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Abstract: A group of 31 rabies viruses (RABVs), recovered primarily from dogs, one deer and one human case, were collected from various areas in China between 1989 and 2006. Complete G gene sequences determined for these isolates indicated identities of nucleotide and amino acid sequences of ≥87% and 93.8%, respectively. Phylogenetic analysis of these and some additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between four distinct groups (I–IV). Several variants identified within the most commonly encountered group I were distributed according to their geographical origins. A comparison of representative Chinese viruses with other isolates retrieved world-wide indicated a close evolutionary relationship between China group I and II viruses and those of Indonesia while China group III viruses formed an outlying branch to variants from Malaysia and Thailand. China group IV viruses were closely related to several vaccine strains. The predicted glycoprotein sequences of these RABVs variants are presented and discussed with respect to the utility of the anti-rabies biologicals currently employed in China. [Copyright &y& Elsevier]

In recent years, Coxsackievirus A2 (CV-A2) has become one of the main serotypes of enterovirus species A associated with hand, foot and mouth disease (HFMD) in China. It has also caused HFMD epidemics in many countries all over the world. Currently, there are no effective, preventive vaccines against it. A CV-A2 strain was isolated in RD cells and then adapted to grow in Vero cells. This is in compliance with guidelines for cell substrates allowed for human vaccines by the Chinese regulatory authority. Groups of newborn Kunming mice were inoculated on day 3 and day 9 using two formulations of candidate vaccines, empty particles and full particles. They were then challenged on day 14 at a lethal dose with a mouse-adapted strain. The mice in the control group all died within 14 days post-challenge whereas most of the mice in the candidate vaccine groups survived. It was found that the titers of neutralizing antibodies was dose-dependent in sera of immunized mice. The results also showed that the vaccine candidates stimulated a strong humoral immune response and protected the mice from disease and death. The virus loads in tissues or organs were significantly reduced and pathological changes were either weak or not observed in the immunized groups compared with those in Al(OH) 3 control group. Preliminary mapping of the nucleotide and amino acid residues potentially related to cell tropism of the vaccine strain and virulence of the challenge strain was performed. The results showed that the RD cell-isolated and Vero cell-adapted CV-A2 strain is a promising vaccine candidate. This active immunization-challenge mouse model mimics the vaccination and then exposure to wildtype viruses, compared with passive immunization-challenge model, and is invaluable for efficacy evaluation in studies on multivalent vaccines containing CV-A2 against HFMD. [ABSTRACT FROM AUTHOR]

Rabies, caused by the rabies virus (RABV), is the most fatal zoonotic disease. It is a neglected tropical disease which remains a major public health problem, causing approximately 59,000 deaths worldwide annually. Despite the existence of effective vaccines, the high incidence of human rabies is mainly linked to tedious vaccine immunisation procedures and the overall high cost of post-exposure prophylaxis. Therefore, it is necessary to develop an effective vaccine that has a simple procedure and is affordable to prevent rabies infection in humans. RABV belongs to the genus Lyssavirus and family Rhabdoviridae. Previous phylogenetic analyses have identified seven major clades of RABV in China (China I–VII), confirmed by analysing nucleotide sequences from both the G and N proteins. This study evaluated the immunogenicity and protective capacity of SYS6008, an mRNA rabies vaccine expressing rabies virus glycoprotein, in mice and cynomolgus macaques. We demonstrated that SYS6008 induced sufficient levels of rabies neutralising antibody (RVNA) in mice. In addition, SYS6008 elicited strong and durable RVNA responses in vaccinated cynomolgus macaques. In the pre-exposure prophylaxis murine model, one or two injections of SYS6008 at 1/10 or 1/30 of dosage provided protection against a challenge with a 30-fold LD 50 of rabies virus (China I and II clades). We also demonstrated that in the post-exposure prophylaxis murine model, which was exposed to lethal rabies virus (China I–VII clades) before vaccination, one or two injections of SYS6008 at both 1/10 and 1/30 dosages provided better protection against rabies virus challenge than the immunization by five injections of commercial vaccines at the same dosage. In addition, we proved that SYS6008-induced RVNAs could neutralise RABV from the China I–VII clades. Finally, 1/10 of the dosage of SYS6008 was able to stimulate significant RABV-G specificity in the T cell response. Furthermore, we found that SYS6008 induced high cellular immunity, including RABV-G-specific T cell responses and memory B cells. Our results imply that the SYS6008 rabies vaccine, with a much simpler vaccination procedure, better immunogenicity, and enhanced protective capacity, could be a candidate vaccine for post-exposure prophylaxis of rabies infections. • SYS6008 can elicit strong and durable RVNA responses in mice and cynomolgus macaques with a simpler vaccination procedure. • In the PrEP murine model, SYS6008 with one injection can provide protection against lethal dose of RABV. • In the PEP murine model, SYS6008 with one injection provide better protection than commercial vaccine with five injections. [ABSTRACT FROM AUTHOR]

