The amino acid variation at hemagglutinin sites 145, 153, 164 and 200 modulate antigenicity andreplication of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus's hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.

Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.

An electrochemical sensor for the rapid detection of zearalenone based on the mimic peptide screened by molecular simulation.

An electrochemical sensor was developed for detecting zearalenone (ZEN) based on the mimic peptide, which was screened from the library and validated by molecular simulation and electrochemical methods. The library of the mimic peptide was constructed according to the structural analysis, molecular docking, molecular dynamics and amino acid mutation. Then, the enhanced electrical signal was attributed to gold nanoparticles (AuNPs) and reduced carboxylated graphene oxide (rGO-COOH). Under the optimal conditions, the detection limit was 0.91 pg·mL-1 (S/N = 3) with a wide linear range from 0.01 ng·mL-1 to 10 ng·mL-1. In grain samples, a good recovery rate of 84% to 105.3% was achieved, while the rate ranged from 82% to 108.8% in the commercial ELISA kits. Additionally, the electrochemical sensor exhibited the remarkable specificity, excellent stability and better reproducibility (RSD = 1.94%). This sensor has great potential for rapidly detecting ZEN in food.

High-throughput mutagenesis and screening approach for the identification of drug-resistant mutations in the rifampicin resistance-determining region of mycobacteria.

Rifampicin is the most powerful first-line antibiotic for tuberculosis, which is caused by Mycobacterium tuberculosis. Although accumulating evidence from sequencing data of clinical M. tuberculosis isolates suggested that mutations in the rifampicin-resistance-determining region (RRDR) are strongly associated with rifampicin resistance, the comprehensive characterisation of RRDR polymorphisms that confer this resistance remains challenging. By incorporating I-SceI sites for I-SceI-based integrant removal and utilizing an L5 swap strategy, we efficiently replaced the integrated plasmid with alternative alleles, making mass allelic exchange feasible in mycobacteria. Using this method to establish a fitness-related gain-of function screen, we generated a mutant library that included all single-amino-acid mutations in the RRDR, and identified the important positions corresponding to some well-known rifampicin-resistance mutations (Q513, D516, S522, H525, R529, S531). We also detected a novel two-point mutation located in the RRDR confers a fitness advantage to M. smegmatis in the presence or absence of rifampicin. Our method provides a comprehensive insight into the growth phenotypes of RRDR mutants and should facilitate the development of anti-tuberculosis drugs.

Virus evolution and reduced viral viability during treatment of persistent COVID-19 Omicron BA.5 infection in an immunocompromised host.

We present the clinical course of a 72-year-old female with COVID-19 and a history of hematologic stem cell transplantation for acute myeloid leukemia. We performed serial analyses of viral load and whole-genome amplification. The virus growth was evaluated by a real-time polymerase chain reaction assay. Neutralizing activity was measured using a chemiluminescence reduction neutralizing test of SARS-CoV-2 pseudotyped virus. After neutralizing antibody therapy, the cycle threshold value of viral genome was 28. Viruses were no longer isolated in a cell culture. K129R, V722I, and V987F of amino acid mutation in spike protein region were identified, although they soon disappeared. Four months after symptom onset, E340K, K356R, R346T, and E484V mutations appeared and persisted. The viability of the virus decreased over time, with the virus at day 145 having a cycle threshold value of 24 and positive virus isolation, but at a slower growth rate. Neutralizing antibody activity for Omicron BA.5 finally appeared about 4 months after infection. In immunocompromised patients, persistent infection with amino acid mutations can occur without neutralizing antibodies. However, the production of neutralizing antibodies reduces the growth rate of the SARS-CoV-2. Moreover, infection control requires attention to viral dynamics and evolution under different conditions.

Identification of critical SARS-CoV-2 amino acids associated with COVID-19 hospitalization rate using machine learning and statistical modeling: An observational study in the United States.

