Background Coxsackievirus B3 (CVB3) is an important pathogen associated with severe diseases such as myocarditis, pancreatitis, hepatitis, and hand, foot, and mouth disease (HFMD), yet no licensed CVB3 vaccine is available. Methods In this study, four clinically isolated CVB3 strains were characterized to identify a suitable vaccine candidate. KM50-W07, belonging to the predominant genotype E circulating in mainland China and exhibiting robust replication in Vero cells (7.75 Log10TCID₅₀/mL), was selected. Results An experimental inactivated vaccine based on KM50-W07 induced high neutralizing antibody titers (9.00–10.58 Log₂) in BALB/c mice and demonstrated broad cross-neutralization against multiple CVB3 genotypes (7.00–9.58 Log₂). Maternal antibodies from immunized female mice provided complete protection in neonatal mice against lethal CVB3 challenge. Conclusion The CVB3 vaccine candidate strain KM50-W07, with high growth titer, good immunogenicity, and genetic stability, was successfully screened, providing an experimental basis for developing inactivated CVB3 vaccines for global and especially Chinese prevention and control efforts.

Background: Coxsackievirus B2 (CVB2) causes a range of diseases, including hand, foot, and mouth disease; myocarditis; acute flaccid paralysis; meningitis; and encephalitis. However, no specific antiviral drugs or vaccines are currently available for CVB2. Methods: We used plaque purification, virus titre determination, and serial passaging to screen and identify an inactivated CVB2 vaccine candidate strain, KM31-C05, which exhibited high viral titres and good genetic stability. Comprehensive biological characterization of this candidate strain was performed, including phylogenetic analysis, virulence assessment in BALB/c mice, one-step growth curve analysis, optimization of the multiplicity of infection, as well as determination of viral load, pathological evaluation, and immunohistochemical analysis in tissues of BALB/c suckling mice post-challenge. An experimental inactivated vaccine was prepared using KM31-C05 to evaluate its immunogenicity and protective efficacy. Results: The viral titres of KM31-C05 reached 108 CCID50/mL. After 20 serial passages, only three amino acid mutations were identified (VP3-G165V, VP1-N84K, and VP1-D129N). Although the two VP1 mutations were located in surface-exposed loops, the strain maintained high neutralizing titres across passages, indicating good genetic stability. However, whether these sites affect virulence and replication requires further investigation. Phylogenetic analysis revealed that this strain belonged to genotype C, which is consistent with the strains circulating in mainland China in recent years. The experimental inactivated vaccine prepared from KM31-C05 induced effective neutralizing antibodies (1:128-1:256) in BALB/c mice and provided complete protection to suckling mice against lethal challenge with this CVB2 strain in maternal antibody protection experiments. Conclusions: KM31-C05 demonstrates potential as a CVB2 vaccine candidate in China and provides a theoretical basis for the development of a CVB2 vaccine.

Coxsackievirus B4 (CVB4) is a major pathogen involved in hand, foot, and mouth disease as well as aseptic meningitis, and is associated with type 1 diabetes mellitus. Currently, no effective antiviral drugs or vaccines are available for CVB4 infections. Drawing on successful experience with inactivated vaccines for enterovirus 71 and poliovirus, we developed an inactivated CVB4 vaccine based on Vero cells. The highly pathogenic CVB4 virus strain 140/CHN/2019, isolated from Vero cells and classified as genotype D, reflecting the circulating strains in China. Through three cycles of plaque assays and successive passages, the clone strain KM140-G01 could adapt to Vero cells and grew to a titer exceeding 10⁷ CCID50/mL. The experimentally inactivated CVB4 vaccine, developed from KM140-G01, effectively elicited high levels of neutralizing antibodies in BALB/c mice, with titers ranging from Log2 7.2 to 11.0 and effectively neutralized various clinical isolates (> Log210). Maternal antibodies provided 100% protection against a lethal challenge with CVB4 isolates in lactating BALB/c mice. Therefore, the KM140-G01 strain in this study is a viable new candidate for the development of a coxsackievirus B4 vaccine. The CVB4-infected neonatal mouse model should substantially facilitate CVB4 vaccine evaluation.

