新发微生物与感染杂志(Emerging Microbio and Infection)在国际上首次报道中国研究团队关于白蛉(Sandfly)微生物组(microbiome)的研究结果，题目为"感染组学研究揭示被忽视的吸血媒介白蛉所携带对人类具有致病性的病毒、细菌和真核微生物"。研究结果显示，我国山西省中部和南部地区自然界采集的白蛉标本中微生物组RNA占标本中总的非核糖体RNA的1.8%，共含有87种病原体，其中70种是新物种。RNA病毒在病原体中占比最多，其次是细菌、DNA病毒和真核微生物。RNA病毒包括15个超群(viral super-groups)，共78种，其中66种是RNA病毒中的新病毒种。在RNA病毒序列信息中包含大量已经在当地白蛉中分离到的白蛉病毒属(Genus Phlebovirus)的Wuxiang病毒(Wuxiang virus, WUXV)和Hedi病毒(Hedi virus, HEDV )两种新病毒的基因组信息。分析中发现2种DNA病毒(Adintovirida和细小病毒科病毒)。细菌基因组信息包括不动杆菌(Acinetobacter)、假单胞菌(Pseudomonas)、立克次体(Rickettsia)和沃尔巴克氏菌(Wolbachia)。在10批白蛉标本中至少5批标本存在利什曼原虫。此外还发现一种锥虫科(Trypanosomatidae)新成员。病毒基因组分子遗传进化分析结果显示，RNA病毒中存在黄病毒属(Flavivirus)病毒的新成员，该病毒与蚊虫相关病毒，如广平病毒(Quang binh virus)、细胞融合病毒(Cell fusing agent virus)和伊蚊黄病毒(Aedes flavivirus)等相聚类。综上结果提示自然界白蛉中不仅携带多种可以在组织培养细胞复制的病毒分离物，还携带病毒以外的多种细菌、立克次体和利什曼原虫等。这一研究首次报道自然界白蛉的微生物感染组学结果，对于了解白蛉携带和传播病原微生物及其所带来的公共卫生危害有了较深入的认识，同时这种应用还可以扩展到其他媒介物种，从而将更有效地发现和表征媒介中的潜在病原体。
In 2008, Japanese encephalitis (JE) vaccine was included as Expanded Program on Immunization (EPI) for children(under 15)in China, and the number of JE cases of children was greatly reduced. However, since 2006, there have been outbreaks of adult JE in northern China, which has formed a huge public health burden on the local area and caused wide attention from the society. In addition, a number of adult JE have appeared in Japan, South Korea, Malaysia and other countries, and adult JE has become a public health concern at abroad. This paper introduces the current situation of adult JE both China and abroad, and discusses the relevant issues of the prevention and control of adult JE.
Objective:To explore the relationship between severe fever with thrombocytopenia syndrome virus (SFTSV) Gc and its N-glycosylation site and viral infectivity, a recombinant pseudovirus containing SFTSV Gc glycosylation site mutant was constructed.Methods:The eukaryotic expression vectors pcDNA3.1(+ )-GC, PCDNA3.1(+ )-GC(N291Q), PCDNA3.1(+ )-GC(N352Q) and PCDNA3.1(+ )-GC (N374Q) were constructed by site-directed mutagenesis and homologous recombination. After their successful expression in 293T cells, we infected VSVΔG-Fluc*G pseudovirus, constructed four recombinant pseudoviruses and tested their effects on the cell force of infection.Results:Double digestion identification and sequence determination confirmed the successful construction of eukaryotic expression vectors pcDNA3.1 (+ )-Gc, pcDNA3.1 (+ )-Gc(N291Q), pcDNA3.1 (+ )-Gc(N352Q) and pcDNA3.1 (+ )-Gc(N374Q). Indirect immunofluorescence and Western Blotting result indicated the successful expression of all the four recombinant plasmids. SFTSV Gc recombinant pseudoviruses are specific for infecting Vero cells. Pseudovirus infection capacity was decreased significantly after the glycosylation site mutation, and the mutant strain with the glycosylation site at position 352 had the lowest level of infectivity ( P<0.001, P=0.001). Conclusions:The glycosylation site of SFTSV Gc may be associated with the infectious effect of the viral infection, and the amino acid mutation at position 352 has the greatest effect on the viral infectivity.
