Associations between genetic mutations in different SARS-CoV-2 strains and negative conversion time of viral RNA among imported cases in Hangzhou: A cross-sectional study.

PURPOSE: Previous studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have focused on factors that influence the achievement of negative conversion of viral RNA. This study aimed to investigate the effects of the genetic mutations in different SARS-CoV-2 strains on the negative conversion time (NCT) among imported cases in Hangzhou, Zhejiang Province, China, in order to provide valuable insights for developing targeted epidemic prevention guidelines. METHODS: This retrospective study involved 146 imported SARS-CoV-2 cases in Hangzhou from 8 April 2021 to 11 June 2022. We compared the SARS-CoV-2-specific indicators, clinical indexes, and NCT among the wild-type (WT), Delta, and Omicron groups. Spearman correlation analysis was used to identify the correlations of NCT with mutation types/frequencies. RESULTS: The mean age of the imported cases was 35.3 (SD: 12.3) years, with 71.92 % males and 28.08 % females. The mean cycle threshold (Ct) values of open reading frame 1ab (ORF1ab) and nucleocapsid (N) RNA were 25.17 (SD: 6.44) and 23.4 (SD: 6.76), respectively. The mutations of SARS-CoV-2 strains were mainly located in N, membrane (M), spike (S), ORF1a, ORF1b, ORF3a, ORF6, and ORF9b genes among the WT, Delta, and Omicron groups. NCT was significantly prolonged in the WT and Delta groups compared to the Omicron group. T lymphocyte, white blood cell, eosinophil, and basophil counts were dramatically higher in the WT group than the Delta group. White blood cell, red blood cell, and basophil counts were significantly lower in the Delta group than the Omicron group. Spearman correlation analysis revealed a significant correlation between the NCT of viral RNA and mutation types of viral genes of WT and Omicron strains. Additionally, NCT was markedly negatively correlated with the frequencies of five mutations in Omicron strains (ORF1b:P1223L, ORF1b:R1315C, ORF1b:T2163I, ORF3a:T223I, and ORF6:D61L). CONCLUSIONS: This study indicates that five mutations in Omicron strains (ORF1b:P1223L/R1315C/T2163I, ORF3a:T223I and ORF6:D61L) shortened NCT in imported SARS-CoV-2 cases.

Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism.

Regulatory Functions of PurR in Yersinia pestis: Orchestrating Diverse Biological Activities.

The bacterium Yersinia pestis has developed various strategies to sense and respond to the complex stresses encountered during its transmission and pathogenic processes. PurR is a common transcriptional regulator of purine biosynthesis among microorganisms, and it modulates the transcription level of the pur operon to suppress the production of hypoxanthine nucleotide (IMP). This study aims to understand the functions and regulatory mechanisms of purR in Y. pestis. Firstly, we constructed a purR knockout mutant of Y. pestis strain 201 and compared certain phenotypes of the null mutant (201-ΔpurR) and the wild-type strain (201-WT). The results show that deleting purR has no significant impact on the biofilm formation, growth rate, or viability of Y. pestis under different stress conditions (heat and cold shock, high salinity, and hyperosmotic pressure). Although the cytotoxicity of the purR knockout mutant on HeLa and 293 cells is reduced, the animal-challenging test found no difference of the virulence in mice between 201-ΔpurR and 201-WT. Furthermore, RNA-seq and EMSA analyses demonstrate that PurR binds to the promoter regions of at least 15 genes in Y. pestis strain 201, primarily involved in purine biosynthesis, along with others not previously observed in other bacteria. Additionally, RNA-seq results suggest the presence of 11 potential operons, including a newly identified co-transcriptional T6SS cluster. Thus, aside from its role as a regulator of purine biosynthesis, purR in Y. pestis may have additional regulatory functions.

Genomic characterization of dengue virus serotype 2 during dengue outbreak and endemics in Hangzhou, Zhejiang (2017-2019).