Background:Spinocerebellar ataxia type 2(SCA2)is a neurodegenerative disease marked by significant clinical and genetic heterogeneity,primarily caused by expanded CAG mutations in the ATXN2 gene.The unstable expansion of CAG repeats disrupts the genetic stability of animal models,which is detrimental to disease research. Methods:In this study,we established a mouse model in which CAG repeats do not undergo microsatellite instability(MSI)across generations.A humanized ATXN2 cDNA with four CAA interruptions within 73 CAG expansions was inserted into the Rosa26 locus of C57BL/6J mice.A 23 CAG control mouse model was also generated to verify ATXN2 integration and expression. Results:In our model,the number of CAG repeats remained stable during transmis-sion,with no CAG repeat expansion observed in 64 parent-to-offspring transmissions.Compared with SCA2-Q23 mice,SCA2-Q73 mice exhibited progressive motor impair-ment,reduced Purkinje cell count and volume(indicative of cell atrophy),and muscle atrophy.These observations in the mice suggest that the behavioral and neuropatho-logical phenotypes may reflect the features of SCA2 patients.RNA-seq analysis of the gastrocnemius muscle in SCA2-Q73 mice showed significant changes in muscle differentiation and development gene expression at 56 weeks,with no significant dif-ferences at 16 weeks compared to SCA2-Q23 mice.The expression level of the Myf6 gene significantly changed in the muscles of aged mice. Conclusion:In summary,the establishment of this model not only provides a stable animal model for studying CAG transmission in SCA2 but also indicates that the lack of long-term neural stimulation leads to muscle atrophy.

Coxsackievirus A6 (CVA6) is a primary pathogen associated with hand, foot, and mouth disease (HFMD) and is typified by fever, rashes or herpetic lesions at distinct locations. Although HFMD patients exhibit mild symptoms, a subset of patients may develop severe complications, such as viral encephalitis, myocarditis, pneumonia, and neurological disorders. However, in addition to rodent models, such as the CVA6-infected mouse model, no definitive nonhuman primate animal model or related research or analysis tool is available, which makes the development of suitable nonhuman primate animal models particularly crucial. In this study, 3- to 4-month-old rhesus monkeys were infected via the respiratory or digestive tract, and the pathogenic, pathological, and immunological alterations following CVA6 infection were investigated. The results revealed that the infected rhesus monkeys exhibited symptoms similar to those of patients, including signs of HFMD, blood cell changes, viremia, viral excretion, and inflammatory reactions during the acute phase (1-11 days). Pathological observations revealed inflammatory reactions in the intestinal and lymph node tissues. Notably, the acute symptoms gradually waned in the recovery phase (12-120 days), and a high level of neutralizing antibodies was sustained. Intriguingly, no significant disparity was observed between the infections initiated via the respiratory or digestive tract in terms of clinical symptoms, hemogram results or virus shedding. Overall, this study yielded a comprehensive dataset regarding the physiological, pathological, and immunological outcomes of CVA6 infection in a primate host, enhancing our comprehension of the mechanism of CVA6 infection and providing essential data for related vaccine and drug development.

Coxsackievirus（CV） B belongs to the species Enterovirus B, genus Enterovirus of the family Picornaviridae. Enterovirus B（EV-B） includes 63 serotypes: CVB1–6; CVA9; echoviruses E1–7, 9,11–21, 24–27, and 29–33; EV-B69, EV-B 73–75, EV-B77–88, EV-B 93, EV-B 97–101, EV-B 106–107,