Objective:A comparison of method for the detection of hepatitis E virus (HEV) in oysters was performed to provide a technical reference for the detection of HEV in oysters.Methods:After pre-treatment of oyster digestive gland specimens artificially contaminated with HEV fecal suspensions by the proteinase K digestion with reference to the European Union ISO/TS 15216-2∶2019, HEV RNA was extracted by four nucleic acid extraction method and assayed by Real time RT-PCR to compare the HEV recoveries; artificially contaminated oyster digestive gland specimens were pretreated by proteinase K digestion, proteinase K digestion + PEG precipitation, and proteinase K digestion + PEG precipitation + chloroform extraction, respectively, and HEV RNA was extracted by the optimal nucleic acid extraction method, which was assayed by real time RT-PCR to compare the HEV recoveries and inhibition rates of the three pretreatment method. The optimal HEV assay was applied to commercially available oyster specimens.Results:The HEV recoveries of the four nucleic acid extraction methods were 1.37%, 2.50%, 4.24% and 7.56%, respectively, with statistically significant differences ( F=847.220, P<0.001); The HEV recoveries for each of the three pre-treatment method were 6.02%, 13.65% and 21.17%, respectively, with statistically significant differences ( F=16.800, P<0.001), and the proteinase K digestion + PEG precipitation + chloroform extraction method had the highest recovery; the inhibition rates of the three method were 13.38%, 20.98% and 8.66%, respectively, and the differences were statistically significant ( F=20.205, P<0.001), with the lowest inhibition rate for the proteinase K digestion + PEG precipitation + chloroform extraction method. One HEV RNA positive specimen was detected in 120 commercially available oyster specimens. Conclusions:In the HEV detection of oyster specimens, pre-treatment with proteinase K digestion + PEG precipitation + chloroform extraction can improve the recovery of HEV from oysters and is more suitable for pre-treatment of oyster specimens; different manufacturers′ viral nucleic acid extraction method have different HEV recoveries and should be compared and screened for superiority before carrying out the assay.
Objective:To establish a real-time, sensitive and specific SIV/SHIV RNA load detection method and compare the repeatability, stability and sensitivity of this method with in vitro transcriptions and chemical synthesis of RNA as reference materials.Methods:SIV/SHIV Gag 91 nucleotide (nt) RNA was synthesized by chemical synthesis, DNA sequence with T7 promoter was obtained by reverse transcription of SHIV SF162 P3 RNA with T7 promoter specific primer, and Gag 385 nt RNA was synthesized by in vitro transcription. Two kinds of RNA were respectively diluted by 10-fold serial dilution, which was investigated by quantitative Real-time reverse transcription PCR (qRT-PCR). Standard curve and amplification curve were determined by qRT-PCR, and the sensitivity and repeatability of the two references were compared by repeated measurements in 10 independent experiments. Amplification curves and Ct values of the two RNA references were detected at 4 ℃ and 25 ℃ for 3 h, 6 h and 9 h, respectively, to compare the stability of the two RNA references. Results:In vitro transcribed RNA group, the amplification curves of multiple wells with the same concentration were highly coincident, and Ct values of each concentration had a good linear relationship. The standard curve equation was Y=-3.709 x+ 49.143, R2=0.999. At the same time, the sensitivity of in vitro transcriptions of RNA was 5.5×10 3copies/ml, and the mean coefficient of variation of 10 repeated measurements was only 2.24%. However, the standard curve for the chemosynthetic RNA group was Y=-4.014 x+ 53.108, R2=0.944, the detection sensitivity was 5.5×10 4 copies/ml, and the mean coefficient of variation for 10 repeated measurements was 5.33%. The data indicated that the method using in vitro transcribed RNA as reference was more stable, repeatable and sensitive. Conclusions:The sensitivity, specificity, repeatability and stability of RNA reference prepared by in vitro transcription method were superior to those of the chemosynthetic RNA reference. The one-step TaqMan probe qRT-PCR method established by this standard reference could be used to analyze SIV/SHIV RNA virus load.