BACKGROUND: The COVID-19 pandemic has put many medical systems on the verge of collapse in the last two years. Virus mutation was one of the important factors affecting the COVID-19 infection severity and hospitalizations. Although over ten thousand SARS-CoV-2 mutations being reported since the beginning of the COVID-19 pandemic, only a small percentage of mutations are likely to affect the virus phenotype and change its severity. Finding out which amino acids have the greatest impact on COVID-19 hospitalization rate is an important research question. METHODS: This observational study used the COVID-19 case hospitalization ratio (CHR) to represent the virus severity related with hospitalization. The database is based on the daily state-level epidemiological and genomic sequential data in the United States from the Alpha wave to the first Omicron wave. The critical amino acids that mostly affected the CHR were determined by using four types of models including extreme gradient boosting decision trees (XGBoost), artificial neural networks (ANNs), logistic regression and Lasso regression models. RESULTS: The XGBoost, ANN, logistic regression, and Lasso regression models all produce excellent results (mean square error for all state-level models does not exceed 0.0008 using the testing dataset). Based on the rank of importance of all covariates, the critical amino acids most affecting the CHR were identified, including T19, L24, P25, P26, A27, A67, H69, V70, T95, G142, V143, Y145, E156, F157, N211, L212, V213, R214, D215, G339, R346, S373, L452, S477, T478, E484, N501, A570, P681, and T716. CONCLUSION: This study identified critical amino acids that are most likely to affect the hospitalization rate, allowing public health workers to monitor these highly risky amino acids and raise an alarm immediately when more severe mutations occur. Furthermore, the methodology and results may be extended to other regions.

The whole-genome sequencing of prevalent DENV-1 strains during the largest dengue virus outbreak in Xishuangbanna Dai autonomous prefecture in 2019.

In 2019, a serious dengue virus (DENV) infection broke out in the Xishuangbanna Dai Autonomous Prefecture, China. Therefore, we conducted a molecular epidemiological analysis in people that contracted DENV serotype 1 (DENV-1) during this year. We analyzed the molecular epidemiology of six DENV-1 epidemic strains in 2019 by full-length genome sequencing, amino acid mutation site analysis, evolutionary tree analysis, and recombination site comparison analysis. Through the analysis of amino acid mutation sites, it was found that DENV-1 strain (MW386867) was different from the other five epidemic DENV-1 strains in Xishuangbanna in 2019. MW386867 had unique mutation sites at six loci. The six epidemic DENV-1 strains in Xishuangbanna in 2019 were divided into two clusters. MW386867 was highly similar to the MG679800 (Myanmar 2017), MG679801 (Myanmar 2017), and KC172834 (Laos 2008), and the other five strains were highly similar to JQ045660 (Vietnam 2011), FJ176780 (GuangDong 2006). Genetic recombination analysis revealed that there was no recombination signal in the six epidemic DENV-1 strains in Xishuangbanna in 2019. We speculate that the DENV-1 epidemic in 2019 has a co-epidemic of local strains and cross-border strains.

Molecular epidemiology and clinical characteristics of respiratory syncytial virus in hospitalized children during winter 2021-2022 in Bengbu, China.

Objective: This study aimed to study the molecular epidemiology and clinical characteristics of respiratory syncytial virus (RSV) infection from hospitalized children with ARTI in Bengbu. Methods: One hundred twenty-four nasopharyngeal swab specimens and clinical data from children with ARTI cases were collected in Bengbu, China, during winter 2021-2022. The samples were detected by qPCR of 13 respiratory viruses. Phylogenetic analysis was constructed using MEGA 7.0. All analyses were performed using SAS software, version 9.4. Results: In winter 2021-2022, URTI, NSCAP, SCAP, and bronchiolitis accounted for 41.03%, 27.35%, 17.09%, and 14.53% of hospitalized children in Bengbu, China. The detection rates of the top three were RSV (41.94%), ADV (5.65%), and FluB (5.65%) in hospitalized children through 13 virus detection. RSV is the main pathogen of hospitalized children under 2 years old. Forty-eight sequences of G protein of RSV were obtained through PCR amplification, including RSV-A 37 strains and RSV-B 11 strains. Phylogenetic analysis showed that all RSV-A and RSV-B were ON1 and BA9 genotypes, respectively. ON1 genotypes were further divided into two clades. The majority of ON1 strains formed a unique genetic clade with T113I, V131D, N178 G, and H258Q mutations. Furthermore, RSV infection was an independent risk factor for ventilator use (OR = 9.55, 95% CI 1.87-48.64). Conclusion: There was a high incidence of RSV among hospitalized children during winter 2021-2022 in Bengbu with ON1 and BA9 being the dominant strains. This study demonstrated the molecular epidemiological characteristics of RSV in children with respiratory infections in Bengbu, China.