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the diagnosis of enteroviruses associated HFMD. In this study, a mAb 1A11 was generated using full particles of CV-A5 as an immunogen. In indirect immunofluorescence and Western blotting assays, 1A11 bound to the viral proteins of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of the Enterovirus A and targeted VP3. It has no cross-reactivity to strains of Enterovirus B and C . By mapping with over-lapped and truncated peptides, a minimal and linear epitope 23 PILPGF 28 was identified, located at the N-terminus of the VP3. A BLAST sequence search of the epitope in the NCBI genus Enterovirus (taxid: 12059) protein database indicates that the epitope sequence is highly conserved among the Enterovirus A species, but not among the other enterovirus species, first reported by us. By mutagenesis analysis, critical residues for 1A11 binding were identified for most serotypes of Enterovirus A . It may be useful for the development of a cost-effective and pan- Enterovirus A antigen detection for surveillance, early diagnosis and differentiation of infections caused by the Enterovirus A species.

Hand, foot and mouth disease (HFMD) is caused by a variety of serotypes in species A of the Enterovirus genus, including recently re-emerged Coxsackievirus A2 (CV-A2), CV-A4 and CV-A5. For development of diagnostic reagents, for surveillance, and the development of multivalent vaccines against HFMD, the antigenicity of HFMD-associated enteroviruses warrants investigation. The purified virions of CV-A4 were inoculated into Balb/c mice and hybridomas were obtained secreting monoclonal antibodies (mAbs) directed against CV-A4 and cross-reacting with other closely related species A enteroviruses. The mAbs were characterized by ELISA, Western blotting and in vitro neutralizing assays. The majority of mAbs was non-neutralizing, with only 2% of the mAbs neutralizing CV-A4 specifically. Most of mAbs bound to linear VP1 epitopes of CV-A4. Interestingly, four types of mAbs were obtained which bound specifically to CV-A4 or were broadly to CV-A4/-A2, CV-A4/-A5 and CV-A4/-A2/-A5, respectively. Mapping with overlapping or single-amino-acid mutant peptides revealed that the four types of mAbs all bound to the first 15 amino acids at the N-terminus of the VP1. This region of picornaviruses is functionally important as it is involved in uncoating and releasing of viral RNA into the cytosol. The binding footprints of four type mAbs are composed of conserved and variable residues and are different from each other. The newly discovered broadly cross-reactive mAbs reflect the high homology of CV-A4/ CV-A2/CV-A5. The results also demonstrate that it is possible and beneficial to develop the diagnostic reagents to detect rapidly the main pathogens of enteroviruses associated with HFMD cause by CV-A4/CV-A2/CV-A5., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Rabies virus (RABV) causes severe neurological disease and death. As an important mechanism for generating genetic diversity in viruses, homologous recombination can lead to the emergence of novel virus strains with increased virulence and changed host tropism. However, it is still unclear whether recombination plays a role in the evolution of RABV. In this study, we isolated and sequenced four circulating RABV strains in China. Phylogenetic analyses identified a novel lineage of hybrid origin that comprises two different strains, J and CQ92. Analyses revealed that the virus 3' untranslated region (UTR) and part of the N gene (approximate 500 nt in length) were likely derived from Chinese lineage I while the other part of the genomic sequence was homologous to Chinese lineage II. Our findings reveal that homologous recombination can occur naturally in the field and shape the genetic structure of RABV populations.