In August 2023, the National Health Science and Education Commission of the National Health Commission issued Notice No.24[2023], officially releasing the " Catalogue of Pathogenic Microorganisms Transmitted to Human Beings". This is to better implement the relevant regulations of the Biosafety Law of the People′s Republic of China and the Biosafety Management Regulations for Pathogenic Microbial Laboratories. With the continuous emergence of new pathogenic microorganisms, updated understanding of existing pathogenic microorganisms, and deepening laboratory biosafety research, the original " List of Pathogenic Microorganisms Transmitted from Human Beings" cannot meet the needs, so the " Catalogue of Pathogenic Microorganisms Transmitted from Human Beings" has been issued. This article aims to provide a detailed interpretation of the virus section of the Catalogue, including its background, principles, processes, and main content, in order to facilitate a better understanding of the Catalogue.
Objective:To characterize the epidemic of influenza in Beijing from 2022 to 2023 and the variation of gene and antigenicity of hemagglutinin (HA) of influenza A H3N2 virus, so as to provide scientific basis for influenza prevention and control in Beijing.Methods:Statistical analysis was carried out on the result of influenza pathogenic monitoring in Beijing from week 14, 2022 to week 20, 2023, and 79 strains of influenza A H3N2 virus were selected at different time and population sources, and their genetic variation and evolution characteristics were analyzed through HA gene amplification sequencing and antigenicity analysis.Results:From week 14, 2022 to week 20, 2023, 24 244 throat swabs of influenza like cases were collected in Beijing, and 4 987 influenza virus nucleic acid positive cases were detected, including 2 749 influenza A H3N2 positive cases, with a detection rate of 11.34%. Among the 79 strains, 50 strains (63.29%) showed low response, 94.44% of the strains from August to November 2022 had low response, and 54.10% of the strains from February to March 2023 had low response, with a statistically significant difference ( χ2=8.079, P=0.004). Compared with the vaccine strain A/Darwin/9/2021, the HA gene sequence of 79 strains of influenza A H3N2 showed nucleotide similarity of 97.47% to 98.47% and amino acid similarity of 97.05% to 98.17%. Genetic evolution analysis showed that the 18 strains isolated from August to November 2022 were all distributed in the 3C.2a1b.2a.1a.1 branch, while the 61 strains isolated from February to March 2023 all belonged to the 3C.2a1b.2a.3a.1 branch. Compared with the vaccine strain, there were multiple site mutations distributed at multiple antigenic determinants and receptor binding sites in A, B, C, D, and E. All strains had potential glycosylation sites of 8NST, 22NGT, 38NAT, 45NSS, 63NCT, 126NWT, 133NGT, 246NST, 285NGS, 483NET, while one strain missed 165NVT glycosylation sites; 55 strains between February and March 2023 missed 122NES glycosylation sites. Conclusions:The HA gene locus of influenza A H3N2 virus detected in Beijing from week 14, 2022 to week 20, 2023 showed multiple mutations, continuous monitoring of this subtype variation is crucial.
Objective:To study the epidemiological characteristics and pathogen status of infection of patients with herpetic pharyngitis in eastern region of Chengdu in 2022, in order to provide experimental basis for scientific prevention and control of herpetic pharyngitis.Methods:Information and fecal specimens were collected from patients diagnosed with herpetic angina at sentinel monitoring hospitals in eastern region of Chengdu from January to December 2022, their epidemiological characteristics were analyzed, real-time fluorescence quantitative PCR (RT-PCR) and semi nested RT-PCR (RT-snPCR) were used to detect and identify enterovirus subtypes.Results:Among the 236 cases of herpetic angina, the age of the affected children was mainly 0-9 years (99.15%), with children aged below 3 years accounting for 75.85% of the total cases, children in the 1-year-old group had the highest constituent ratio, accounting for 47.88% of the total cases; The virus detection rate in the 2-year-old group was higher than that in the 0-year-old group ( χ2=5.945, P=0.015). There were 142 male infected cases (60.17%) and 94 female cases (39.83%); there was a statistically significant difference in the virus detection rate between the male group (91.55%) and the female group (78.72%) ( χ2=7.938, P=0.005). In the seasonal distribution, the summer group had the highest constituent ratio (40.25%), while the winter group had the lowest constituent ratio (11.86%), The composition ratio of the summer group was significantly higher than that of the other groups, and the differences were statistically significant ( χ2=10.393, 19.199, 49.358, all P<0.001). The virus detection rate in the summer group was significantly higher than that in the spring group and the winter group ( χ2=16.951, 4.592, both P<0.05). The RT-PCR result showed that a total of 204 out of 236 children with herpetic angina were virus positive, with a detection rate of 86.44% (204/236), including coxsackievirus A (CV-A) 6 (22.55%), CV-A16 (5.88%), CV-A2 (2.94%), enterovirus type A71 (EV-A71) (2.94%), and other enteroviruses (65.69%). The RT-snPCR result showed that five viruses were identified from other enteroviruses, including CV-A4 (8.33%), CV-A5 (6.86%), CV-A10 (12.25%), CV-A12 (24.02%), and coxsackievirus B3 (CV-B3) (6.86%); another 15 positive samples of enterovirus were not successfully classified. We selected 9 strains of the virus in this study and 30 reference sequences from the NCBI database to. Conclusions:Herpetic angina in eastern region of Chengdu in 2022 is mainly found in male children under 3 years of age, and was concentrated between April and June. It has been confirmed that CV-A12 and CV-A6 were more common in intestinal viruses, and the 9 strains isolated in this study were homologous to multiple isolates.
