Norovirus-like particle (VLP) vaccine is promising against human norovirus infection. Unfortunately, genetic diversity of norovirus hindered the development of this vaccine. In this study, the immunogenicity of norovirus VLPs induced by the endemic GII.4 and the epidemic GII.17 genotypes, and the cross-reactivity between them as well as GI.1 and GII.3 VLPs were evaluated in mice by using serum IgG and histo-blood group antigen (HBGA) blocking antibodies as index. Results showed well immunogenicity of both GII.4 and GII.17 VLPs in mice. Serum IgG GMT (Geometric Mean Titer) were 3.63 (GII.4) and 3.88 (GII.17) respectively, and sustained to the 15th week. The HBGA blocking antibodies were 130 (GII.4) and 360 (GII.17) respectively at the end of the 4th week. Additionally, there was a dramatically statistical difference found in the cross-reactivity within genogroup (GII.3, GII.4 and GII.17) (p < .001), and also showed similar difference between genogroups (GI.1 vs. GII.3, GII.4 and GII.17) (p < .001). Summarized the pPICZapichi pichiaexpression system showed a potential to be the alternative for expression of norovirus VLPs in secretion form, and the little cross-reactivity found between the endemic strain and the epidemic strain provides an evident for the consideration of selecting candidates of norovirus vaccine strains.

Objective:To screen the neutralizing epitope of enterovirus 71 (EV71) and determine the specific minimum amino acid sequence that triggers immunity for providing a theoretical basis for the development of synthetic peptide vaccines.Methods:EV71 neutralizing antibody-specific binding clones were panned and sequenced using a phage display random 12-peptide library to obtain the key sequences of neutralizing epitopes. A series of peptides containing the key sequences with N-terminal acetylation (AC) and C-terminal linking to Keyhole limpet hemocyanin (KLH) were synthesized. Serum samples were collected after immunizing mice with the modified peptides. Then the immunogenicity of the peptides and the neutralizing activity of serum samples were analyzed by Western blot, ELISA and neutralization test.Results:After three rounds of panning, cloning and sequencing, KQEKDL was identified as the key motif. The serum samples collected from the mice immunized with the modified series of peptides containing key motifs had different degrees of binding ability to EV71 and VP1 protein. The serum samples of mice immunized the synthetic peptide containing only the minimum key motif (AC-KQEKDL-KLH) had the strongest response to the other three peptides and EV71 and the highest neutralizing titer.Conclusions:The EV71 neutralizing epitope was successfully screened using the phage display random peptide library. The key motif of KQEKDL might be the specific minimum amino acid sequence that triggered the immune system. This study provides a theoretical basis for better understanding the immune response mechanism, evaluating the immunogenicity of the antigens and further research and development of polypeptide vaccines.

目的 探讨诺如病毒(Norovirus,NoV)VP1蛋白的病毒样颗粒(virus-like particle,VLP)在毕赤酵母表达系统中分泌表达的可行性.方法 用pPICZa-A表达载体和X-33毕赤酵母株进行NoV GⅡ.4和GⅡ.17型VP1蛋白的分泌表达,并对阳性菌株进行Mut表型筛选.同时优化起始pH(5.0、6.0、7.0、8.0)、甲醇终浓度(0.5％、1.0％、1.5％、2.0％)及发酵时间(24、48、72、96、120 h)3个表达条件.取原始及传代10代菌株,采用优化条件进行发酵,于诱导0 ～60 h间,通过双标准曲线实时荧光定量PCR法测定外源基因拷贝数.发酵产物经蔗糖密度梯度离心纯化后,检测其浓度及纯度,并观察VLP的形态.结果 表达目的 蛋白的重组酵母菌株均为Mut+型.最佳发酵pH为7.0,甲醇终浓度为1％,发酵时间为72 h.菌株传代后未发生基因缺失,整合基因在10代内稳定性良好.GⅡ.4及GⅡ.17型VP1纯度均约85％,浓度分别为133和165 μg/mL,VP1蛋白均可自发组装成直径约40 nm的VLP颗粒,形态与天然病毒相似.结论 用毕赤酵母表达系统进行NoV VP1蛋白VLP分泌表达是可行的.