Introduction: Dengue fever (DF) is a mosquito-borne viral disease caused by the dengue virus (DENV). In recent years, Hangzhou has undergone a DF epidemic, particularly in 2017, with an outbreak of 1,128 patients. The study aimed to investigate the genetic diversity and molecular evolution among the DF clinical isolates during and after the outbreak to aid in mapping its spread. Methods: To understand the genetic diversity, 74 DENV-2 strains were isolated from DF epidemic cases between 2017 and 2019. Combining whole genome sequencing (WGS) technology, additional phylogenetic, haplotype, amino acid (AA) substitution, and recombination analyses were performed. Results: The results revealed that strains from 2017 were closely related to those from Singapore, Malaysia, and Thailand, indicating an imported international transmission. Local strains from 2018 were clustered with those recovered from 2019 and were closely associated with Guangzhou isolates, suggesting a within-country transmission after the significant outbreak in 2017. Compared to DENV-2 virus P14337 (Thailand/0168/1979), a total of 20 AA substitutions were detected. Notably, V431I, T2881I, and K3291T mutations only occurred in indigenous cases from 2017, and A1402T, V1457I, Q2777E, R3189K, and Q3310R mutations were exclusively found in imported cases from 2018 to 2019. The recombination analysis indicated that a total of 14 recombination events were observed. Conclusion: This study may improve our understanding of DENV transmission in Hangzhou and provide further insight into DENV-2 transmission and the local vaccine choice.

Molecular epidemiological characteristics of SARS-CoV-2 in imported cases from 2021 to 2022 in Zhejiang Province, China.

Backgrounds: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a global threat since 2020. The emergence of the Omicron variant in 2021, which replaced Delta as the dominant variant of concern, has had a significant adverse impact on the global economy and public health. During this period, Zhejiang Province implemented dynamic zeroing and focused on preventing imported cases. This study aimed to gain clear insight into the characteristics of imported COVID-19 cases in Zhejiang Province. Methods: We conducted a systematic molecular epidemiological analysis of 146 imported cases between July 2021 and November 2022 in Zhejiang Province. Virus samples with cycle threshold (Ct) value less than 32 were performed next generation sequencing. Basing the whole genome sequence obtained after quality control and assembly of reads, the whole genome variation map and phylogenetic tree were constructed and further analyzed. Results: Our study identified critical months and populations for surveillance, profiled the variation of various lineages, determined the evolutionary relationships among various lineages of SARS-CoV-2, and compared the results in Zhejiang with those obtained worldwide during this period. Conclusion: The continuous molecular epidemiological surveillance of imported cases of COVID-19 in Zhejiang Province during 2021 to 2022 is consistent with the global epidemic trend.

Importation of a novel Indian Ocean lineage carrying E1-K211E and E2-V264A of Chikungunya Virus in Zhejiang Province, China, in 2019.

The chikungunya virus (CHIKV) is widespread. In Zhejiang province, China, CHIKV infection is often associated with travelers from tropical and subtropical countries. In the present study, three CHIKV isolates from serum samples of travelers in Zhejiang province in 2019 were sequenced, and phylogenetically analyzed to study their molecular characteristics. Sequence analysis showed that the non-structural protein and the structural protein had 37 and 28 amino acid mutations, respectively; no mutation site was found at the E1-A226 residue, which could increase the adaptability of CHIKV to Aedes albopictus. All three samples carried two mutations, namely, E1-K211E and E2-V264A, which were introduced to Bangladesh around late 2015 and Thailand in early 2017. Phylogenetic analysis revealed that these three CHIKVs were Indian Ocean lineage of the East Africa/Central/South Africa genotype (ECSA) and that the MF773566 strain from Bangladesh (Australia/Bangladesh 2017) had the closest evolutionary relationship. The three CHICKs imported into Zhejiang province in 2019 belonged to the ECSA genotype and had multiple amino acid variation sites. The variation in the three samples provides a certain reference for the subsequent research on CHIKV evolution.

The role of long non-coding RNAs in breast cancer microenvironment.

The tumor microenvironment (TME), which includes tumor cells, fibroblasts, endothelial cells, immune cells, and blood vessels, can affect tumor growth and metastasis. Studies have shown that tumor cells, fibroblasts, and macrophages can promote the development of tumors, while T and B cells can inhibit tumor progression. The crosstalk among different cells within the TME needs further study. Long non-coding RNAs (lncRNAs) are involved in biological processes, including cell proliferation, migration, and differentiation. The abnormal expression of certain lncRNAs is correlated with the progression of breast cancer and has been proven as diagnostic markers in various cancers, including breast cancer. In breast cancer, recent studies have shown that tumor cell- and non-tumor cell-derived lncRNAs can affect various facets of tumor progression, including growth, proliferation, and migration of tumor cells. Interestingly, in addition to being regulated by lncRNAs derived from tumor and non-tumor cells, the TME can regulate the expression of lncRNAs in tumor cells, fibroblasts, and macrophages, influencing their phenotype and function. However, the detailed molecular mechanisms of these phenomena remain unclear in the breast cancer microenvironment. Currently, many studies have shown that TME-associated lncRNAs are potential diagnostic and therapeutic targets for breast cancer. Considering that TME and lncRNAs can regulate each other, we summarize the role of lncRNAs in the breast cancer microenvironment and the potential of lncRNAs as valuable diagnostic markers.