Objective:This study contrasts the immune efficacy of the varicella zoster virus glycoprotein E (VZV gE)using Al/CpG combined adjuvants and AS01 adjuvant in BALB/c mice.Methods:BALB/c mice were immunized at 0 and 21 days respectively, and serum antibodies were detected using enzyme-linked immunosorbent assay. Detection of neutralizing antibodies in mouse serum using varicella zoster virus; enzyme-linked immunosorbent spot assay was used to detect cellular immune response.Results:Following two intramuscular immunizations, mice in the experimental groups (Shingrix, gE+ Al/CpG, and gE+ AS01) demonstrated elevated neutralizing antibody titers and an augmented count of lymphocytes releasing IFN-γ and IL-4. The gE+ Al/CpG group displayed the highest neutralizing antibody titer (1943), yet the AS01-adjuvanted groups (Shingrix and gE+ AS01) showed increased lymphocyte counts secreting IFN-γ and IL-4 compared to the Al/CpG group (gE+ Al/CpG). In comparison to the AS01 adjuvant, Al/CpG adjuvants triggered a humoral immune response favoring Th2 in mice. The proportions of CD4 + T and CD8 + T cells were not significantly different among the experimental groups. Conclusions:Al/CpG adjuvant combined with gE protein resulted in high neutralizing antibody titers, while the intensity of the induced cellular immune response was inferior to that of AS01 adjuvant.
Human papillomavirus (HPV) is the most common sexually transmitted virus, of which high-risk HPV mainly infects mucosal epithelial cells causing malignant transformation of the cells. HPV infection could ultimately develop to malignant tumors including cervical, head and neck, anal, penile, vulvar, and vaginal cancers. The burden of cervical cancer is highest among these cancers and high-risk HPV persistent infection is considered an etiologic factor in its development. Therefore, it is crucial to identify early infected cases and determine HPV infection status by HPV testing. HPV testing can also be used for prophylactic HPV vaccine efficacy assessment, triage of women with cytological abnormalities, prediction of post-treatment efficacy and identification of HPV-attributable malignant lesions. There is the scarcity of studies elucidating the existing HPV detection methodologies. Through providing a comprehensive landscape of the currently available HPV detection methodologies, including their principles, applications, and clinical use, the ultimate objective is to provide information for the clinical practice of HPV detection, so as to reduce the incidence and mortality rates of HPV-associated diseases.
Objective:To establish a rapid method for the detection of varicella-zoster virus (VZV) by recombinase-aid amplification (RAA).Methods:The whole genome sequences of VZV were downloaded from the global shared database for comparison and analysis. Specific primers and probe were designed for the four conserved genes respectively and the optimal combination was selected. The optimal reaction system was selected through the concentration gradient of primers and probes, and a fluorescence RAA detection method was established. The sensitivity of the method was evaluated with VZV positive plasmid standard and clinical samples with gradient dilution, the repeatability of the method was evaluated with the lowest detectable limit concentration of positive plasmid standard, and the specificity of other viral nucleic acid method was evaluated. At the same time, this method and quantitative real-time PCR (qPCR) were used to detect clinical samples and the result were compared.Results:The optimal combination of primer pair F2/R2 and probe P2 targeting open reading frame (ORF) 28 gene was selected. Considering the cost factor, the optimal primer concentration was set at 500 nmol/L and the optimal probe concentration was 280 nmol/L. The minimum detection limit was 10 1 copies/μL, and the minimum clinical positive samples with a Ct value of 36.027 could be detected, and the result of repeated experiments were consistent. The method has no cross-reaction with other viral nucleic acids. The detection rate of clinical positive samples was 93.33%, which was almost identical to that of qPCR. Conclusions:This method is simple to operate with high sensitivity, strong specificity, low requirements for experimental conditions, visual detection result, and can detect VZV nucleic acid in samples within 20 minutes, which is a rapid VZV detection method that can be considered for clinical use for detection.