In order to study phylogeography, population dynamics and molecular evolution of rabies viruses (RABVs) isolates from China, especially spatio-temporal dynamics, the timescale of RABVs evolution and its pattern of migration, we performed an extensive comparative analysis of RABV N gene sequence data, representing 167 isolates sampled from 20 provinces in a 78-year period (from 1931 through 2009). The available Chinese isolates could be divided into two distinct clades:Phylogroup clades I comprised Chinese group 1-4; Phylogroup clades II contained Chinese group 5-8. We found no evidence for positive selection (dN/dS>1) acting at any codon and found strong selective constraints for N gene. Bayesian Markov Chain Monte Carlo (MCMC) analysis suggested that the Chinese rabies viruses originated within the last 2000 years and the mean rates of nucleotide substitution for the N gene were approximately 4 x 10(-4) substitutions per site per year. The analyses of the spatial and spatio-temporal evolution indicated that RABV isolates from China migrated among different Provinces.

Objective: Based on sequencing the genomes of glycoprotein (GP) gene of rabies viruses isolated in Zhejiang, we analyzed the properties of rabies viruses genetic variation in molecular level, and to compare with those of other representative vaccine strains and street virus strains, get the information about rabies viruses variation., Methods: Suckling mice against rabies virus were selected. Overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses of the GP genes., Results: The fourteen full-length genomes were completely sequenced and they had the same genetic structure with 1575 nts and deduced protein with 524 aa. Genetic analysis revealed that the nucleotide and amino acid homologies of GP gene from Zhejiang strains and other vaccine strains or street virus strains were 82.3% - 99.9% and 85.1% - 99.8%. The fourteen strains were genotype 1 according to the phylogenetic analyses. The GP amino acids of Zhejiang strains rabies virus strains without any recombination occurred in GP and no larger variation appeared in the major antigenic sites., Conclusion: The comprehensive analysis based on the first-level structure of GP demonstrated that it was possible that some advantageous antigenic epitopes existed in certain areas and potential antigenic determinants. It was evident that the GP gene of Zhejiang strains appear to be stable and their sequence similarity with the representative strains of street virus in China were higher than those of other vaccine strains. Some differences showed in the genetic structure and evolution relationship among Zhejiang strains, other street strains in other regions and vaccine strains.

Based on sequencing the full-length genomes of four Chinese Ferret-Badger and dog, we analyze the properties of rabies viruses genetic variation in molecular level, get the information about rabies viruses prevalence and variation in Zhejiang, and enrich the genome database of rabies viruses street strains isolated from China. Rabies viruses in suckling mice were isolated, overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses from Chinese Ferret-Badger, dog, sika deer, vole, used vaccine strain were determined. The four full-length genomes were sequenced completely and had the same genetic structure with the length of 11, 923 nts or 11, 925 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions(IGRs), 423 nts-Pseudogene-like sequence (psi), 70 nts-Trailer. The four full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by BLAST and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the four full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so the nucleotide mutations happened in these four genomes were most synonymous mutations. Compared with the reference rabies viruses, the lengths of the five protein coding regions had no change, no recombination, only with a few point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the four genomes were similar to the reference vaccine or street strains. And the four strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessed the distinct district characteristics of China. Therefore, these four rabies viruses are likely to be street viruses already existing in the natural world.