Case Report: A case of Chlamydia psittaci infection in an HIV patient.

Chlamydia psittaci is the pathogen of psittacosis and infects a wide range of birds and even humans. Human infection occurs most commonly in those with a history of contact with birds or poultry. We describe a case of psittacosis in a human immunodeficiency virus infected patient in Zhejiang Province for the first time. C. psittaci infection was confirmed by nested polymerase chain reaction (PCR) and Real-Time PCR. Phylogenetic analysis revealed that the sequences from the patient's samples clustered with genotype A in the same branch. Our study highlights the possibility of diagnosing psittacosis in patients with a chronic disease such as HIV-infected patients, and should increase awareness and surveillance of psittacosis in China.

An external quality assessment for the molecular testing of the SARS-CoV-2 virus genome in Zhejiang Province, China.

The COVID-19 pandemic has necessitated the rapid expansion of laboratories that conduct SARS-CoV-2 tests. A provincial external quality assessment (EQA) scheme on SARS-CoV-2 tests was organized by Zhejiang Provincial CDC to assess the accuracy of the tests in individual CDC municipal and county laboratories in Zhejiang Province, China. Three positive samples in high, medium, and low concentrations, respectively, were prepared using the serial dilutions from the culture with the viral titer concentration of 1×106.3 TCID50/mL, and one negative sample were included. A total of 93 laboratories participated, contributing results from 36 distinct combinations of nucleic acid extraction methods and PCR reagents. There was 100% concordance among all laboratories for all EQA samples, and no false-positive or false-negative results were observed. The EQA survey provides confidence in the identification of infected individuals or asymptomatic populations and assurance for clinical and public health decision-making based on test results.

A cluster of Psittacosis cases in Lishui, Zhejiang Province, China, in 2021.

Introduction: Psittacosis, caused by Chlamydia psittaci, is widespread throughout the world. In humans, C. psittaci infection may lead to severe conditions and complications, including sepsis and multiple organ failure. We report a cluster of cases caused by C. psittaci in Zhejiang Province, 2021, which led to one death and three cases of hospitalization. Methods: The cases were confirmed by nest-PCR, RT-PCR, and mNGS. Results: The four cases were related and the sequences obtained from the samples were closely correlated with those from Taiwan. Discussion: This study is the first to report on the case of death from psittacosis in Zhejiang Province, and our results help to assess the disease and recommend effective measures to prevent further spread of C. psittaci.

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.

Simultaneous Detection of Ebola Virus and Pathogens Associated With Hemorrhagic Fever by an Oligonucleotide Microarray.

Ebola virus infection causes severe hemorrhagic fever, and its mortality rates varied from 25 to 90% in the previous outbreaks. The highly infectious and lethal nature of this virus highlights the need for reliable and sensitive diagnostic methods to distinguish it from other diseases present with similar clinical symptoms. Based on multiplex polymerase chain reaction (PCR) and oligonucleotide microarray technology, a cost-effective, multipathogen and high-throughput method was developed for simultaneous detection of Ebola virus and other pathogens associated with hemorrhagic fever, including Marburg virus, Lassa fever virus, Junin virus, Machupo virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, malaria parasite, hantavirus, severe fever with thrombocytopenia syndrome virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A virus, and influenza B virus. This assay had an excellent specificity for target pathogens, without overlap signal between the probes. The limit of detection was approximately 103 pathogen copies/µl. A total of 60 positive nucleic acid samples for different pathogens were detected, a concordance of 100% was observed between microarray assay and real-time PCR analysis. Consequently, the described oligonucleotide microarray may be specific and sensitive assay for diagnosis and surveillance of infections caused by Ebola virus and other species of hemorrhagic fever pathogens.

Factors associated with spatial distribution of severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) was firstly identified in mainland China in 2009 and the geographic distribution has expanded in recent years. In this study, we constructed ecological niche models (ENM) of SFTS with meteorological factors, environmental factor, and density of domestic animals using MaxEnt. We found four significant associated factors including altitude, yearly average temperature, yearly accumulated precipitation, and yearly average relative humidity which accounted for 94.1% percent contribution. SFTS occurrence probability was high when altitude was between -100 m and 100 m, and the probability was nearly 0 when altitude was beyond 3000 m. Response curves of SFTS to the yearly average temperature, yearly accumulated precipitation, and yearly average relative humidity were all reversed V-shape. SFTS occurrence probability was high where the yearly average temperature, yearly accumulated precipitation, and yearly relative humidity were 12.5-17.5 °C, 700-2250 mm and 63-82%, respectively. ENMs predicted that the potential high-risk areas were mainly distributed in eastern areas and central areas of China. But there were some predicted potential high-risk areas where no SFTS case was reported up to date. More researches should be done to make clear whether SFTS case had occurred in these areas.

