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.

Epidemiological investigation of Mycoplasma Synoviae in native chicken breeds in China.

BACKGROUD: Mycoplasma synoviae (M. synoviae) is widely distributed around the world, and leads to serious economic losses in the world every year. Nevertheless, the incidence and epidemiology of M. synoviae infection in China have remained unclear. RESULTS: In this study we demonstrate that over 9773 broiler chicken flocks in 16 Chinese provinces were affected by M. synoviae between 2010 and 2015. Our epidemiological study revealed that M. synoviae was widely prevalent in multi-aged Chinese native breeder chickens, and the prevalence of M. synoviae in embryos of breeders reached up to 16.29%. In addition, our data showed that chickens aged 14 days or younger carried simultaneously high levels of maternal antibody against M. synoviae and high M. synoviae infection (10%), and low M. synoviae antibody levels in breeders and high proportion of M. synoviae infection in embryos could increase the chances of incidence in the offspring. Finally, our results also indicated that 3- to 7-week-old chickens might be most the susceptible to M. synoviae and, therefore, might play a key role in the horizontal transmission of M. synoviae. CONCLUSION: Our findings suggest that M. synoviae is widely circulating in Chinese native chickens, accordingly, effective control measures are urgently needed to control the spread.

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.

Apolipoprotein D facilitates rabies virus propagation by interacting with G protein and upregulating cholesterol.

Rabies virus (RABV) causes a fatal neurological disease, consisting of unsegmented negative-strand RNA, which encodes five structural proteins (3'-N-P-M-G-L-5'). Apolipoprotein D (ApoD), a lipocalin, is upregulated in the nervous system after injury or pathological changes. Few studies have focused on the role of ApoD during virus infection so far. This study demonstrated that ApoD is upregulated in the mouse brain (in vivo) and C8-D1A cells (in vitro) after RABV infection. By upregulating ApoD expression in C8-D1A cells, we found that ApoD facilitated RABV replication. Additionally, Co-immunoprecipitation demonstrated that ApoD interacted with RABV glycoprotein (G protein). The interaction could promote RABV replication by upregulating the cholesterol level. These findings revealed a novel role of ApoD in promoting RABV replication and provided a potential therapeutic target for rabies.

Phylogenetic analysis of Bombyx mori nucleopolyhedrovirus polyhedrin and p10 genes in wild isolates from Guangxi Zhuang Autonomous Region, China.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a severe pathogen that seriously impacts the sericulture industry. In this study, 45 wild BmNPV isolates were collected from different silkworm-raising regions in China's Guangxi Zhuang Autonomous Region. Two highly expressed very late genes from each isolate, polyhedrin and p10, were sequenced and subjected to phylogenetic analysis. The polyhedrin gene was found to be highly conserved, while the p10 gene was more variable frequently harboring point mutations and displaying variations in codon use without obvious codon bias. The BmNPV isolates from Guangxi were separated into three main clades, I, II, and III, according to the p10 gene phylogenetic tree. The geographical distribution of clade I isolates in Guangxi showed a concentrated pattern and that of clade II isolates showed a connected pattern. Clade III isolates were irregularly scattered throughout Guangxi. Local transmission of this pathogen clearly occurred in the silkworm-raising regions in Guangxi. This study may provide some data on BmNPV transmission in the silkworm-raising regions and be helpful in devising strategies for the prevention and control of BmNPV disease.

Lyssavirus surveillance in bats of southern China's Guangxi Province.

Although rabies virus is widely distributed in the world, and has been the subject of extensive investigations with the objective of its ultimate prevention, control, and management, there is much less knowledge of the characteristics, distribution, and infectivity of other lyssaviruses. Since bats are known animal vectors for all but one of the known lyssavirus genotypes, we have performed an extensive survey of bats in the Guangxi Province to provide information on lyssavirus distribution in southern China. The lyssavirus nucleoprotein gene was detected in brains of 2.86 % of 2,969 bats. Nucleotide sequence homologies among isolates were 86.9-99.6 %, but only 70.0-85.0 % for lyssaviruses in GenBank. These infected bats were detected from a wide area, essentially forming a band running from the south-west to the north-east of Guangxi, and it appears that infection by new lyssaviruses is widespread in this region.

Molecular epidemiology of PRRSV from China's Guangxi Province between 2007 and 2009.