近年来，RNA被广泛应用于药物和疫苗等的研发之中，RNA在应用研究中遇到的困难可以尝试在合成RNA的过程中加入经过修饰的核苷酸来克服。基于酶合成的方法主要包括体内转录和体外转录，体内转录可以使用工程支架来促进RNA产物环状化，提高RNA的稳定性，相对经济也是其优点之一，而广泛的下游加工则是该方法的弊端；体外转录是目前RNA合成的最常用的方法，需要设计携带特异性噬菌体启动子片段(S6、T3、T7)的DNA模板，以便RNA聚合酶结合和提供转录启始信号，获得的寡核苷酸长度范围大，生产成本相对较低，可重复性强，但由于3′端存在异质性，可能导致产物末端不均匀。该方法中，仅T7 RNA聚合酶可以接受修饰的核苷酸。获得含有核苷酸类似物的短RNA分子的最佳方法是化学合成，常用的是磷酸酰胺固相合成法，主要步骤为脱三苯甲基(使用三氯或二氯乙酸溶液去除DMT基团)、缩合反应(游离的5′-羟基与活化的核苷3′-磷酸酰胺反应形成新的核苷酸间键)、加帽(使用乙酸酐/鲁替丁/N-甲基咪唑混合物将游离的5′-OH转化为乙酰酯)、氧化(不稳定的亚磷酸盐-P(III)氧化为P(V)磷酸)。化学合成可获得任何短RNA(<20 nt)的准确片段，便于引入修饰的核苷酸，但对于合成长RNA(>100 nt)效率低下，设备非常昂贵。核苷酸修饰可以调节RNA的功能，包括调控基因表达，参与一些病理过程或影响RNA的结构。获得含有修饰核苷酸的RNA分子最方便的方法是化学合成，优点是可以在RNA片段的每个位置加入修饰的核苷酸，将较多种类的修饰结合到两条RNA链中。每一种修饰都可以调节RNA的性质，这严格依赖于核苷酸单位内修饰的位置，包括核碱基、磷酸主链、核糖环等。
Rotavirus isolation and culture in vitro is the most basic and important technology and means in pathogen detection and research. Currently, rotavirus isolation and culture have achieved great success and has been widely used. In this review, the related studies on rotavirus isolation and culture were organized to provide a basis for further research on rotavirus disease identification, antiviral drug and vaccine development.
Objective:To compare the differences between suspension and adherent cells of MDCK cell line in the isolation of influenza virus, and to explore the application prospects of MDCK cell suspension.Methods:Determination of viable cell density and cell specific growth rate were recorded by cell count. The WHO recommended vaccine strains were used for virus infection experiments. After five passages, hemagglutination titers were detected, while the sequencing analysis of their HA and NA genes revealed the mutation frequency.Results:The 24-hour and 48-hour viable cell density of the cell suspension was more stable than that of adherent cells. The cell suspension achieved an HA titer of 1∶256 or higher in the third generation, while adherent cells had no titer. In the fourth and fifth generations, one amino acid site mutation was found in the HA gene of H3N2 and BV subtypes of influenza virus cultured in the cell suspension, while no gene mutation was found in adherent cells in two passages. There were no mutations in the whole NA gene.Conclusions:Suspension of MDCK cells have more stable growth and higher efficiency in virus isolation than adherent cells, meanwhile there was a low rate of virus mutation during continuous passage. This study demonstrated the feasibility of this suspension of MDCK cells for influenza vaccine production based on cell culture technology.
