Molecular Epidemiology and Sequencing of the G-L Intergenic Region of Rabies Viruses Isolated in China

一组 25 个狂犬病病毒(RABV ) ，从 24 条狗和一个人的盒子恢复了，在在 2004 和 2006 之间的中国从各种各样的区域被收集。G-L intergenic 区域的基因、种系发生的分析在 25 街 RABV 被执行孤立， CTN 疫苗 7 拉紧代。学习基于 519 bp 核苷酸顺序的比较，包含 G-L intergenic 区域。中国街紧张的核苷酸顺序相同从 95.5% ～ 100% 。种系发生的分析证明中国的所有孤立清楚地在 Lyssavirus 遗传型 1 支持了所有中国病毒的放置，他们根据他们的地理起源是分布式的。所有仔细中国紧张被联系，但是他们能仍然被划分成二个组：一些街紧张和一些 CTN 紧张。这研究基于 G-L Intergenic 区域的序列关于狂犬病病毒的分子的传染病学介绍细节。关键词狂犬病病毒 - 分子的传染病学 - G-L intergenic 区域 - 中国 CLC 数字 R373.33 基础条款：第 10 国家 five-year-plan (2004BA718 b03 ) 的关键技术 R&D

Pathogenicity of Rabies Viruses Isolated in China: Two Fixed Strains and a Street Strain

Objective To investigate the virulence characteristics of two fixed strains （CTN and aG） and a street strain （HNIO） of rabies viruses isolated in China, Methods ICR mice of different age groups were via the intracracerebral （i.c.） or intramuscular （i.m inoculated with CTN, aG and HN10 rabies virus strains ） routes, and observed for 20 days. Results The CTN strain was pathogenic to 7-day-old suckling mice that received i.c. inoculations and 3-day-old suckling mice that received i.m. inoculations. The aG strain was pathogenic to 4-week-old mice that received i.c. inoculations and 7-day-old suckling mice that received i.m. inoculations. The HN10 strain was pathogenic to mice of all age groups via both inoculation routes. In moribund mice, the viruses had spread to most regions of the brain. The CTN and HN10 strains had similar dissemination patterns in the brain; both viral antigens could be found in the dentate gyrus （DG）, whereas few viral antigens were present in the DG from specimens that had been infected with the aG strain. Conclusion A comprehensive sequence analysis of the G protein suggested that differences in gene sequences may be responsible for producing strain-specific differences in pathogenicity and distribution in the brain.

Epidemic and Maintenance of Rabies in Chinese Ferret Badgers (Melogale moschata) indicated by Epidemiology and the Molecular Signatures of Rabies Viruses

An epidemic of Chinese ferret badger-associated human rabies was investigated in Wuyuan county, Jiangxi province and rabies viruses isolates from ferret badgers in different districts in Jiangxi and Zhejiang provinces were sequenced with their nucleotides and amino acids and aligned for epidemiological analysis. The results showed that the human rabies in Wuyuan are only associated with ferret badger bites; the rabies virus can be isolated in a high percentage of ferret badgers in the epidemic areas in Jiangxi and Zhejiang provinces; the isolates share the same molecular features in nucleotides and have characteristic amino acid signatures, i.e., 2 sites in the nucleoprotein and 3 sites in the glycoprotein, that are distinct from virus isolates from dogs in the same region. We conclude that rabies in Chinese ferret badgers has formed an independent transmission cycle and ferret badgers may serve as another important rabies reservoir independent of dog rabies in China.

Screening of Serum Dilution and Determination of Normal Range of Antibodies against Rabies Viruses in Dog Sera

[ Objective] To screen a suitable serum dilution and determine the normal range of antibodies against rabies viruses （RV） in dog serum. [Method]A sensitive, specific and suitable serum dilution was screened. A total of 812 dog serum samples were collected from Changchun, Nanning and Uuzhou regions, and then they were diluted with above screened serum dilution and evaluated by indirect enzyme-linked immunosorbont assay （ELISA）. The positive and negative standard dog sara from the World Organization for Animal Health （OIE） were used as controls. The serum samples with different A450 were randomly selected and the anti-RV serum titers were detected by rapid fluorescent focus inhibition test （RFFIT）. According to the A450 of dog sera, the normal A450 range of negative serum and 95% confidence intervals were calculated with SPSS 10.0 software, and the regression equation of OIE standards was established. [ Result] The screened serum dilution was sensitive and specific; at least 756 negative serum samples were obtained; the normal range and 95% confidence interval of negative serum were 0.127 3 ± 0.059 8 and 0.078 1 -0.172 3, respectively; and the regression equation was A450 = 1.139 serum titer （IU） ±0.470. [ Conclusion] These results lay a foundation for thecontrolling of dog rabies endemic and the development of ELISA kits of dog antibodies against RV.