Porcine reproductive and respiratory syndrome (PRRS) is considered to be one of the most important infectious diseases affecting livestock. This study used gene sequence analysis of ORF5 and Nsp2 to determine the molecular epidemiology of PRRSV in different parts of the Guangxi province of China. These genes were selected due to their extensive variation within the genome. Out of 189 samples from animals suspected to have PRRS, 145 were PRRSV RNA positive. ORF5 and Nsp2 gene sequence analysis of 31 of these samples showed that all of the Guangxi isolates were of type 2. A phylogenetic tree analysis based on ORF5 showed that the Guangxi isolates were divided into two groups. Most of these were closely related to highly pathogenic strains, showing a 30 amino acid deletion at positions 481 and 533-561 of Nsp2, but an additional unique isolate (GXNN06) possessed a further four amino acid deletion at positions 485-488 of Nsp2.

Host Desmin Interacts with RABV Matrix Protein and Facilitates Virus Propagation.

Microfilaments and microtubules, two crucial structures of cytoskeletal networks, are usurped by various viruses for their entry, egress, and/or intracellular trafficking, including the Rabies virus (RABV). Intermediate filaments (IFs) are the third major component of cytoskeletal filaments; however, little is known about the role of IFs during the RABV infection. Here, we identified the IF protein desmin as a novel host interactor with the RABV matrix protein, and we show that this physical interaction has a functional impact on the virus lifecycle. We found that the overexpression of desmin facilitates the RABV infection by increasing the progeny virus yield, and the suppression of endogenous desmin inhibits virus replication. Furthermore, we used confocal microscopy to observe that the RABV-M co-localizes with desmin in IF bundles in the BHK-21 cells. Lastly, we found that mice challenged with RABV displayed an enhanced expression of desmin in the brains of infected animals. These findings reveal a desmin/RABV-M interaction that positively regulates the virus infection and suggests that the RABV may utilize cellular IFs as tracks for the intracellular transport of viral components and efficient budding.

Genetic, biological and epidemiological study on a cluster of H9N2 avian influenza virus infections among chickens, a pet cat, and humans at a backyard farm in Guangxi, China.

During an investigation in October 2018, two people with diarrhoea, mild abdominal pain, and mild arthralgia symptoms in Guangxi, China, were identified as infected by H9N2 avian influenza virus (AIV). Four H9N2 AIVs were isolated from one of two patients, a pet cat, and a dead chicken (two respective isolates from its lung and kidney tissues) bred by the patients at a backyard farm. Epidemiological investigation indicated that the newly bought chicken died first, and clinical syndromes appeared subsequently in the two owners and one cat. Furthermore, the two individuals possessed high H9N2-specific hemagglutination inhibition and microneutralization antibodies. Shared nucleotide sequence identity (99.9% - 100%) for all genes was detected in the four H9N2 isolates, and hemagglutinin (HA) T138A located on the receptor binding domain (RBD), resulted from nucleotide polymorphisms that were exclusively found in the isolate from the female patient. Moreover, HA K137N on the RBD was found in isolates from these three host species. Importantly, these four H9N2 isolates presented an exclusive binding preference for the human-type receptor (α2-6-SA), and could replicate and cause pathological changes in mice. Phylogenetic analyses showed that these four isolates clustered together and belonged to clade C1.2, lineage Y280. In addition, H9N2 viruses of human origin are genetically divergent and interspersed with the widespread poultry-origin H9N2 AIVs. All these results indicate a high risk of H9N2 AIVs in public health, and effective prevention and control measures against H9N2 AIVs should be considered and performed for both animal and human health.

Transcriptomic Analysis of mRNA Expression Profiles in the Microglia of Mouse Brains Infected with Rabies Viruses of Varying Virulence.

Rabies is a lethal encephalitis caused by the rabies virus (RABV) with a fatality rate near 100% after the onset of clinical symptoms in humans and animals. Microglia are resident immune cells in the central nervous system. Few studies have been conducted on the functional role of microglia in RABV infection. Here, we performed a transcriptomic analysis of mRNA expression profiles in the microglia of mouse brains intracerebrally infected with RABV. We successfully isolated single microglial cells from the mouse brains. The survival rate of dissociated microglial cells was 81.91%-96.7%, and the purity was 88.3%. Transcriptomic analysis revealed 22,079 differentially expressed mRNAs identified in the microglia of mouse brains infected with RABV strains (rRC-HL, GX074, and CVS-24) of varying virulence at 4 and 7 days post-infection (dpi) compared to the control group. The numbers of DEGs versus the control at 4 and 7 dpi in mice infected with rRC-HL, GX074, and CVS-24 were 3622 and 4590, 265 and 4901, and 4079 and 6337. The GO enrichment analysis showed that response to stress, response to external stimulus, regulation of response to stimulus, and immune system process were abundant during RABV infection. The KEGG analysis indicated that the Tlr, Tnf, RIG-I, NOD, NF-κB, MAPK, and Jak-STAT signaling pathways were involved in RABV infection at both 4 and 7 dpi. However, some phagocytosis and cell signal transduction processes, such as endocytosis, p53, phospholipase D, and oxidative phosphorylation signaling pathways, were only expressed at 7 dpi. The involvement of the Tnf and Tlr signaling pathways prompted us to construct a protein-protein interaction (PPI) network of these pathways. The PPI revealed 8 DEGs, including Mmp9, Jun, Pik3r1, and Mapk12. Notably, Il-1b interacted with Tnf and Il-6 with combined scores of 0.973 and 0.981, respectively. RABV causes significant changes in mRNA expression profiles in the microglia in mice. 22,079 differentially expressed mRNAs were identified in the microglia of mice infected with RABV strains of varying virulence at 4 and 7 dpi. The DEGs were evaluated using GO, KEGG, and PPI network analysis. Many immune pathways were up-regulated in RABV-infected groups. The findings will help elucidate the microglial molecular mechanisms of cellular metabolism dysregulated by RABV and may provide important information for investigating RABV pathogenesis and therapeutic methods.