Because of deficiencies of traditional potency tests in rotavirus detection, a one‐step TaqMan probe‐based quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) assay combined with cell‐based method was established to determine the infectious potency of the target virus in multivalent live rotavirus vaccines in vitro. Series dilutions of rotavirus samples were inoculated into Vero cells and cultured for 24 hours. The cells were lysed and the potency was detected by RT‐qPCR. The reference standards with a known titer (lgCCID50/mL) were assayed in parallel, and the potencies of each sample were determined using parallel line method. The specificity, precision and accuracy of the assay were evaluated, respectively. The results showed that messenger RNA produced during rotavirus replication was the primary template of RT‐qPCR and the primers and probes were specific to each strain. The coefficient of variation of different wells and different working days did not exceed 6% and the results of the assay were proved to be concordant with those of cell culture infective dose 50% with a relative deviation less than 5%. This assay is a more rapid, cost‐effective and high‐throughput way for detecting multivalent rotavirus vaccine, and will be a valuable tool in the quality control and stability monitoring of live multivalent rotavirus vaccine.

Background: A randomized, double-blind, placebo-controlled multicenter trial was conducted in healthy Chinese infants to assess the efficacy, immunogenicity and safety of a novel trivalent live human-lamb reassortant rotavirus vaccine (LLR3) against rotavirus gastroenteritis (RVGE). Methods: Healthy children aged 6-13 weeks were enrolled and randomized (1:1) to either 3 oral doses of LLR3 or placebo according to a 0, 1, 2 month schedule. The objectives were to evaluate vaccine efficacy (VE) against RVGE of any-severity, severe RVGE (sRVGE) and inpatient caused by rotavirus serotypes contained in the vaccine and not contained in the vaccine after the third dose. Immunogenicity was also assayed in a subgroup. All adverse events (AEs) were collected from 30 min after each dose for immediate reaction, even to the entire study period, including the serious AEs (SAEs) and intussusception. Results: VE against RVGE of any-severity, sRVGE and inpatient caused by any serotype was 56.6% (95% CI: 50.7, 61.8), 70.3% (95% CI: 60.6, 77.6) and 74.0% (95% CI: 57.5, 84.1) respectively. VE against RVGE of any-severity, sRVGE caused by serotypes not contained in vaccine were 54.2% (95% CI: 47.5, 60.1) and 70.4% (95% CI: 60.4, 77.9). The rate of seroconversion and four-fold increase of rotavirus serotype G2-, G3-, and G4-specific IgA is 60.8%, 58.0%, and 60.6% in vaccine group, which was higher than 21.35%, 22.7%, and 23.1% in placebo group (p < 0.0001 for G2, G3, G4), as well as the Geometric Mean Titer (GMT). Through the entire trial, 65.91% and 67.79% of participants reported at least one AE, and 0.02% and 0.02% reported SAEs in the vaccine and placebo groups, respectively. Two intussusception cases were reported both in vaccine and placebo group. Conclusions: In Chinese infants, LLR3 provided a substantial protection against RVGE of any-severity, sRVGE and inpatient caused by any serotype, and showed well immunogenicity and safety. (C) 2020 The Authors. Published by Elsevier Ltd.

目的 考察口服Ⅰ型Ⅲ型脊髓灰质炎减毒活疫苗在不同温度和pH环境下的稳定性.方法 将6批疫苗分别于2～8℃放置6个月、22～25℃放置7d、37℃放置7d、-20℃放置27、30、33个月,或反复冻融5次;将疫苗pH调至3.0～10.0.采用细胞培养半数感染量(cell culture infective dose 50％,CCID50)法检测病毒总滴度及分型滴度.结果 口服二价脊髓灰质炎减毒活疫苗在2～8℃保存6个月,22～25℃保存5d,37℃保存2d,反复冻融5次,-20℃保存27个月,pH3.0～10.0环境下,疫苗效力均符合质量标准.结论 疫苗的保存温度及时间对其稳定性均有影响.

Abstract Rotavirus (RV) vaccines show distinct immunogenicity in dozens of clinical trials, which is associated with multiple host and environmental factors. Previous research has demonstrated that the highly polymorphic human leukocyte antigen (HLA) system plays an essential role in regulating immune response to a variety of vaccines. This study aims to investigate the relationship between HLA polymorphisms and immunogenicity of RV vaccine. A nested case-control study was carried out among infants enrolled in phase III clinical trial of trivalent human-lamb reassortant vaccine (RV3) in Henan province, China. Serum RV specific immunoglobulin A (RV-IgA) was detected before and after a 3-dose vaccination series, followed by calculation of seroconversion rates. Seroconversion was defined as a 4-fold or greater increase in RV-IgA titers between pre-vaccination and 1-month post-dose 3 vaccination. The infants who seroconverted were defined as responders, and the others without seroconversion were considered as non-responders. Their HLA genotypes were obtained by using the sequence-based typing method. The HLA allele and supertype frequencies of 2 groups were analyzed statistically. Eighty-three of 133 infants seroconverted after vaccination. Twenty-one HLA-A, 45 HLA-B, 24 HLA-Cw, 29 HLA-DRB1 and 16 HLA-DQB1 distinct alleles were detected. The frequency of HLA-B∗4001 (corrected P = .01, adjusted OR = 0.152, 95% CI = 0.048–0.475) in non-responder group was significantly higher than that in responder group. Furthermore, significant association was found between HLA-B44 supertype (corrected P = .02, adjusted OR = 0.414, 95% CI = 0.225–0.763) and RV non-response. Certain HLA allele (HLA-B∗4001) and supertype (HLA-B44) are potentially associated with non-response after immunization with the novel RV3 vaccine in Chinese infants.