Distal myopathies are a group of rare heterogeneous diseases that are mostly caused by genetic factors. At least 20 genes have been associated with distal myopathies. We performed whole-exome sequencing to identify the genetic cause of disease in a family with distal myopathy. Following the American College of Medical Genetics and Genomics (ACMG) guidelines, we analyzed the sequencing results and screened suspicious mutations based on mutation frequency, functional impact, and disease inheritance pattern. The harmfulness of the mutations was predicted using bioinformatics methods, and the pathogenic mutations were determined. We identified a novel amino acid mutation (NP_005467.1:p.S663L) on the GNE gene that may cause familial distal myopathy. This mutation is the result of the simultaneous mutation of two adjacent nucleotides (c.1988C > T, c.1989C > A) in the codon. First, we measured the mRNA and protein expression of the GNE gene in the lymphoblastoid cell lines (LCLs) of the probands and their family members. Second, GNE vectors carrying the novel mutation, two other known pathogenic mutations, and the wild-type gene were constructed and transfected into HEK293T cells. The enzymatic activity of these GNE variants was investigated and showed that the p.S663L mutation significantly reduced the activity of the bifunctional GNE enzyme without altering the expression level of the GNE protein. Furthermore, the mutation may also alter the immunogenicity of the 3' end of the GNE protein, potentially affecting its oligomer formation. In this study, a novel GNE gene mutation that may cause distal myopathy was identified, expanding the spectrum of genetic mutations associated with this disease.

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection.

Coxsackievirus B3 (CVB3) is the pathogen causing hand, foot and mouth disease (HFMD), which manifests across a spectrum of clinical severity from mild to severe. However, CVB3-infected mouse models mainly demonstrate viral myocarditis and pancreatitis, failing to replicate human HFMD symptoms. Although several enteroviruses have been evaluated in Syrian hamsters and rhesus monkeys, there is no comprehensive data on CVB3. In this study, we have first tested the susceptibility of Syrian hamsters to CVB3 infection via different routes. The results showed that Syrian hamsters were successfully infected with CVB3 by intraperitoneal injection or nasal drip, leading to nasopharyngeal colonization, acute severe pathological injury, and typical HFMD symptoms. Notably, the nasal drip group exhibited a longer viral excretion cycle and more severe pathological damage. In the subsequent study, rhesus monkeys infected with CVB3 through nasal drips also presented signs of HFMD symptoms, viral excretion, serum antibody conversion, viral nucleic acids and antigens, and the specific organ damages, particularly in the heart. Surprisingly, there were no significant differences in myocardial enzyme levels, and the clinical symptoms resembled those often associated with common, mild infections. In summary, the study successfully developed severe Syrian hamsters and mild rhesus monkey models for CVB3-induced HFMD. These models could serve as a basis for understanding the disease pathogenesis, conducting pre-trial prevention and evaluation, and implementing post-exposure intervention.

Ventricular septal defect (VSD) is the most common congenital heart disease. Although a small number of genes associated with VSD have been found, the genetic factors of VSD remain unclear. In this study, we evaluated the association of 10 candidate single nucleotide polymorphisms (SNPs) with isolated VSD in a population from Southwest China. Based on the results of 34 congenital heart disease whole-exome sequencing and 1000 Genomes databases, 10 candidate SNPs were selected. A total of 618 samples were collected from the population of Southwest China, including 285 VSD samples and 333 normal samples. Ten SNPs in the case group and the control group were identified by SNaPshot genotyping. The chi-square (χ2) test was used to evaluate the relationship between VSD and each candidate SNP. The SNPs that had significant P value in the initial stage were further analysed using linkage disequilibrium, and haplotypes were assessed in 34 congenital heart disease whole-exome sequencing samples using Haploview software. The bins of SNPs that were in very strong linkage disequilibrium were further used to predict haplotypes by Arlequin software. ViennaRNA v2.5.1 predicted the haplotype mRNA secondary structure. We evaluated the correlation between mRNA secondary structure changes and ventricular septal defects. The χ2 results showed that the allele frequency of FLT4 rs383985 (P = 0.040) was different between the control group and the case group (P < 0.05). FLT4 rs3736061 (r2 = 1), rs3736062 (r2 = 0.84), rs3736063 (r2 = 0.84) and FLT4 rs383985 were in high linkage disequilibrium (r2 > 0.8). Among them, rs3736061 and rs3736062 SNPs in the FLT4 gene led to synonymous variations of amino acids, but predicting the secondary structure of mRNA might change the secondary structure of mRNA and reduce the free energy. These findings suggest a possible molecular pathogenesis associated with isolated VSD, which warrants investigation in future studies.