Objective:The result of influenza etiology surveillance in Chuxiong prefecture were analyzed to master the epidemic characteristics of influenza virus and provide scientific basis for influenza prevention and control.Methods:Real-time PCR assay and MDCK cell culture method were used to detect and separate influenza virus nucleic acids from influenza-like cases (ILI) and suspected influenza cases from 2017 to 2022 come from the sentinel hospitals.Results:From 2017 to 2022, a total of 7 302 cases of ILI and 1 079 cases were detected positive, with a positive rate of 14.78%. The positive rate in the six years was 14.71%、15.44%、20.11%、5.04%、10.90% and 21.08%, and the positive rate in 2020 was the lowest. Among them, the positive rate of influenza A was higher than that of influenza B, and higher in spring and winter than in summer and autumn., One H9N2 human infected with avian influenza virus was detected positive.The outbreak cases were mainly influenza B, followed by influenza A H3N2, and students were susceptible to it. The proportion of influenza-like cases in the 0~5 year old group was the highest (64.16%), and the protortion in 60~year old group was the lowest (0.05%).Conclusions:From 2017 to 2022, influenza A was predominant in Chuxiong prefecture, with high prevalence in winter and spring. The influenza strains presented diversity and each strain circulated alternately. The implementation of COVID-19 prevention and control measures can effectively reduce the infection rate of influenza virus .In addition, there are human cases of avian influenza infection in Chuxiong prefecture. So the surveillance of influenza-like cases should be strengthened an improved, and to expand the monitoring of human and avian influenza.
Objective:To explore the effect of significant variation in non-structural protein 1 (NSP1) of 2019 novel coronavirus (2019-nCoV) on binding to 5′UTR, and to provide clues for the development of antiviral drugs and vaccines.Methods:The bioinformatics analysis of 2019-nCoV genome database was conducted to select the amino acid variation sites (T12A, R124L, N128I, K141A, GHVMV82-86DEL and KSF141-143DEL) that may affect the structure of NSP1 and the ability binding to 5′UTR. PSIPRED online tool was used to analyze the secondary structure of the variants, mCSM-NA was used to predict their binding ability to RNA, and DynaMut webserver was used to analyze the influence of variants on protein stability. The variant plasmids were constructed and transfected into HEK-293T cells. The dual luciferase reporter gene assay and RNA binding protein immunoprecipitation (RIP) assay were used to detect the binding ability of the NSP1 variant for viral 5′UTR.Results:Bioinformatics analysis predicted that except for R124L mutation, the other five variants could change the secondary structure of protein, and the mutations of T12A, R124L, N128I and K141A could reduce the binding ability of RNA, while the mutations of T12A, R124L and N128I reduced the stability of protein. The experimental results showed that R124L, N128I, GHVMV82-86DEL and KSF141-143DEL significantly weakened the binding ability of NSP1 to 5′UTR.Conclusions:Some mutations or deletion of NSP1 amino acids could change the secondary structure and significantly weaken the binding ability of NSP1 to the 5′UTR, suggesting that the pathogenicity of the virus may be changed, which could provide a theoretical basis for the development of antiviral drugs and vaccines.
To promote the reasonable development of reference materials in the field of health care in China, on the basis of introducing reference materials and certified reference materials, we summarize the current management system and management norms of reference materials in our country, summarize the development status and trends of reference materials in the field of health care at home and abroad, analyze and discuss the development status, development trend and current challenges of standard materials in the fields of environmental health, medical sanitation and virus detection in China, and look forward to the major demand for reference materials in the field of health in China.