In vitro infection with classical swine fever virus inhibits the transcription of immune response genes.

BACKGROUND: Classical swine fever virus (CSFV) can evade the immune response and establish chronic infection under natural and experimental conditions. Some genes related to antigen processing and presentation and to cytokine regulation are known to be involved in this response, but the precise mechanism through which each gene responds to CSFV infection remains unclear. RESULTS: In this study, the amplification standard curve and corresponding linear regression equations for the genes SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-α, and IFN-ß were established successfully. Real-time RT-PCR was used to quantify the immune response gene transcription in PK-15 cells post CSFV infection. Results showed that: (1) immune response genes were generally down-regulated as a result of CSFV infection, and (2) the expression of SLA-2, SLA-DR, Ii and CD80 was significantly decreased (p < 0.001). CONCLUSION: We conclude that in vitro infection with CSFV inhibits the transcription of host immune response genes. These findings may facilitate the development of effective strategies for controlling CSF.

Detection of rabies virus via exciton energy transfer between CdTe quantum dots and Au nanoparticles.

Rabies is a fatal encephalitis caused by the rabies virus. The diagnosis of the disease depends in large part on the exposure history of the victim and clinical manifestations of the disease. Rapid rabies diagnosis is an important step in its prevention and control. Therefore, for accurate and timely diagnosis and prevention of rabies, we developed nanomaterials for a novel photoelectrochemical biosensing approach (PBA) for the rapid and reliable diagnosis of rabies virus. This approach uses high-efficiency exciton energy transfer between cadmium telluride quantum dots and Au nanoparticles and is low cost, and easy to miniaturize. By constructing PBA, rabies virus can be detected quickly and with a high degree of sensitivity and specificity; the minimum detection concentration limit for rabies virus is approximately 2.16 ffu/mL of rabies virus particles, or 2.53 × 101 fg/mL of rabies virus RNA. PBA could also detect rabies virus in the brain and lung tissue from rabid dogs and mice with better sensitivity than RT-PCR.

Phylogenetic analysis of the E2 gene of classical swine fever virus from the Guangxi Province of southern China.

In this study, suspected classical swine fever (CSF) samples from the Guangxi Province of China were obtained from pigs with acute CSF, aborted fetuses, newborn pigs that died at 1-2 days of age, tonsils of healthy pigs, and leukocytes of immunized sows during 2001-2009. About 92 of 775 samples were found to be positive by RT-PCR, and 41 isolates were obtained. Phylogenetic analysis was performed on the 31 isolates by sequencing the E2 gene, and the isolates were found to cluster into two groups: (1) isolates from aborted fetuses (except GXGZ02), deceased newborn baby pigs, tonsils of healthy pigs, and leukocytes of immunized sows belonged to group 1.1, along with vaccine strain, HCLV, and standard virulent strain, Shimen, of China, and (2) 20 isolates from pigs with acute CSF belonged to group 2.1, 13 of which were clustered into subgroup 2.1b with isolates from other provinces of China, and 7 of which were clustered into subgroup 2.1a with isolates from Italy and Germany.

Design, Synthesis, Antibacterial Evaluation, and Induced Apoptotic Behaviors of Epimeric and Chiral 18ß-Glycyrrhetinic Acid Ester Derivatives with an Isopropanolamine Bridge against Phytopathogens.