目的 建立叙利亚金黄地鼠CVB1(Coxsackievirus B1,CVB1)感染动物模型.方法 取叙利亚金黄地鼠经鼻腔滴注方式呼吸道感染CVB1,感染剂量每只为107.25 CCID50.观察14 d体重、体温、精神状态、皮肤黏膜变化、行为、粪便状态、是否有神经症状等临床情况;每天采集咽拭子、鼻灌洗液以及粪便进行病毒载量检测,感染第7天取3只实施安乐死,采血进行病毒载量及生化检测;同时采集脑、心脏、肝等多个组织样品进行病毒载量、组织病理学和IHC检测.结果 感染CVB1病毒的动物在14 d内均出现不同程度精神萎靡、体温下降,以及口唇部出现典型的红疹及疱疹等类似人类手足口病临床表现.咽拭子、鼻灌洗液、粪便以及血液中能检测到病毒;组织中检测到病毒载量并观察到病毒抗原,同时伴有炎症、增生、出血等病理改变;血清酶中肝功、心肌酶升高.结论 CVB1病毒经滴鼻方式感染叙利亚金黄地鼠建立的感染模型,表现出心肌和肝等组织器官的病理损伤特征,可用于人类手足口病的研究.

Coxsackie virus B1 (CVB1) is an enterovirus that presents as hand, foot and mouth disease (HFMD), which may cause long-term symptoms in some patients. However, CVB1 animal models, detoxification cycles, and long-term tissue tropism have not been systematically characterized. In this study, a CVB1 respiratory infection model was established in rhesus macaques, and clinical symptoms, viral load, and immune marker levels were observed in the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also quantified the distribution, replication, and pathology of CVB1 during the long-term recovery period using 35 postmortem rhesus macaques tissue samples at 30 days post infection (d.p.i), thus revealing the long-term tissue tropism of the virus. The results showed that the respiratory tract of rhesus macaques was susceptible to CVB1 and showed HFMD symptoms, detoxification phenomena, changes in cytokine levels, and neutralizing antibodies. Autopsy viral load results were positive in heart, spleen, pancreas, pharyngeal flat, and olfactory bulb tissues, and HE staining revealed pathological damage to the liver, spleen, lung, pharyngeal flat, palatal flat, and tracheal epithelium. Viral antigens were present in visceral, immune, respiratory, and muscle tissues but not detected in intestinal and neural tissues. Brain tissue was dissected and showed viral meningitis-like changes, and CVB1 antigen expression was observed in occipital, pontine, cerebellar, and spinal cord tissues. This study first provides a basis for elucidating CVB1 pathogenesis in a nonhuman primate HFMD model and then confirms the CVB1 tissue tropism pattern after long-term infection.

Echovirus 3 (E3) belongs to the species Enterovirus B. Currently, three nearly whole-genome sequences of E3 are available in GenBank in China. In this study, we determined the whole genomic sequences of six E3 strains isolated from the stools of patients with hand-foot-and-mouth disease in Southwest China in 2022. Their nucleotide and amino acid sequences shared 82.1%-86.4% and 96.6%-97.2% identity with the prototype Morrisey strain, respectively, and showed 87.1% and 97.2% mutual identity. The six E3 strains are not clustered with other Chinese strains and formed a novel subgenotype (C6) with the recent American and British strains. Recombination analyses revealed that intertype recombination had occurred in the 2 C and 3D regions of the six E3 strains with coxsackieviruses B5 and B4, respectively. This study augments the nearly whole-genome sequences of E3 in the GenBank database and extends the molecular characterization of this virus in China.