In 2010, Rotarix was found to be contaminated with infectious porcine circovirus type 1 (PCV1). In China, the Lanzhou lamb rotavirus (LLR) vaccine is the only vaccine used to prevent rotavirus disease. From 2006 to September 2014, more than 54 million doses of LLR vaccines have been lot released. It is a safety issue whether PCV1 is present in the LLR vaccine. Although the cell substrate of LLR, bovine kidney (BK), is different from that of Rotarix, we have investigated the cell's permissivity for PCV1 by both infectivity and full-length PCR analysis. We have assessed the LLR using a quantitative PCR (qPCR) assay. A total of 171 random batches of LLR final products over a period of 5 years were tested, and no PCV1 was detected (0/171). Infectivity studies showed that two strains of PCV1, the PCV1-prototype, which was derived from PK-15 cells, and the mutant, PCV1-GSK, which was isolated from Rotarix, were capable of replicating in BK cells over a wide m.o.i. ranging from 10 to 0.01. After culture for 6 days, copies of PCV1-prototype DNA were higher than those of PCV1-GSK on average. The genome of the virus was detected at 6 days post-infection. In summary, the LLR vaccine is free of PCV1. Nevertheless, because PCV1 can replicate in the BK cell substrate, manufacturers need to be vigilant in monitoring for this adventitious agent.

Objective To develop detection methods for porcine circovirus type 1 (PCV1) — the adventitious agent in rotavirus vaccine contamination incident,in African green monkey kidney cell (Vero cell).Methods Six days after PCV1 infection,virus replication and proliferation in Vero cells were tested with qualitative PCR,quantitative PCR (qPCR),electron microscopy (EM) and in situ hybridization (ISH).Results PCR products amplified from PCV1-infected Vero cell DNA extracts presented specific band in agarose gel electrophoresis.According to qPCR,PCV1 copy number increased to 794.3 times of the original level.The detection limits of qualitative PCR and qPCR were both 102.0 copies/ml.Characteristic viral particles were observed by EM.ISH showed dark positive signal of PCV1 in infected cells.Conclusion The replication and proliferation of PCV1 in infected Vero cells can be detected by the 4 methods used.

Mouse is one of the infection animal models for rotavirus. Since the optimal age of mouse sensitive to rotavirus infection thus far has not been unified, we elucidated clinical symptoms, immune responses and pathological changes of mice in different ages after challenged by murine rotavirus wild strain EDIM (Epidemic Diarrhea of Infant Mice) to provide data for the estimation. One-week-old, two-week-old, and three-week-old BALB/c mice were inoculated with EDIM in the challenge dose of 235 ID50, 470 ID50 and 705 ID50 respectively and were compared to mock-infected controls. Diarrhea illness, mobility, bodyweight were recorded, viral shedding and immune responses including serum IgA, fecal sIgA were detected, and small intestine tissue was evaluated for virus distribution and pathological changes. All the mice in one-week-old and two-week-old groups were completely unavoidable to be infected by EDIM and have been found to be malaise, activity reduced and even diarrhea, while three-week-old mice partly resist the challenge with 40% mice free from diarrhea. Meanwhile, EDIM infection has greater impact to the bodyweight of two-week-old group than those of one-week-old, three-week-old (0.9860 vs 1.2340, 1.2375g/day). One peak of virus shedding in three groups was observed in day 1-2 post infection, but the duration shortened with age increase. Feces sIgA in both two-week-old and three-week-old groups began to increase in day 4, 2-3days earlier than that in one-week-old group, and grow to the peak in day 8, which is about 2 fold of that in one-week-old group. Stronger serum IgA response was found in two-week-old group, it increased to the peak in day 15 and the level was 2 fold of three-week-old group and 4 fold of one-week-old group. The pathological changes included vacuolar degeneration, edema and congestion of intestinal wall, integrity destruction of enteric epithelium, and the changes relieved with the increase of age. Besides, rotavirus particles were found in small intestine tissues, especially in the surface and crypt of villi. In conclusion, the two-week-old mice were more sensitive to EDIM infection and initiated more effective immune response. In combination with that 14days old mice equals to 2 months infant when the first dose of rotavirus vaccine should be administrated, two-week-old mice is preferred to be used as infection model for the study of pathogenicity and immunogenicity of rotavirus.