Molecular Characterization and Antibiotic Resistant Profiles of Campylobacter Species Isolated From Poultry and Diarrheal Patients in Southeastern China 2017-2019.

Campylobacter is a zoonotic pathogen that causes foodborne diarrheal illness globally. To better understand health risks in Southeastern China, Campylobacter spp. were surveyed in humans and representative poultry products over 3 years. One hundred and ninety-five representative isolates (n = 148, Campylobacter jejuni; n = 45, Campylobacter coli; n = 2 Campylobacter hyointestinalis) were examined for genetic relatedness and antimicrobial susceptibility. Nearly all Campylobacter isolates (99.0%, 193/195) were resistant to at least one class of antimicrobials, and 45.6% (89/195) of the isolates exhibited multidrug resistance. Genotypic analysis revealed high diversity among tested strains. Multilocus sequence typing (MLST) displayed 120 sequence types (STs) including 42 novel STs being added to the PubMLST international database. Sixty-two STs belonged to 16 previously characterized clonal complexes (CCs), of which CC-21, CC-45, CC-464, CC-574, CC-353, and CC-828 were most frequently identified. In addition, pulsed-field gel electrophoresis (PFGE) fingerprinting resulted in 66 PFGE SmaI patterns among the 125 isolates, with eight patterns shared between human and poultry sources. Subtyping data did not correlate with antimicrobial resistance phenotypes. Taken together, this large-scale surveillance study highlights high antimicrobial resistance and molecular features of Campylobacter isolates in Southeastern China.

Climate factors and the East Asian summer monsoon may drive large outbreaks of dengue in China.

OBJECTIVE: To investigate the relationship between climate variables, East Asian summer monsoon (EASM) and large outbreaks of dengue in China. METHODS: We constructed ecological niche models (ENMs) to analyse the influence of climate factors on dengue occurrence and predict dengue outbreak areas in China. Furthermore, we formulated a generalised additive model (GAM) to quantify the impact of the EASM on dengue occurrence in mainland China from 1980 to 2016. RESULTS: Mean Temperature of Coldest Quarter had a 62.6% contribution to dengue outbreaks. Southern China including Guangdong, Guangxi, Fujian and Yunnan provinces are more vulnerable to dengue emergence and resurgence. In addition, we found population density had a 68.7% contribution to dengue widely distribution in China using ENMs. Statistical analysis indicated a dome-shaped association between EASM and dengue outbreak using GAM, with the greatest impact in the South-East of China. Besides, there was a positive nonlinear association between monthly average temperature and dengue occurrence. CONCLUSION: We demonstrated the influence of climate factors and East Asian summer monsoon on dengue outbreaks, providing a framework for future studies on the association between climate change and vector-borne diseases.

Characterization of the vaginal microbiome during cytolytic vaginosis using high-throughput sequencing.

BACKGROUND: Cytolytic vaginosis (CV) is a common disease that results in pruritus, dyspareunia, and vulvar dysuria. However, the pathological mechanisms of the disease are still unclear. Compared to traditional methods, high-throughput sequencing can obtain more accurate qualitative and quantitative information on the microbiome. METHODS: We collected 75 samples from 32 healthy women (average age 44 ± 8) and 43 patients with CV (average age 38 ± 8). We used high-throughput sequencing of the 16S rRNA V3-V4 region to characterize and compare the vaginal microbiota of patients with CV and healthy women and to identify potential biomarkers for CV. RESULTS: The vaginal pH of patients with CV was ≤3.8, and the vaginal concentration of H2 O2 was ≥2 µmol/L. Colony densities of Lactobacillus spp. in patients with CV ranged from +++ (5-30) to ++++ (>30) and were significantly higher than those in healthy women. High-throughput sequencing showed that Lactobacillus was the most prominent genus both in patients with CV and in healthy women, with abundances of 83.8% and 97.2%, respectively (P < 0.001). Lactobacillus crispatus was more abundant in patients with CV, whereas Lactobacillus sp. L-YJ was more abundant in healthy women, with area under the curve (AUC) values of 0.9375 and 0.8379, respectively. CONCLUSION: The abundance of Lactobacillus spp. in CV patients was significantly different from that of healthy patients. Two suitable biomarkers, L. crispatus and Lactobacillus sp. L-YJ, were identified. These results will be useful for the identification of women at risk of serious illness before they develop obvious symptoms.

