We wished to understand the genetic characteristics of enteric cytopathic human orphan (ECHO) virus type 6 (ECHO6) circulating in China. First, the partial VP1 coding region of six strains of the ECH-O6 virus isolated from cases of hand, foot and mouth diseases during routine surveillance in Hunan Province (China) from 2009 to 2014 were sequenced. Those sequences were analyzed along with 138 sequences of ECHO viruses covering five provinces of China and countries outside China retrieved from the GenBank database. A phylogenetic tree based on partial VPI was constructed, and it indicated that Chinese strains of the ECHO virus could form two distinct evolutionary branches: branch 1 and branch 2. All isolates of the ECHO virus from Hunan Province belonged to the 2c subranch, which revealed that they may share a common evolutionary origin. ECHO strains in branch 2 may be the predominant strains in China due to their wide geographic distribution and long period of circulation. We used nucleotide differences of >30%o as the basis of cluster division. ECHO, viruses could be divided into four clusters (A-D). Cluster D could be divided further into ten subclusters on the basis of nucleotide differences of 15%-30%. All ECHO6 isolates from Hunan Province belonged to the D7 subcluster. These data showed that the ECHO6 strains that circulated in Hunan Province in 2009-2014 were closely related to each other, and probably shared a common evolutionary origin. In addition, at least four distinct lineages of ECHO viruses have circulated in China.
Amino Acid Sequence ; China ; epidemiology ; Echovirus 6, Human ; chemistry ; classification ; genetics ; isolation & purification ; Echovirus Infections ; epidemiology ; virology ; Evolution, Molecular ; Female ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny ; Sequence Homology ; Viral Proteins ; chemistry ; genetics ; Young Adult

Objective:To evaluate the value of measles IgG antibody avidity assay in identifying the measles cases.Methods:Data from the Measles Surveillance Information System was used to collect laboratory confirmed or discarded cases in 2013-2015, and then tracing back the blood specimens from all measles network laboratories in Tianjin. Measles antibody avidity assay was used to detect and to redefine cases from the discarded ones.Results:A total of 326 measles cases including 267 laboratory-confirmed and 59 discarded cases were enrolled into this study, with 92.33% (301/326) of them aged ≥20 years. Result from the measles IgG antibody avidity assay showed that the ratio of high-avidity was 91.23%(52/57) of the discarded cases, which was significantly higher than 66.95% (158/236) of the laboratory confirmed cases ( χ2=13.33, P<0.001). According to the case criterion, 15.25% (9/59) of the discarded cases were redefined as measles cases. Eight out of the nine cases were high-avidity with measles containing vaccine (MCV) vaccination history that named as SVF cases. One in nine cases with low-avidity was with typical clinical symptomatic measles but with no vaccination history of MCV. Conclusion:Measles IgG antibody avidity assay could provide reference serological evidence to reduce the error from those discarded cases caused by false negative results on IgM antibody, when diagnosing the measles cases.

Objective To study the application of measles specific IgM and IgG antibody detection in classification of primary vaccination failure (PVF) and secondary vaccination failure (SVF). Methods Measles surveillance information system was used to collect measles confirmed cases in Tianjin, 2013-2015, and their blood specimens were collected, totally 284 cases were enrolled. Measles IgM and IgG were detected with enzyme?linked immunosorbent assay (ELISA), and the relative avidity index (RAI) was used to express the result of measles avidity. Measles IgM, IgG and IgM/IgG was analyzed with receiver operating characteristic (ROC) curve, the area under the ROC curve (AUC) as evaluation indicators. In addition, compared with a measles outbreak (26 cases) of a middle school in Tianjin in 2016, for making further verification on the diagnostic value of vaccination failure with IgM, IgG and IgM/IgG. Results The age of cases ranged was 0-58 years old, the interval median (P25, P75) of serum collection after rash onset was 2 (1, 4) days. The positive rate of measles IgM and IgG in acute phase specimens were 76.06% (216 cases) and 88.38% (251 cases). According to the ROC curve analysis, the area under the ROC curve (AUC) of IgM, IgG and IgM/IgG were 0.753, 0.891 and 0.952, indicating that IgM/IgG was the best index to distinguish PVF and SVF. The best cut off value for IgM/IgG was 0.06, the sensibility and specificity were 88.75% and 86.63%. When IgM/IgG>1, 96.30% cases were low?avidity (RAI<40%), only 1 case was equivocal response (RAI: 40%-60%). 97.14% cases were high?avidity (RAI >60%) when IgM/IgG <0.01, only 3 cases were equivocal response (RAI 40%-60%). The threshold of IgM/IgG was used to verify the measles outbreak of a middle school in Tianjin, 2016. In the acute phase specimens, 100% (26 cases) of IgM/IgG were <0.06, 84.62% (22 cases) of IgM/IgG were<0.01. Conclusion The detection of measles IgM and IgG with ELISA, and IgM/IgG is a valuable diagnostic tool to distinguish PVF and SVF.