Intestinal ischemia reperfusion (IR) injury is a critical problem, which can cause intestinal injury locally and acute lung injury (ALI) distally by inflammatory responses and oxidative stress. Toll-like receptor 4 (TLR4) is involved in innate immune and inflammatory responses. This study was to determine whether TLR4 mutant can attenuate intestinal and lung injuries after intestinal IR. Wild type (WT) and TLR4 mutant mice were submitted to intestinal IR by occluding the superior mesenteric artery. Histological assessment of the intestine and the lung were conducted by HE staining. The levels of proinflammatory cytokines, oxidative stress markers, apoptotic index and other mediators were measured. In addition, a 24-hour survival study was performed. Histological assessment showed that intestinal IR caused serious injuries in the intestine and the lung, corroborated by increased proinflammatory cytokines in the circulation. TLR4 mutant suppressed the histological injuries as demonstrated by significantly decreased pathological scores. Consistent with the morphological results, the TLR4 mutant mice exhibited remarkably lowered cytokine expressions in the intestine (TNF-α, IL-6, IL-1β, and NF-κB) and the lung (NO, iNOS, MCP-1, MIP-2, NF-κB, and Caspase-3). ALT and creatinine were also significantly dampened in the liver and kidney, respectively. Furthermore, the survival rate over the course of 24 hours was significantly improved. Collectively, the findings reveal that TLR4 mutant significantly abated the intestinal IR injury and ALI at least in part by alleviating the inflammatory response and oxidative stress.

Enteric virus-induced disease, even death, greatly affects the life quality of children <5 years of age in China, and imposes a heavy sociometric burden on family and government. The infectious diarrhea diseases and hand-foot-mouth disease (HFMD) have been enrolled in the list of notifiable infectious diseases in China. Fortunately, the highly valued rotavirus-induced diarrhea and enterovirus 71-induced HFMD can be prevented by the commercial vaccines. However, the underrated norovirus vaccine is still in progress, even norovirus was considered as the leading cause of acute gastroenteritis in children <5 years of age in the world, partially due to the inability to cultivate norovirus. Besides, the broad genetic and serotypic diversity of norovirus also complicates selection of candidate vaccine strain(s). Therefore, the primary objective of this article is to review norovirus epidemics in China recur to the data published in recent five years.

目的:研究柯萨奇病毒A 16型候选疫苗株(CA 16-KM)经灭活后免疫小鼠,对病毒攻击的保护效果.方法:用Vero细胞进行CA 16-KM株培养,病毒收获后经浓缩、蔗糖密度梯度离心进行纯化及β-丙内酯灭活,用酶联免疫吸附法(ELISA法)对灭活后的CA 16-KM病毒液进行抗原含量的测定并在Vero细胞上盲传3代对灭活后CA 16-KM毒株的残余毒力进行检测.灭活后的病毒加入氢氧化铝佐剂(质量浓度为1 mg· mL-1),经离心及PBS重悬,制成实验性灭活疫苗.分别于第0天和第14天经肌肉注射免疫8周龄ICR母鼠,对照组用等体积PBS代替.分娩后24h内的新生乳鼠用100 LD50的CA 16强毒株(CA16-CC)经颅内注射进行攻毒,攻毒后连续21 d观察其临床症状,研究该灭活疫苗对新生乳鼠的保护效果.结果:经β-丙内酯灭活后的CA 16-KM在Vero细胞上盲传3代均无细胞病变效应(CPE)出现,灭活后的CA 16-KM抗原含量为320 EU·mL-1.经注射实验性疫苗的乳鼠攻毒后全部存活,表明CA 16-KM株制备的灭活疫苗诱导的免疫反应可对新生乳鼠产生100％的保护作用.结论:β-丙内酯对CA 16-KM毒株的灭活效果较好;CA 16-KM疫苗候选株灭活后具有良好的免疫原性,可以保护新生乳鼠抵抗CA 16病毒的攻击,也许是研制CA 16灭活疫苗的理想毒株.