Sequence Analyses on N Gene of Rabies Viruses Isolated from Guangdong Province

[ Objective] To find the dominant rabies virus strains that are epidemic in Guangdong Province and thus to better prevent and control rabies. [ Method] Canine brains and salivary glands collected from several regions of Guangdong Province were detected by direct imrnunofluorescence assay and nested RT-PCR. The isolates were further identified by intracranial inoculation in suckling mice. The N gene was amplified by RTPCR and the sequences were aligned with those of prevalent strains in Guangxi region and vaccine strains. [ Result] Two strains were isolated from specimens collected from Guangdong Province. Their N gene sequences had a high similarity of 97.9% -99.4% to those of Guangxi strains and also had a similarity of 87.7% -88.1% to those of vaccine strains of rabies virus. [ Conclusion] Apparently healthy dogs may carry rabies virus, and the prevalence of rabies virus shows obvious regional differences.

A Single-chain Antibody for One-pot Fabrication of Luminescent Gold Nanoclusters and Rabies Virus Imaging in Cells

The fragile antibody leads to a great challenge as a scaffold to fabricate the luminescent metal nanoclusters using one-pot method.This study presents a stable single-chain anti-body(scFv57R-ATS)for the fabrication of luminescent gold nanoclusters(AuNCs@scFv57R-ATS)and a quick,sensitive rabies virus detection in living cells.In this paper,AuNCs@scFv57R-ATS was designed to specifically recognize antigen RV in modified HeLa cells,which promoted the demonstration of metal nanocluster fluorescent probes for antigen targeting and therapy.

A rabies virus-based toolkit for efficient retrograde labeling and monosynaptic tracing

Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases.Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing,making these vectors powerful candidates for the dissection of synaptic inputs.Although several attenuated rabies viral vectors have been developed,their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors.To overcome these limitations,we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus.Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably:(1)N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro,and the efficiency was six times higher than that of rAAV9-Retro;(2)the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2–3 times higher than that of oG-mediated SAD-B19-ΔG;(3)CVS-N2c-ΔG could delivery modified genes for neural activity monitoring,and the time window during which this was maintained was 3 weeks;and(4)CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination.These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.

Brain-wide N2cG compensation permits glycoprotein-deleted rabies virus to trace neural circuits across multiple synapses

Rabies-viruses-based retrograde tracers can spread across multiple synapses in a retrograde direction in the nervous system of rodents and primates,making them powerful tools for determining the structure and function of the complicated neural circuits of the brain.However,they have some limitations,such as posing high risks to human health and the inability to retrograde trans-synaptic label inputs from genetically-de¯ned starter neurons.Here,we established a new retrograde trans-multi-synaptic tracing method through brain-wide rabies virus glycoprotein(RVG)compensation,followed by glycoprotein-deleted rabies virus(RV-△G)infection in specific brain regions.Furthermore,in combination with the avian tumor virus receptor A(TVA)controlled by a cell-type-specific promoter,we found that EnvA-pseudotyped RV-△G can mediate e±cient retrograde trans-multi-synaptic transduction from cell-type-specific starter neurons.This study provides new alternative methods for neuroscience researchers to analyze the input neural networks of rodents and nonhuman primates.

Molecular Characterization of Viral G Gene in Emerging and Re-emerging Areas of Rabies in China, 2007 to 2011

In recent years (2007 to 2011), although the overall number of rabies cases in China has decreased, there is evidence of emerging or re-emerging cases in regions without previous rabies cases or with low incidence of rabies. To investigate the origin and the factors affecting the spread of rabies in China, specimens were collected from 2007 to 2011 from provinces with emerging and re-emerging cases and tested for the presence of the rabies virus. Positive specimens were combined with sequences from GenBank to perform comparisons of homology and functional sites, and to carry out phylogenetic analyses. Out of these regions, five provinces had 9 positive specimens from canine and cattle, and 34 canine or human specimens were obtained from previously high-incidence provinces. Complete sequences of G gene were obtained for these samples. Homology of the sequences of these 43 specimens was 87%-100% at the nucleotide level and 93.7% -100% at the amino acid level. These G gene sequences were combined with reference sequence from GenBank and used to construct a phylogenetic tree. The results showed that 43 specimens were all assigned to China clade I and clade II, with all specimens from emerging and re-emerging areas placed within clade I. Specimens isolated from Shanxi and Inner Mongolia in 2011 were distinct from previously-isolated local strains and had closer homology to strains from Hebei, Beijing and Tianjin whereas new isolates from Shanghai were tightly clustered with strains isolated in the 1990s. Finally, Shaanxi isolates were clustered with strains from adjacent Sichuan. Our results suggest that the rabies cases in emerging and re-emerging areas in China in the last 5 years are a consequence of the epidemic spreading from of neighboring provinces and regions experiencing a serious epidemic of rabies.