Epidemiological Characterization of the 2017 Dengue Outbreak in Zhejiang, China and Molecular Characterization of the Viruses.

Dengue, a mosquito-borne disease caused by the dengue virus (DV), has been recognized as a global public health threat. In 2017, an unexpected dengue outbreak occurred in Zhejiang, China. To clarify and characterize the causative agent of this outbreak, data on dengue fever cases were collected from the China Information System for Disease Control and Prevention in Zhejiang province for subsequent epidemiological analysis. A total of 1,229 cases were reported, including 1,149 indigenous and 80 imported cases. Most indigenous cases (1,128 cases) were in Hangzhou. The epidemic peak occurred in late August and early September, and the incidence rate of elderly people (4.34 per 100,000) was relatively high. Imported cases were reported all year round, and most were from South-East Asia and Western Pacific regions. Young people and men accounted for a large fraction of the cases. Acute phase serums of patients were collected for virus isolation. And 35 isolates (including 25 DV-2, 8 DV-1, 1 DV-3, and 1 DV-4) were obtained after inoculation and culture in mosquito C6/36 cells. The E genes of the 35 new DV isolates and the complete genome of a DV-2 isolate (Zhejiang/HZ33/2017), and the E gene of a DV-2 isolate from Ae. albopictus (Zhejiang/Aedes-1/2017) were determined. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. Phylogenetically, DVs of all four serotypes with multiple genotypes (mainly including 21 Cosmopolitan genotype DV-2, 4 Asian I genotype DV-2, 6 genotype I DV-1, and 2 genotype V DV-1) were present in the indigenous and imported cases in Zhejiang during the same period. Most of the isolates probably originated from South-East Asia and Western Pacific countries. The imported cases, high density of mosquito vector, and missed diagnosis might contribute to the 2017 outbreak in Zhejiang.

Complete genome sequence of the Vibrio vulnificus strain VV2014DJH, a human-pathogenic bacterium isolated from a death case in China.

BACKGROUND: Vibrio vulnificus, an opportunistic pathogen, is the causative agent of life-threatening septicemia and severe wound infections. However, the pathogenicity and virulence factors of V. vulnificus are not fully understood. Here we report the complete genome sequence of V. vulnificus VV2014DJH, which was isolated from a death case. RESULTS: The genome of the V. vulnificus VV2014DJH contains two circular chromosomes with a mean G+C content of 46.8%, but does not consists of any plasmids. The chromosome I and chromosome II consist of 3,303,590 and 1,770,972 bp, respectively. In addition, the genome consists of 4617 protein coding genes, 172 RNA genes and type I, II and III secretion systems were predicted. CONCLUSIONS: In this study, the genomic information of the V. vulnificus VV2014DJH has been described. The information would contribute to the increasing scope and depth of Vibrio genome database, and provide insights into the pathogenicity and virulence factors of V. vulnificus.

F199E substitution reduced toxicity of Clostridium perfringens epsilon toxin by depriving the receptor binding capability.

Epsilon toxin (ETX), a potent toxin, is produced by types B and D strains of Clostridium perfringens, which could cause severe diseases in humans and domestic animals. Mutant rETXF199E was previously demonstrated to be a good vaccine candidate. However, the mechanism concerned remains unknown. To clarify how F199E substitution reduced ETX toxicity, we performed a series of experiments. The results showed that the cell-binding and pore-forming ability of rETXF199E was almost abolished. We speculated that F199E substitution reduced toxicity by depriving the receptor binding capability of ETX, which contributed to the hypothesis that domain I of ETX is responsible for cell binding. In addition, our data suggested that ETX could cause Ca2+ release from intracellular Ca2+ stores, which may underlie an alternate pathway leading to cell death. Furthermore, ETX induced crenation of the MDCK cells was observed, with sags and crests first appearing on the surface of condensed MDCK cells, according to scanning electron microscopy. The data also demonstrated the safety and potentiality of rETXF199E as a vaccine candidate for humans. In summary, findings of this work potentially contribute to a better understanding of the pathogenic mechanism of ETX and the development of vaccine against diseases caused by ETX, using mutant proteins.

Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX(Y196E)-C confers strong protection in mice.

Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETX(Y196E)-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:10(5) in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 µg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 µg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 µg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 µg) of rETX challenge in mice. These data suggest that recombinant protein rETX(Y196E)-C is a potential vaccine candidate for future applied researches.