Objective To investigate haplotype frequencies of HLA using Arlequin software .Methods The genetic type data of alleles in human leukocyte antigen (HLA) A and B loci were transformed to the file with .arp .Then the HLA haplotypes were cal‐culated by Arlequin software .Results The frequencies of alleles and haplotypes were shown in table directly .A total of 19 HLA‐A alleles ,39 HLA‐B alleles and 128 HLA A‐B haplotypes were observed .Conclusion In conclusion ,the HLA haplotypes frequencies could be calculated by Arlequin software ,which is a simple ,convenient ,fast ,universal and operable way to analyze the haplotype .

Lanzhou Lamb derived Rotavirus (RV) Vaccine (namely LLR) for children is only used in China. Since there were no reports on evaluation of LLR, even the data of phase IV clinical trial, we proceed the evaluation of LLR through focusing on T-cell to investigate whether LLR could induce the potential function involving in protection as a vaccine. Four groups of nude mice were transfused with CD4(+)/CD8(+) T-cells isolated from LLR-immunized (primed) and LLR-unimmunized (naïve) mice via intraperitonea (i.p.) respectively. Consequently, the adoption mice were challenged with mice-origin wild rotavirus EDIM (Epizootic Diarrhea of Infant Mice) by intragastric administration. Series of fecal/serum samples were collected and viral shedding, then serum IgA/IgG and secreted IgA were assayed. Compared to the mice transfused with T lymphocytes from naïve mice, the nude mice transfused with CD4(+) T lymphocytes from primed mice induce fecal and serum IgA increasing more rapidly, and have a shorter duration of virus shedding too. Whereas, no significant difference in virus clearance was found between the mice transfused with CD8(+) T lymphocytes isolated from primed and naïve mice. Therefore, we cleared the distinct roles of transfused CD4(+)/CD8(+) T lymphocytes for rotavirus clearance in nude mice, that the viral clearance conducted by CD4(+) T lymphocytes. Meanwhile, it has ability to help induction of LLR specific immunogenicity. Comparing with the transfusion of cell from primed and naïve mice, LLR can induce CD4(+) T lymphocytes memory which is a potential index to reflect the immunogenicity and protection, while CD8(+) T lymphocytes remove rotavirus by CTL with little memory ability.

目的 分析河南汉族人群HLA基因多态性和超型分布. 方法 采用测序法对111名志愿者的血样进行HLA-A、-B、-C、-DRB1和-DQB1五个座位的基因分型,分别根据5个研究组的HLA超型分型标准进行HLA Ⅰ类和Ⅱ类超型的分析统计. 结果 总共检测出130个HLA等位基因,其中HLA-A座位为21个等位基因,-B座位为41个,-C座位为24个,-DRB1座位为27个,-DQB1座位为17个.依据不同的分型方法,共得到86个超型,其中HLA Ⅰ类51种,Ⅱ类35种.按照5个研究组的分型标准,HLA Ⅰ类的优势超型型别基本相似,其中HLA-A2和-B7的分布差异无统计学意义(P＞0.05).但对于HLAⅡ类分子,超型名称分类标准在不同的研究组均不同. 结论 获得河南汉族人群的超型分布特征,HLA Ⅰ类分子的优势超型为HLA-A3、-A2、-B7、-B44和-B62,但HLAⅡ类分子的超型分类标准尚不统一,有待进一步研究.

Objective To establish the national reference for detecting infective titer of coxsackievirus A16 (CA16),in order to provide the national reference for viral titer control during CA16 vaccine production and immune effect evaluation of CA16 vaccine.Methods The qualified CA16 culture was divided into 0.5 ml aliquots as candidate reference.Candidate reference was collaboratively calibrated by 3 independent laboratories.The infective titer of CA16 was detected by cell culture method,and 50％ cell culture infective does (CCID50) of CA16 was calculated by Behrens-K(a)rber method.Meanwhile,CA16 candidate reference was stored at-60 ℃ for 12 months,at-20 ℃ for 6 months,at 4 ℃ for 28 days,at 22-25 ℃ (room temperature) for 7 days and at 37 ℃ for 7 days or frozen and thawed repeatedly,and the stability under different circumstances was evaluated.Results The viral titer of CA16 candidate reference was (6.80 ± 0.95) lgCCID50/ml.The viral titers of CA16 candidate reference had no significant reduction after keeping in storage at-60 ℃ for 12 months,-20 ℃ for 6 months,and 4 ℃ for 28 days,indicating high stability at these conditions.The viral titer loss rate of CA16 candidate reference at 22-25 ℃ (room temperature) and 37 ℃ were 3.4％ and 4.4％ per day respectively,and respective titer loss after 27 and 21 d storage was not significant.The viral titer loss rate of CA16 candidate reference was 4.8％ per time under repeated freeze-thaw condition.Conclusion CA16 candidate reference meets the requirement as a national reference,and its valuation was (6.80 ± 0.95) lgCCID50/ml.

