviruses in water buffalo for the first time and is thus far among the largest epidemiological investigations of BAstV infection in cattle conducted in China.

A recombinant rabies virus expressing a phosphoprotein-eGFP fusion is rescued and applied to the rapid virus neutralization antibody assay.

Rabies remains a worldwide concern, and dogs are a major vector for rabies virus (RABV) transmission. Vaccination is used in China to control the spread of rabies in dogs, a practice which necessitates effective, efficient, and high-throughput methods to confirm vaccination. The current rapid fluorescent focus inhibition test (RFFIT) method to measure virus-neutralizing antibody titers in the serum involves multiple steps, and more efficient methods are needed to match the increasing demand for this type of monitoring. In this study, based on the parental rRC-HL strain, a recombinant RABV rRV-eGFP expressing enhanced green fluorescent protein (eGFP) fused with RABV P protein was generated by a reverse genetic technique. The rRV-eGFP grew stably and successfully expressed P-eGFP fusion in Neuro-2A (NA) host cells. Furthermore, the P protein was shown to co-localize with eGFP in rRV-eGFP-infected NA cells. Since eGFP is easily detected in infected cells under a fluorescence microscope, rRV-eGFP could be used to establish a more rapid virus-neutralizing antibody titers assay based on RFFIT, designated as the RFFIT-eGFP method. From 69 canine serum samples, the RFFIT-eGFP method was shown to be as specific and as sensitive as the RFFIT method, suggesting that it might represent a faster tool than conventional RFFIT for measuring RABV virus-neutralizing antibody titers in canine sera without sacrificing accuracy.

Complete genome sequence of a rabies virus isolate from cattle in guangxi, southern china.

A street rabies virus (RV) isolate, GXHXN, was obtained from brain tissue of rabid cattle in the Guangxi Zhuang Autonomous Region of China in 2009. GXHXN is the first isolate from cattle in China with its entire genome sequenced and is closely related to BJ2011E from horse in Beijing, WH11 from donkey in the Hubei Province, and isolates from dogs in the Guangxi and Fujian Provinces, with homologies of 97.6% to 99.6%. It is more distantly related to isolates from domestic cat, pig, Chinese ferret badger, and vaccine strains, with homologies of 83.1% to 88.0%.