Development and application of a multiplex qPCR assay for the detection of duck circovirus, duck Tembusu virus, Muscovy duck reovirus, and new duck reovirus.

A multiplex qPCR assay was developed to simultaneously detect duck circovirus (DuCV), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and novel duck reovirus (NDRV), but it did not amplify other viruses, including duck virus enteritis (DVE), infectious bursal disease virus (IBDV), avian reovirus (ARV), H5 avian influenza virus (H5 AIV), H7 avian influenza virus (H7 AIV), H9 avian influenza virus (H9 AIV), Newcastle disease virus (NDV), and Muscovy duck parvovirus (MDPV), and the detection limit for DuCV, DTMUV, MDRV, and NDRV was 1.51 × 101 copies/µL. The intra- and interassay coefficients of variation were less than 1.54% in the repeatability test with standard plasmid concentrations of 1.51 × 107, 1.51 × 105, and 1.51 × 103 copies/µL. The developed multiple qPCR assay was used to examine 404 clinical samples to verify its practicability. The positivity rates for DuCV, DTMUV, MDRV, and NDRV were 26.0%, 9.9%, 4.0%, and 4.7%, respectively, and the mixed infection rates for DuCV + DTMUV, DuCV + MDRV, DuCV + NDRV, MDRV + NDRV, DTMUV + MDRV, and DTMUV + NDRV were 2.7%, 1.2%, 1.2%, 1.0%, 0.5%, and 0.7%, respectively.

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.

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.

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.

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.

Re-emergence of rabies in the Guangxi province of Southern China.

BACKGROUND: Human rabies cases in the Guangxi province of China decreased from 839 in 1982 to 24 in 1995, but subsequently underwent a sharp increase, and has since maintained a high level. METHODOLOGY/PRINCIPAL FINDINGS: 3,040 brain samples from normal dogs and cats were collected from 14 districts of Guangxi and assessed by RT-PCR. The brain samples showed an average rabies virus (RV) positivity rate of 3.26%, but reached 4.71% for the period Apr 2002 to Dec 2003. A total of 30 isolates were obtained from normal dogs and 28 isolates from rabid animals by the mouse inoculation test (MIT). Six representative group I and II RV isolates showed an LD50 of 10-5.35/ml to 10-6.19/ml. The reactivity of monoclonal antibodies (MAbs) to group I and II RV isolates from the Guangxi major epidemic showed that eight anti-G MAbs showed strong reactivity with isolates of group I and II with titers of ≥10,000; however, the MAbs 9-6, 13-3 and 12-14 showed lower reactivity. Phylogenetic analysis based on the G gene demonstrated that the Guangxi RV isolates have similar topologies with strong bootstrap values and are closely bonded. Alignment of deduced amino acids revealed that the mature G protein has four substitutions A96S, L132F, N436S, and A447I specific to group I, and 13 substitutions T90M, Y168C, S204G, T249I, P253S, S289T, V332I, Q382H, V427I, L474P, R463K Q486H, and T487N specific to group II, coinciding with the phylogenetic analysis of the isolates. CONCLUSIONS: Re-emergence of human rabies has mainly occurred in rural areas of Guangxi since 1996. The human rabies incidence rate increased is related with RV positive rate of normal dogs. The Guangxi isolates tested showed a similar pathogenicity and antigenicity. The results of phylogenetic analysis coincide with that of alignment of deduced amino acids.

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%.