BACKGROUND:Echovirus 30 is prone to cause hand-foot-and-mouth disease in infants and children. However, molecular epidemiologic information on the spread of E30 in southwestern China remains limited. In this study, we determined and analyzed the whole genomic sequences of E30 strains isolated from the stools of patients with hand-foot-and-mouth disease in Yunnan Province, China, in 2019.METHODS:E30 isolates were obtained from fecal samples of HFMD patients. The whole genomes were sequenced by segmented PCR and analyzed for phylogeny, mutation and recombination. MEGA and DNAStar were used to align the present isolates with the reference strains. The VP1 sequence of the isolates were analyzed for selection pressure using datamonkey server.RESULTS:The complete genome sequences of four E30 were obtained from this virus isolation. Significant homologous recombination signals in the P2-3'UTR region were found in all four isolates with other serotypes. Phylogenetic analysis showed that the four E30 isolates belonged to lineage H. Comparison of the VP1 sequences of these four isolates with other E30 reference strains using three selection pressure analysis models FUBAR, FEL, and MEME, revealed a positive selection site at 133rd position.CONCLUSIONS:This study extends the whole genome sequence of E30 in GenBank, in which mutations and recombinations have driven the evolution of E30 and further improved and enriched the genetic characteristics of E30, providing fundamental data for the prevention and control of diseases caused by E30. Furthermore, we demonstrated the value of continuous and extensive surveillance of enterovirus serotypes other than the major HFMD-causing viruses.

Hand, foot, and mouth disease (HFMD) is a common pediatric infectious illness caused by enteroviruses (EVs). EV-A serotypes are the main pathogens associated with HFMD. In this study, 213 stool samples from 213 children with severe HFMD in Yunnan, China in 2013, 2015, and 2016 were further analyzed retrospectively for EV-B infection. A total of 70.0% of the specimens tested positive for EV.20 EV serotypes were detected. The predominant serotype was enterovirus A71 (EV-A71, 27.7%), followed by coxsackievirus B4 (CV-B4, 16.4%), CV-A16 (9.9%), CV-B5 (6.6%), and Echovirus 9 (E-9,4.7%). EV-A and EV-B accounted for 45.1% and 41.3%, respectively. Among the positive specimens, 28.6% were CV-Bs. Co-infection was present in 19.3% of these cases. In the study, CV-B5 and the majority of CV-B4 isolates belonged to genotypes VI and C3, respectively. This result indicates that EV-B, especially CV-Bs, might be the important agents associated with HFMD and this knowledge will contribute to the prevention and treatment of the disease.

Coxsackievirus B1 (CVB1) is one of the significant pathogens causing viral myocarditis, hand, foot, and mouth disease (HFMD), and aseptic meningitis, and it has been associated with type 1 diabetes (T1DM). No effective antiviral drugs against CVB1 infection or preventive vaccines are available. Due to the success of two inactivated vaccines against enterovirus 71 and poliovirus, an inactivated Vero cell-based CVB1 vaccine could be developed. In this study, we isolated a high-growth CVB1 virus strain KM7 in Vero cells and developed a Vero-adapted vaccine candidate strain KM7-X29 via three rounds of plaque purification and serial passages. The KM7-X29 strain was grouped into the GII sub-genotype, which belonged to the Chinese epidemic strain and grew to a titer of more than 107 CCID50/ml in Vero cells. The inactivated CVB1 vaccine produced by the KM7-X29 strain induced an effective neutralizing antibody response in BALB/c mice, and maternal antibodies were able to provide a 100% protective effect against lethal challenges with a CVB1 strain in suckling BALB/c mice. Thus, the KM7-X29 strain might be used as a new candidate coxsackievirus B1 vaccine strain. The neonatal murine model of CVB1 infection will contribute to the development of the CVB1 vaccine.

目的 分析柯萨奇病毒A组10型毒株在人胚肺二倍体细胞(KMB17)上增殖的遗传稳定性.方法 将V6-19/XY/CHN/2017毒株适应于KMB17细胞,并连续传代培养至15代.分别提取适应前及适应后第5、10、15代病毒RNA,分段对全基因组序列进行RT-PCR扩增、测序及拼接,利用Maga 7.0、Geneious 6.1.4等软件进行系统进化及同源性分析.结果 命名该KMB17细胞适应株为K6-19/XY/CHN/2017,各子代病毒感染性滴度为7.25～7.75 lgCCID50/mL.适应前后4个子代病毒的全基因核苷酸和氨基酸序列同源性分别为99.99％～100％和99.95％～100％.适应前与适应后第5、10代毒株均无核苷酸和氨基酸序列突变,第15代毒株VP4区域发生1个核苷酸突变(VP4 172 A→G)及1个氨基酸突变(VP4 58 Ile→Val).结论 CV-A10能较好地适应KMB17细胞,K6-19/XY/CHN/2017在KMB17细胞上适应后可保持较高的病毒滴度和遗传稳定性,为后续疫苗研发奠定了实验基础.