Two-Period Study Results from a Large Italian Hospital Laboratory Attesting SARS-CoV-2 Variant PCR Assay Evolution.

In keeping with the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 causative agent, PCR assays have been developed to rapidly detect SARS-CoV-2 variants, which have emerged since the first (Alpha) variant was identified. Based on specific assortment of SARS-CoV-2 spike-protein mutations (ΔH69/V70, E484K, N501Y, W152C, L452R, K417N, and K417T) among the major variants known to date, Seegene Allplex SARS-CoV-2 Variants I and Variants II assays have been available since a few months before the last (Omicron) variant became predominant. Using S gene next-generation sequencing (NGS) as the SARS-CoV-2 variant identification reference method, we assessed the results of SARS-CoV-2-positive nasopharyngeal swab samples from two testing periods, before (n = 288, using only Variants I) and after (n = 77, using both Variants I and Variants II) the appearance of Omicron. The Variants I assay allowed correct identification for Alpha (37/37), Beta/Gamma (28/30), or Delta (220/221) variant-positive samples. The combination of the Variants I and Variants II assays allowed correct identification for 61/77 Omicron variant-positive samples. While 16 samples had the K417N mutation undetected with the Variants II assay, 74/77 samples had both ΔH69/V70 and N501Y mutations detected with the Variants I assay. If considering only the results by the Variants I assay, 6 (2 Beta variant positive, 1 Delta variant positive, and 3 Omicron variant positive) of 365 samples tested in total provided incorrect identification. We showed that the Variants I assay alone might be more suitable than both the Variants I and Variants II assays to identify currently circulating SARS-CoV-2 variants. Inclusion of additional variant-specific mutations should be expected in the development of future assays. IMPORTANCE Omicron variants of SARS-CoV-2 pose more important public health concerns than the previously circulating Alpha or Delta variants, particularly regarding the efficacy of anti-SARS-CoV-2 vaccines and therapeutics. Precise identification of these variants highly requires performant PCR-based assays that allow us to reduce the reliance on NGS-based assays, which remain the reference method in this topic. While the current epidemiological SARS-CoV-2 pandemic context suggests that PCR assays such as the Seegene Variants II may be dispensable, we took advantage of NGS data obtained in this study to show that the array of SARS-CoV-2 spike protein mutations in the Seegene Variants II assay may be suboptimal. This reinforces the concept that initially developed PCR assays for SARS-CoV-2 variant detection could be no longer helpful if the SARS-CoV-2 pandemic evolves to newly emerging variants.

The Changes in Canine Parvovirus Variants over the Years.

Canine parvovirus (CPV-2) is one of the most important pathogens in dogs, and despite the continual development of vaccines against CPV-2, CPV-2 is still circulating in the canine population. The CPV-2a/2b/2c variant has replaced the original CPV-2 virus and seems to exhibit accelerated transmission. Although CPV-2 infection has been frequently reported, no studies have summarized information of CPV-2 variants currently circulating worldwide. To track the evolution of CPV-2, we downloaded and analyzed all VP2 sequences from the NCBI database (from 1978 to 2022). We found that CPV-2c shows a tendency to replace CPV-2a as the new dominant variant in Asia, South America, North America and Africa. Additionally, CPV-2c, which is prevalent in most regions of Asia, carries two special mutations in VP2, A5G and Q370R, and has become a dominant mutation with spillover already occurring. In conclusion, this summary of the types of global epidemic variants provides new insight into the evolution of CPV-2 and raises awareness for blocking the spread of this virus. The spread of Asian-derived CPV-2c urgently needs to be further under surveillance.

A methodology for creating mutants of G-protein coupled receptors stabilized in active state by combining statistical thermodynamics and evolutionary molecular engineering.

We challenged the stabilization of a G-protein coupled receptor (GPCR) in the active state solely by multiple amino-acid mutations without the agonist binding. For many GPCRs, the free energy of the active state is higher than that of the inactive state. When the inactive state is stabilized through the lowering of its free energy, the apparent midpoint temperature of thermal denaturation Tm exhibits a significant increase. However, this is not always the case for the stabilization of the active state. We constructed a modified version of our methodology combining statistical thermodynamics and evolutionary molecular engineering, which was recently developed for the inactive state. First, several residues to be mutated are determined using our statistical-thermodynamics theory. Second, a gene (mutant) library is constructed using Escherichia coli cells to efficiently explore most of the mutational space. Third, for the mutant screening, the mutants prepared in accordance with the library are expressed in engineered Saccharomyces cerevisiae YB14 cells which can grow only when a GPCR mutant stabilized in the active state has signaling function. For the adenosine A2A receptor tested, the methodology enabled us to sort out two triple mutants and a double mutant. It was experimentally corroborated that all the mutants exhibit much higher binding affinity for G protein than the wild type. Analyses indicated that the mutations make the structural characteristics shift toward those of the active state. However, only slight increases in Tm resulted from the mutations, suggesting the unsuitability of Tm to the stability measure for the active state.