中国已从自然界采集的蚊虫标本中分离出多株正布尼亚病毒，包括Simbu血清群的Cat Que、曼扎尼拉和阿卡斑病毒，以及加利福尼亚血清群的Tahyna病毒。但自20世纪80年代以来，还未从蚊虫以外的吸血节肢动物中分离出正布尼亚属的病毒。在本研究中，JXLC1806-2病毒于2018年夏天从中国东部江西省黎川县采集的库蠓中分离，该病毒分离物在接种哺乳动物细胞(BHK-21)48小时内显示出显著的细胞病变作用，病毒滴度为1×10 5.6 pfu/mL。JXLC1806-2病毒不仅在哺乳动物细胞中引起CPE，而且在乳鼠中也可引起发病和死亡，但在C6/36细胞中不引起CPE，并且RT-PCR未检测到复制，提示该病毒为动物病毒。核苷酸和氨基酸序列分析表明，JXLC1806-2病毒基因组由S、M和L三个片段组成。系统发育分析表明，JXLC1806-2病毒的S、M和L基因属于正布尼亚病毒属Tete病毒组，但与Tete血清组的其他成员形成了独立的进化支。结果表明，JXLC1806-2病毒为Tete病毒组的一个新成员，命名为Lichuan病毒，这是中国首次分离到Tete病毒组病毒，也是首次从蚊虫标本之外的蠓虫标本中分离到正布尼亚病毒属病毒。由于该病毒是从养牛场的库蠓中分离，加强该病毒在当地牛群中的监测和检测，以及该病毒是否会在当地其他动物中引起感染和疾病的调查具有十分重要的公共卫生意义。
Objective:To express and purify VP7 protein of group A rotavirus (RVA) G1P[8]. The VP7 polyclonal antibody was prepared and its function was evaluated.Methods:The G1 VP7 protein was expressed by baculovirus expression system and purified by affinity chromatography. Polyclonal antibody against G1 VP7 was obtained by immunizing rabbits with G1 VP7 protein. The function of the G1 VP7 polyclonal antibody was verified by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay.Results:The soluble G1 VP7 protein of human RVA G1P[8] was obtained using the baculovirus expression system and the VP7 protein was mainly in trimer state. The G1 VP7 polyclonal antibody was prepared and displayed relatively high binding titer to G1 VP7 protein by ELISA. The VP7 polyclonal antibodies could recognize multiple G-type RVAs by WB and ELISA. Immunofluorescence assay further demonstrated that G1 VP7 polyclonal antibody can bind to different RVAs, including Wa (genotype G1P[8]), DS-1(genotype G2P[4]), SA11 (genotype G3P[2]), and human G9P[8] RV strains. In addition, double sandwich ELISA showed that VP7 polyclonal antibody could be used to detect rotavirus in clinical samples.Conclusions:The soluble G1 VP7 protein was successfully expressed and VP7 antibody was obtained. The G1 VP7 polyclonal antibody could bind to a variety of G-type rotaviruses, which lays a foundation for the establishment of detection method of different G type rotaviruses.
Objective:To study the inactivation effects of microwave on human adenovirus 2 (HAdV-2) in simulated infectied wastes, and to explore its molecular mechanism.Methods:25 μl of HAdV-2 virus suspension was dripped with medical disposable gloves, masks and cotton swabs to simulate infectied wastes, and irradiated under different microwave conditions: gloves and masks were irradiated for 30 s, 60 s, and 90 s at 300 W, 500 W, and 700 W, respectively. Cotton swabs irradiate 60 s, 90 s, 120 s at 500 W and 700 W respectively. Temperature changes were recorded, and the inactivation logarithmic values were calculated by the 50% endpoint method to evaluate the microwave inactivation effects, and the proliferation ability of the virus was detected by qPCR. The damage of Penton, Fiber, Hexon and E2 B genes was detected by PCR. The virus was treated with the highest temperature of 76 ℃ during microwave irradiation to study whether there was non-thermal effect during microwave disinfection. Results:After microwave irradiation of infectied waste, the temperature of masks and gloves carriers rises rapidly, with the highest temperature of 76 ℃. The temperature of the cotton swab carriers rose slowly, and the highest temperature is 65 ℃. The inactivation effect of microwave on HAdV-2 was positively correlated with microwave power and irradiation time. In the mask and glove group, microwave power of 700 W irradiated for 60 seconds, and the inactivation logarithm value could reach 3.0, In the cotton swab group, microwave power of 700 W irradiated for 120 seconds, and the inactivation logarithm value was still less than 3.0. This indicated that there were differences in the conditions for microwave inactivation of the virus in different carriers. The qPCR result showed that microwave irradiation could weak the proliferation ability of the virus. Microwave irradiation had no effect on the virus's Penton and Fiber genes, but caused some damage to the Hexon and E2 B genes. The inactivation effect of individual heat treatment on HAdV-2 was weaker than that of microwave irradiation, and there was no damage to the Hexon, Penton, Fiber, and E2 B genes. This indicated the presence of non thermal effects during the microwave inactivation process. Conclusions:Microwave irradiation can inactivate HAdV-2 in simulated infectied wastes through thermal and non-thermal effects, and its damage to viral DNA is one of the mechanisms of virus inactivation.