Objective To compare the relationship between the enzyme‐linked immunosorbent assay(ELISA) reagent and West‐ern blot(WB) confirmation reagent for analyzing the quality lever of human T‐cell lymphotropic virus(HTLV) detection reagent . Methods The WB confirmation reagent was used to detect anti‐HTLV antibody in 156 human serum samples of ELISA prelimina‐ry screening positive .The ELISA cut‐off value(optimal value) was selected by using the two‐graph receiver operating characteristics (TG‐ROC) analytical method .The two‐by‐two table analysis was constructed to analyze the consistency of results detected by the two methods ,moreover the McNemar test was used to evaluate the consistency of detection results .The quality level of HTLV de‐tection reagent was comprehensively evaluated .Results Among 156 serum samples of ELISA preliminary screening positive ,only 40 samples were positive by the WB confirmation ,and other 116 samples were negative .The sensitivity and specificity of ELISA de‐tection reagent obtained by TG‐ROC analysis were 97 .5% and 45 .7% respectively ,the TG‐ROC test also indicated that the detec‐tion results had significant difference between ELISA and WB(P<0 .05) .By adjusting the cut‐off value ,the sensitivity and specific‐ity of ELISA were increased to 88 .8% (parametric method) .In the comparison of the parametric method and the non‐parametric method ,the obtained areas under the curve(AUC) was 0 .923 5(parametric method) ,their results were basically consistent .Conclu‐sion Although above results indicate that the detection results of ELISA reagent are different from those of WB ,but adjusting the cut off value can increase its sensitivity and specificity ,thus increases the reliability of diagnosis result .

The challenges posed by norovirus infections to global health are increasing accompa-nied by the rapid rate of the genetic and antigenic evolution of circulating noroviruses. Due to lack of in vitro culture cells and small animal models, norovirus vaccine cannot be prepared by using traditional techniques. With the in-depth understanding and study of norovirus, the subunit vaccines against norovirus infection based on P particles have been developed and presented the characteristics of easily expressed, low cost, high immunogenicity, stable structure and so on. In addition, norovirus P particle has been used as a subvi-ral nanoparticle for vaccine development against other viruses and for antibody production against chronic dis-ease ( Alzheimer′s disease) , which benefits from the accommodation of foreign antigens in the three loops of P particle. In this review, we describe the progresses in the field of P particle related vaccines for providing suggestions about the research and development of multivalent vaccines in China.

Objective To investigate effects of different storage conditions and time on stability of HCV RNA. Methods Totally 22 plasma samples were stored at different conditions as bellow :-20 ℃ for 4 weeks ;4 ℃for 12 days ;room temperature(20-25 ℃) for 7 days ;5 freeze‐thaw cycles. HCV RNA was determined by RT‐PCR to investigate the stability of it. Results The average decline of HCV RNA concentration was less than or equal to 0. 14 lg and ± 95% CI ≤0. 20 lg when samples were stored at -20 ℃ for 4 weeks. The average decline of HCV RNA concentration was less than or equal to 0. 39 lg and ± 95% CI ≥ -0. 50 lg when samples were stored at 4 ℃for 12 days. But ± 95% CI≤0. 24 lg when samples were stored at 4 ℃ less than 12 days. The average decline of HCV RNA concentration was less than or equal to 0. 40 lg and ± 95% CI≥ -0. 53 lg when samples were stored at room temperature for 7 days. But ± 95% CI≤0. 29 lg when samples were stored at room temperature less than 7 days. The average decline of HCV RNA concentration was less than or equal to 0. 10 lg and ± 95% CI≤0. 15 lg when samples were frozen and thawed for 5 times. Conclusion HCV RNA is stable in plasma samples stored at -20 ℃ for 4 weeks, 4 ℃ for 9 days, room temperature for 5 days or after 5 freeze‐thaw cycles. But HCV RNA concentration de‐clined dramatically when samples were stored at 4 ℃ for 12 days and room temperature for 7 days. The storage and transportation should be avoided in these conditions.