Molecular determinants for the chemical activation of the warmth-sensitive TRPV3 channel by the natural monoterpenoid carvacrol.

Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC50 of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology.

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage.

Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements.

Identification of a new amino acid mutation in the HN protein of NDV involved in pathogenicity.

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are viral entry proteins and are recognized as the major virulence determinants. Previously, a lentogenic NDV virus (CE16) was derived from a mesogenic strain (CI10) through sequential passages in chick embryos. Whole-genome sequence analysis revealed that the two homologous strains shared the same F protein but differed in HN with two amino acid (aa) substitutions (A215G and T430A). To elucidate the molecular reasons for virulence attenuation, two original plasmids (HN-CI10 and HN-CE16) and two single-point mutants (G215A and A430T) reverse-mutated from HN-CE16 were constructed to analyse the known biological functions of HN. The results showed that the A430T substitution significantly weakened the haemadsorption (HAd) activity, increased the neuraminidase (NA) activity, improved the fusion-promoting activity, and enhanced the cleavage-promoting activity of HN-CE16. However, G215A failed to induce obvious functional changes. Therefore, the aa residue HN430 may play a key role in determining virulence. To test this hypothesis, further studies on A430T were conducted through reverse genetics using an infectious cDNA clone. At the viral level, the A430T-mutated virus showed dramatic promotion of viral plaque formation, propagation, and pathogenicity in vitro and in vivo. This study demonstrates a new virulence site associated with HN protein functions, viral propagation, and pathogenicity. All these findings could lay a foundation for illuminating the molecular mechanism of NDV virulence.

Molecular Characterization of Dengue Virus Type 1 in Zhejiang in 2019.

Dengue fever (DF) is a mosquito-borne viral disease caused by the dengue virus (DENV), which is considered one of the most important arboviruses in the world. This study aimed to determine the molecular, epidemiological, and phylogenetic characterization of 174 DENV-1 (132 indigenous cases and 42 imported cases) isolated from nine municipalities of Zhejiang province in 2019. The analyses of phylogenetics, haplotypes, and amino acid substitutions were conducted based on the full envelope (E) gene sequences. Sixty-four haplotypes were clustered into two main clades, with isolates from Wenzhou and Taizhou mainly clustered into clade I and Hangzhou and Ningbo cases clustered into clade II. Six sites of amino acid substitutions including A88T, F96L, M297V, T339S, I378L, and V436I were only observed in strains isolated from Ningbo and Hangzhou, while two sites of amino acid substitutions including V312L and V380I were observed in strains from Taizhou and Wenzhou. In our study, strains were in high homology with the strains from Southeast Asian countries, thus cases in Zhejiang were probably imported from Southeast Asian countries. The strains from different regions in Zhejiang were clustered in the same branch which may be caused by the continuous import of cases in the same country at different time periods. After the continuous outbreak in Zhejiang province, some sites of the dengue gene have mutated, and the effects need further study.

A single amino acid mutation in the mouse MEIG1 protein disrupts a cargo transport system necessary for sperm formation.

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.

Comparative genomic analysis of different sexes and diet-specific amino acid mutation identification in Ancherythroculter nigrocauda.

Determining the sex and controlling the sex ratio are essential aspects of fish genetics that can assist in developing successful fish breeding programs. High quality genome assembly and annotations are prerequisites to determine sex-specific genes and their expression. In addition, analysis of resequencing data can identify genomic difference between male and female fishes. In this study, we performed chromosome-level genome assembly in female Ancherythroculter nigrocauda fish having low heterozygosity using PacBio reads. High-throughput chromatin conformation capture (HiC) yielded a genome of size 1054.05 Mb, with a contig N50 length of 3.40 Mb and a scaffold N50 length of 42.68 Mb. In addition, we sequenced 5 female and 5 male A. nigrocauda samples and identified sex-specific regions on LG20 Furthermore, diet-specific amino acid mutation were found on 582 genes between herbivorous and carnivorous fishes, with 26 of them exhibiting significantly different expression patterns in the liver tissue of these two types of fishes. The chromosome-level genome assembly of A. nigrocauda provides valuable resources for conducting in-depth comparative genomic studies with immense applications in fish genetic breeding and farming. Similarly, the diet-specific amino acid mutations are useful in the breeding of new strains of carnivorous fishes with an herbivorous diet.