The Full-length Genome Analysis of a Street Rabies Virus Strain Isolated in Yunnan Province of China

The epidemic of rabies has rapidly increased and expanded in Yunnan province in recent years. In order to further analyze and understand the etiological reasons for the rapid expansion of rabies in Yunnan, a strain of rabies virus CYN1009D in Yunnan was isolated, and the complete genomic sequencing was carried out, and the bioimfomative analysis on genes/encoded proteins and phylogeny with reference to sequences in GenBank was performed. The complete genome of CYN1009D was 11923 nt in length and belonged to genotype I. The genes encoding different structural proteins were all conserved in their lengths, in comparison to other strains in China. The amino acid sequence was conserved at different antigen sites of NP, but the variation was detected at the secondary phosphorylation site of position 375; variations were also detected in the phosphorylation sites at positions 63-63 and 162 of PP; the sites playing important roles in virus synthesis, budding and viral morphology in MP were conserved; two glycosylation sites were detected at Asn37 and Ash319 in GP, the neutralizing antigen sites in GP were conserved; the initial amino acid of LP （ML） was different from that of most of the strains in China （MM）; the variations in G-L region in the intergenic region were significant. The phylogenic tree showed that CYN1009D has a closer genetic relationship to the strains in Southeast Asia, indicating that prevention and control on rabies in borderland areas should be reinforced meanwhile efforts are made to control rabies in China.

Current Status of Canine Rabies in China

Abstract The number of human rabies cases acquired from dog bites constitutes a high proportion of the total rabies cases in China, although the number of human rabies cases has gradually decreased in recent years. The pivotal role of dogs in the spread of rabies indicates that controlling and preventing canine rabies could be a key step in eradicating human rabies in China. The primary aims of this review are to discuss the properties and pathogenesis of the rabies virus, the clinical signs and diagnosis of canine rabies, threshold host density and vaccination of dogs, and the prevention and control of canine rabies in China.

Preparation and Initial Application of a Monoclonal Antibody Specific for a Newly Discovered Conserved Linear Epitope of Rabies Virus Nucleoprotein

Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate （FITC） after purification and used in fluorescent antibody test （FAT）. Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line （BSR）. Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies

Rabies Virus Neutralizing Activity,Safety,and Immunogenicity of Recombinant Human Rabies Antibody Compared with Human Rabies Immunoglobulin in Healthy Adults

Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.

Regulation of innate immune responses by rabies virus

Rabies virus(RABV)is an infectious and neurotropic pathogen that causes rabies and infects humans and almost all warm-blooded animals,posing a great threat to people and public safety.It is well known that innate immunity is the critical first line of host defense against viral infection.It monitors the invading pathogens by recognizing the pathogen-associated molecular patterns and danger-associated molecular patterns through pattern-recognition receptors,leading to the production of type I interferons(IFNα/β),inflammatory cytokines,and chemokines,or the activation of autophagy or apoptosis to inhibit virus replication.In the case of RABV,the innate immune response is usually triggered when the skin or muscle is bitten or scratched.However,RABV has evolved many ways to escape or even hijack innate immune response to complete its own replication and eventually invades the central nervous system(CNS).Once RABV reaches the CNS,it cannot be wiped out by the immune system or any drugs.Therefore,a better understanding of the interplay between RABV and innate immu-nity is necessary to develop effective strategies to combat its infection.Here,we review the innate immune responses induced by RABV and illustrate the antagonism mechanisms of RABV to provide new insights for the control of rabies.

Molecular Characterization of China Human Rabies Vaccine Strains

To understand the molecular characteristics of China human rabies vaccine strains, we report the full-length genome of the aG strain and present a comprehensive analysis of this strain and almost all available lyssavirus genomes （58 strains） from GenBank （as of Jan 6, 2011）. It is generally considered that the G protein plays a predominant role in determining the pathogenicity of the virus, to this end we predicted the tertiary structure of the G protein of aG strain, CTN 181 strain and wild type strain HN 10 based on the crystal structure of Vesicular stomatitis virus （VSV） G. The predicted RABV G structure has a similar topology to VSV G and the ectodomain can be divided into 4 distinct domains DI - DIV. By mapping the characterized mutations to this structure between China vaccine strains and their close street strains, we speculate that the G303（P-H） mutations of CTN181 and HN10 causing D II 3D change may be associated with the attenuated virulence in both strains. Specifically, the two signature mutations （G165P and G231P） in the aG strain are withinβsheets, suggesting that both sites are of structural importance.