对2016年云南省昆明市手足口病相关的柯萨奇病毒B组5型分离株V1641/YN/CHN/2016(简称V1641)全基因组进行测序,并分析其分子变异和进化特点.设计针对V1641的引物,提取病毒RNA,RT-PCR扩增和测序,拼接获得的全基因组序列.利用MEGA7.0.26、Geneious9.1.4和SimPlot3.5.1软件分析全基因序列.V1641毒株基因组全长7 392nt,5'-UTR和3'-UTR分别长744nt和90nt,编码区长6 558nt,与其他CVB5相比未见核苷酸的插入和缺失,编码一个2185aa的多聚蛋白.CVB5流行株大体上分为两个基因组,中国大陆流行株同原型株Faulkner-起分布于Genogroup Ⅰ分支,外国流行株大多分布于Genogroup Ⅱ分支.在GenBank中,V1641与KY303900-417/JS-CHN-2013最为同源,相似性为97.86％.V1641与其他中国大陆流行株的全基因组序列的核苷酸和氨基酸相似性分别为85.1％～97.8％和97.1％～99.6％.V1641在P1、P2和P3区段均与EV-B不同血清型的原型株聚类,经SimPlot3.5.1软件验证,提示有重组事件发生.云南CVB5分离株V1641同中国大陆流行株一样属于Genogroup Ⅰ基因群,在进化过程中可能发生了重组.中国大陆可能存在多条CVB5传播链.

Coxsackievirus A10 (CV-A10) is one of the etiological agents associated with hand, foot and mouth disease (HFMD) and also causes a variety of illnesses in humans, including pneumonia, and myocarditis. Different people, particularly young children, may have different immunological responses to infection. Current CV-A10 infection animal models provide only a rudimentary understanding of the pathogenesis and effects of this virus. The characteristics of CV-A10 infection, replication, and shedding in humans remain unknown. In this study, rhesus macaques were infected by CV-A10 via respiratory or digestive route to mimic the HFMD in humans. The clinical symptoms, viral shedding, inflammatory response and pathologic changes were investigated in acute infection (1-11 day post infection) and recovery period (12-180 day post infection). All infected rhesus macaques during acute infection showed obvious viremia and clinical symptoms which were comparable to those observed in humans. Substantial inflammatory pathological damages were observed in multi-organs, including the lung, heart, liver, and kidney. During the acute period, all rhesus macaques displayed clinical signs, viral shedding, normalization of serum cytokines, and increased serum neutralizing antibodies, whereas inflammatory factors caused some animals to develop severe hyperglycemia during the recovery period. In addition, there were no significant differences between respiratory and digestive tract infected animals. Overall, all data presented suggest that the rhesus macaques provide the first non-human primate animal model for investigating CV-A10 pathophysiology and assessing the development of potential human therapies.

目的 建立柯萨奇病毒B组1型(coxsackievirus B1,CV-B1)特异的TaqMan 一步法荧光定量RT-PCR检测方法.方法 根据CV-B1的VP1序列,设计特异性引物和TaqMan探针.将目的基因插入pMD19TM载体,在DH5a感受态细胞中扩增,体外转录后获得RNA标准品,建立标准曲线.并对检测方法的灵敏度、特异性、重复性进行评价.结果 该方法在102～1011拷贝/μl的模板范围内具有良好的线性关系(R2=1.000),扩增效率E=107.5％.灵敏度达1×102拷贝/μl,只对CV-B1有特异性扩增曲线,且重复性较好.结论 建立的实时荧光定量RT-PCR方法具有较高的灵敏度、特异性和重复性,可作为CV-B1的快速检测和绝对定量分析.