A Heat-Induced Mutation on VP1 of Foot-and-Mouth Disease Virus Serotype O Enhanced Capsid Stability and Immunogenicity.

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals that causes a significant economic burden globally. Vaccination is the most effective FMD control strategy. However, FMD virus (FMDV) particles are prone to dissociate when appropriate physical or chemical conditions are unavailable, such as an incomplete cold chain. Such degraded vaccines result in compromised herd vaccination. Therefore, thermostable FMD particles are needed for use in vaccines. This study generated thermostable FMDV mutants (M3 and M10) by serial passages at high temperature, subsequent amplification, and purification. Both mutants contained an alanine-to-threonine mutation at position 13 in VP1 (A1013T), although M3 contained 3 additional mutations. The selected mutants showed improved stability and immunogenicity in neutralizing antibody titers, compared with the wild-type (wt) virus. The sequencing analysis and cryo-electron microscopy showed that the mutation of alanine to threonine at the 13th amino acid in the VP1 protein (A1013T) is critical for the capsid stability of FMDV. Virus-like particles containing A1013T (VLPA1013T) also showed significantly improved stability to heat treatment. This study demonstrated that Thr at the 13th amino acid of VP1 could stabilize the capsid of FMDV. Our findings will facilitate the development of a stable vaccine against FMDV serotype O. IMPORTANCE Foot-and-mouth disease (FMD) serotype O is one of the global epidemic serotypes and causes significant economic loss. Vaccination plays a key role in the prevention and control of FMD. However, the success of vaccination mainly depends on the quality of the vaccine. Here, the thermostable FMD virus (FMDV) mutants (M3 and M10) were selected through thermal screening at high temperatures with improved stability and immunogenicity compared with the wild-type virus. The results of multisequence alignment and cryo-electron microscopy (cryo-EM) analysis showed that the Thr substitution at the 13th amino acid in the VP1 protein is critical for the capsid stability of FMDV. For thermolabile type O FMDV, this major discovery will aid the development of its thermostable vaccine.

Genetic Characterisation and Local Genotypes of Canine Parvovirus Strains Collected from Pet Dogs in Central and Eastern China During 2018-2019.

INTRODUCTION: Canine parvovirus type-2 (CPV-2) causes acute infectious diseases in puppies, which show high morbidity and mortality. Better effect of vaccination against these diseases could be achieved with deeper knowledge of CPV-2 genotype dissemination and mutation history. This study investigated CPV-2-positive samples collected recently over a wide region of China. MATERIAL AND METHODS: A total of 118 faecal samples from dogs identified as CPV-positive were collected from veterinary clinics in central and eastern China. Overall, 16 strains collected from Anhui, 29 from Henan, and 16 from Zhejiang Province were sequenced to determine the genotypic composition of CPV-2 and mutational complexity of CPV-VP2. RESULTS: The CPV-2a, CPV-2b, and CPV-2c genotypes were detected in Anhui and Henan Provinces, while CPV-2c alone was detected in Zhejiang Province. Sequence analysis of all strains showed 98.5%-99.8%, 98.3%-99.9%, and 98.7%-99.8% identity among the 16 Anhui, 29 Henan, and 16 Zhejiang strains, respectively. Strains collected from Anhui and Henan Provinces showed lower identity (97.0%), suggesting greater genetic divergence in central China. The mutation rates of Henan and Anhui strains were lower than that of Zhejiang strains. Major amino acid mutations occurred at sites 5, 370, 426, and 440. Epitope and entropy analyses implied these sites' likely conformance to the principles of mutation tendency, complexity, and diversity. CONCLUSION: The findings for the evolutionary structure of CPV-2 strains collected from three provinces in central and eastern China advance trend monitoring of the genetic variation in canine parvovirus and point to its implications in the development of novel vaccines.