Research Advances of Epidemiological Characteristics and Diagnosis Technology of Rabies Virus

Rabies is a zoouosis caused by rabies virus, which has been characterized by infection of central nervous system. Rabies virus, which can cause fatal in- fections in humans and other mammals, has the characteristic of neurotropism, and the mortality caused by rabies is almost 100%. Routine diagnosis of rabies in- clude clinical symptoms, while a final diagnosis depends on laboratory diagnostic methods, such as etiological diagnosis, serological diagnosis and molecular biologi- cal diagnosis. As a zoouodc infectious disease, rabies presents a global distribution situation, and the deaths caused by rabies virus infection in China ranks the sec- ond place in the world. Therefore, strengthening the recognition of disease etiology and epldemiology is very important to comprehensive prevention measures of rabi- es. The author mainly introduced the research progress on pathogen, epidemiology and diagnosis technology of rabies virus at home and abroad. In addition, the advantages and disadvantages of various methods were compared respectively, in order to provide a scientific basis for further study and rapid diagnosis of rabies.

Emerging New Phylogenetic Groups of Rabies Virus in Chinese Ferret Badgers

Chinese ferret badger（FB）-transmitted rabies is a serious threat to public health in southeast China. Although mostly associated with dogs, the rabies virus（RABV） presents genetic diversity and has a significantly wide host range in China. Instead of the dog-and wildlife-associated China ⅠI lineage in the past decades, the China Ⅰ lineage has become the main epidemic group hosted and transmitted by dogs. In this study, four new lineages, including 43 RABVs from FBs, have been classified within the dog-dominated China Ⅰ lineage since 2014. FBRABVs have been previously categorized in the China Ⅱ lineage. Moreover, FB-hosted viruses seem to have become the main independent FB-associated clade in the phylogenetic tree. This claim suggests that the increasing genetic diversity of RABVs in FBs is a result of the selective pressure from coexisting dog rabies. FB transmission has become complicated and serious with the coexistence of dog rabies. Therefore, apart from targeting FB rabies, priority should be provided by the appropriate state agencies to perform mass immunization of dog against rabies.

Preparation and Identification of Anti-rabies Virus Monoclonal Antibodies

To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents.

Whole Genome Sequencing and Comparisons of Different Chinese Rabies Virus Lineages Including the First Complete Genome of an Arctic-like Strain in China

Objective To learn the rabies genome molecular characteristics and compare the difference of China rabies lineages. Methods The complete genomes of 12 strains from different China rabies lineages were amplified and sequenced, and all the China street strain genomes （total 43）, Arctic and Arctic-like genomes were aligned using ClustalX2, the genome homologies were analyzed using MegAlign software, and the phylogenetic trees were constructed by MEGA 5. Results First Arctic-like rabies genome in China （CO, H1202D） was reported, and we supplemented the rabies genome data of China, ensuring at least one genome was available in each China lineage. The genome size of China V （11908nt） is obviously shorter than other lineages＇ （11923-11925nt） for the difference of N-P non-coding regions. Among different lineages, the genome homologies are almost under 90%. CQH1202D （China IV lineage） has close relationship with strains from South Korea and they share about 95% genome similarities. Conclusion The molecular characteristics of 6 different China rabies lineages were compared and analyzed from genome level, which benefits for continued comprehensive rabies surveillance, rabies prevention and control in China.

Comparative Analysis of the Pathogenic Mechanisms of Street Rabies Virus Strains with Different Virulence Levels

Objective To characterize two strains of street rabies virus （RABV） isolated from the brain tissue of cattle from Inner Mongolia. Differences in the histopathological and ultrastructural changes in the brain tissue of infected mice were determired to reveal variation in the pathogenesis of infection between street rabies virus strains. Methods Ten-day-old mice were intracranially inoculated with one of three virus strains and brain tissue harvested when the mice were moribund. Various histopathological and ultrastructural markers of disease were then compared between the groups. Results Infection with the street virus strain CNMllO1C resulted in severe neuronal dendrites damage, but only mild cell apoptosis, T lymphocyte infiltration and microglial activation. Infection with the other street virus strain, CNMll03C, was characterized by cell apoptosis, T lymphocyte infiltration and microglial activation as well as dendrites damage. However, in comparison, infection with the attenuated virus strain CTN caused ~evere T lymphocyte infiltration, microglial activation and cell apoptosis, but left the neuronal dendrites intact. Conclusion The two street rabies virJs strains isolated from cattle from Inner Mongolia had different levels of virulence and caused distinct pathological changes in infected mice. Therefore, we concluded that different pathogenic mechanisms exist between different RABV strains.