Because only a handful of agrochemicals can manage bacterial infections, the discovery and development of innovative, inexpensive, and high-efficiency antibacterial agents targeting these infections are challenging. Herein, a series of novel epimeric and chiral 18ß-glycyrrhetinic acid (GA) ester derivatives with various tertiary amine pendants were designed, synthesized, and screened for pharmacological activity. Results showed that some of the title compounds were conferred with significantly enhanced antibacterial activity toward phytopathogens Xanthomonas oryzae pv oryzae (A2, B1-B3, and C1, EC50 values within 3.81-4.82 µg/mL) and Xanthomonas axonopodis pv citri (B1, EC50 = 3.18 µg/mL; B2, EC50 = 2.76 µg/mL). These activities are superior to those of GA (EC50 > 400 µg/mL), thiodiazole copper, and bismerthiazol. Pharmacophore studies revealed that the synergistic combination of GA skeleton and tertiary amine scaffolds contributed to the biological actions. In vivo experiments displayed their promising applications in controlling bacterial infections. Antibacterial mechanism studies revealed that the title compounds could trigger apoptosis in the tested pathogens, evident by bacteria morphological changes observed in scanning electron microscopy images. This outcome should motivate the development of various apoptosis inducers against plant bacterial diseases by a novel mode of action compared to that of existing agricultural chemicals.

Phosphorylation and dephosphorylation of threonine 188 in nucleoprotein is crucial for the replication of influenza A virus.

Nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex that is responsible for viral replication, transcription and packaging of influenza A virus. Phosphorylation of NP plays an important role during viral infection. In the present study, we identified threonine 188 (T188) as a novel phosphorylated residue in the NP of influenza A virus by using mass spectrometry. T188 is located within nuclear export signal 2 (NES2) which is chromosome region maintenance 1 (CRM1)-independent. We observed that the phosphorylation and dephosphorylation of residue T188 regulated viral replication by controlling NES2-dependent NP nuclear export and the polymerase activity of the vRNP complex. Our findings provide further insights for understanding the replication of influenza A virus.

IRF1 up-regulates isg15 gene expression in dsRNA stimulation or CSFV infection by targeting nucleotides -487 to -325 in the 5' flanking region.

Interferon (IFN)-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is heavily involved in immune response elicitation. As an important member of interferon regulatory factor (IRF) family, IRF1 can activate the expression of multiple genes, including the human optineurin gene (Sudhakar et al., 2013). In this study, a sequence in the promoter region of the optineurin gene was compared to the 5' flanking region of the porcine isg15 gene. Porcine IRF1 also possesses antiviral activity against several swine viruses (Li et al., 2015), but the mechanism is not well understood. Herein, we report that porcine IRF1 and ISG15 were up-regulated in porcine kidney (PK-15) cells following stimulation with double-stranded RNA (dsRNA) or classical swine fever virus (CSFV) infection. We also found that siRNA-mediated knockdown of IRF1 expression resulted in lower ISG15 expression in response to polyinosinic:polycytidylic acid [poly(I:C)] or CSFV infection. The overexpression of IRF1 resulted in ISG15 up-regulation. IRF1 was shown to translocate to the nucleus in response to dsRNA stimulation. To further identify the functional domain of the isg15 gene that promotes IRF1 transcriptional activity, firefly luciferase and ISG15 reporter systems were constructed. The results of the firefly luciferase and ISG15 reporter assay suggested that IRF1 mediates the up-regulation of ISG15. Nucleotides -487 to -325, located in the 5' flanking region of the isg15 gene, constituted the promoter region of IRF1. ChIP assay indicated that IRF1 protein was able to interact with the DNA in the 5'fr of isg15 gene in cells. As an innate immune response protein with broad-spectrum antiviral activity, the up-regulation of ISG15 mediated by IRF1 in porcine cells is reported for the first time. These results warrant further investigation into the antiviral activity of porcine IRF1 against reported swine viruses.

Molecular epidemiology of rabies in Guangxi Province, south of China.

BACKGROUND: Surveillance data for rabies in Guangxi Province in China showed that human rabies cases have gradually increased since 1996. OBJECTIVE: To evaluate the epidemiology of rabies at the molecular level and provide suggestions for effective prevention of rabies in Guangxi. STUDY DESIGN: Since 2000, 1569 brains from suspected rabid animals were collected from different areas of Guangxi. Rabies virus was isolated from 42 samples. RT-PCR was used to amplify a 455 nucleotide segment of the 3'-terminal of the N gene. The sequencing data from that segment was used for phylogenetic analysis. RESULTS: Nucleotide homology comparisons and phylogenetic tree analysis based on this sequence indicated that all the rabies virus isolates from Guangxi belonged to genotype 1 and could be divided into four groups. Groups I, II and IV included 23, 10 and 8 isolates, respectively. These had nucleotide homologies of 97.1-100%, 98.2-100% and 99.1-99.6%, respectively. Only the GXN119 strain belonged to group III. Group I had two group-specific mutations: T90N and E110D. Group II had one group-specific mutation of T42S. CONCLUSIONS: This study showed that rabies virus isolates from Guangxi have a close genetic relationship and topographical distribution.

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.