Objective To establish an indirect ELISA method for detection of measles virus antibody based on the nucleoprotein of measles virus expressed in prokaryotic system selected as the coating antigen.Methods The working conditions were screened and optimized to determine the operation process of indirect ELISA.The anti-measles virus IgG antibody in 157 sera of healthy children and mothers of newborn infants was detected and compared with the commercial measles virus ELISA IgG antibody detection kit.Results The result showed that the coefficient of variation of intra and inter assay repeatability of the indirect ELISA was less than 10％.Compared with the result of the commercial kit,the total concordance rate of serum samples was 95.5％,and the sensitivity and specificity were 94.8％ and 98.3％,respectively.There was no significant difference between the two methods (x2 =0.313,P＞0.05;x2 =0.000,P＞ 0.05) in the positive rate of measles virus IgG antibody in 85 healthy children at the age of 0～ 15 years.The level of measles virus antibody detected by ELISA established in this study showed the same increasing and decreasing trend as that detected by the commercial kit in different age groups.The correlation coefficient r of the quantitative result was 0.893 (P＜ 0.001),which showed the quantitative potential of the method.Conclusions The ELISA method established in this study has good stability,high sensitivity and specificity,and there was no significant difference between the detection results of commercial kit and the ELISA method.It can be used for the seroepidemiological investigation of measles virus and the evaluation of antibody level of the population after vaccination.

Objective To study the application of measles specific IgM and IgG antibody detection in classification of primary vaccination failure (PVF) and secondary vaccination failure (SVF). Methods Measles surveillance information system was used to collect measles confirmed cases in Tianjin, 2013-2015, and their blood specimens were collected, totally 284 cases were enrolled. Measles IgM and IgG were detected with enzyme?linked immunosorbent assay (ELISA), and the relative avidity index (RAI) was used to express the result of measles avidity. Measles IgM, IgG and IgM/IgG was analyzed with receiver operating characteristic (ROC) curve, the area under the ROC curve (AUC) as evaluation indicators. In addition, compared with a measles outbreak (26 cases) of a middle school in Tianjin in 2016, for making further verification on the diagnostic value of vaccination failure with IgM, IgG and IgM/IgG. Results The age of cases ranged was 0-58 years old, the interval median (P25, P75) of serum collection after rash onset was 2 (1, 4) days. The positive rate of measles IgM and IgG in acute phase specimens were 76.06% (216 cases) and 88.38% (251 cases). According to the ROC curve analysis, the area under the ROC curve (AUC) of IgM, IgG and IgM/IgG were 0.753, 0.891 and 0.952, indicating that IgM/IgG was the best index to distinguish PVF and SVF. The best cut off value for IgM/IgG was 0.06, the sensibility and specificity were 88.75% and 86.63%. When IgM/IgG>1, 96.30% cases were low?avidity (RAI<40%), only 1 case was equivocal response (RAI: 40%-60%). 97.14% cases were high?avidity (RAI >60%) when IgM/IgG <0.01, only 3 cases were equivocal response (RAI 40%-60%). The threshold of IgM/IgG was used to verify the measles outbreak of a middle school in Tianjin, 2016. In the acute phase specimens, 100% (26 cases) of IgM/IgG were <0.06, 84.62% (22 cases) of IgM/IgG were<0.01. Conclusion The detection of measles IgM and IgG with ELISA, and IgM/IgG is a valuable diagnostic tool to distinguish PVF and SVF.

Objective:The purpose of this study was to investigate the characteristics of viral pathogen spectrum and the epidemiological characteristics of each viral pathogen in hospitalized cases associated with severe acute respiratory infection (SARI) in Luohe City, Henan Province from 2017 to 2019.Methods:Based the SARI Case Surveillance Platform, SARI cases were collected in Central Hospital of Luohe City, Henan Province from November 2017 to February 2019. In the end, 783 SARI cases were included, whose throat swabs were taken within 24 h of admission, as well as their demographic characteristics, onset time, clinical characteristics and other information recorded. At the same time, viral identification was performed, and the age and time distribution of each virus were analyzed.Results:The age of 783 SARI cases shown as M ( P 25, P 75) was 3 (1, 5) years old, ranging from 1 month to 95 years old. Children under 5 years old were the majority (71.01%). The males (61.81%) were more than females (38.18%). Among the 783 SARI cases, a total of 9 kind of viruses were identified with 64.88% (508/783) of the throat swabs tested positive for at least one virus. The positive rate of influenza virus and human respiratory syncytial virus were both 20.18% (158 cases), which was the highest among all the detected respiratory virus. The co-infection rate was 15.84% (124/783), among which double infection was the most common, accounting for 85.48% (106/124) of the co-infected cases. And human respiratory syncytial virus, human rhinovirus and influenza virus were the most common pathogen in co-infection cases. Moreover, the viral positive rate was 68.71% in children aged 5 years and 63.27% in people aged 60-95 years. Influenza and human respiratory syncytial virus dominated in winter and spring, while human parainfluenza virus was the main infection in summer. Conclusion:Influenza virus and human respiratory syncytial virus were the main viruses in throat swabs of SARI cases from 2017 to 2019 in Luohe City, Henan Province. There were differences in the age and seasonal epidemiological characteristics of each virus.

Objective:To establish a Plaque-reduction Neutralization Test (PRNT) for the detection of neutralizing antibody titers of Human Respiratory Syncytial Virus (HRSV) and optimize the conditions for preliminary application.Methods:The CHO expression system was used to produce palivizumab monoclonal antibody (palivizumab) and the influencing factors such as cell type, cell culture duration, fixation and permeabilization protocols, and blocking agents. The reproducibility of the method was verified and its correlation was verified with conventional PRNT. Finally, the optimized PRNT assay was further used to determine neutralizing antibody titers against HRSV subtypes A and B in BALB/c mouse serum (immunized by intramuscular injection of HRSV fusion proteins).Results:Palivizumab was expressed at approximately 50 mg/L. The optimal working conditions for PRNT were as follows: culturing HEp-2 cells for 2 days, fixing with 4% (V/V) paraformaldehyde at room temperature for 15 min followed by 0.2% (V/V) Triton X-100 permeabilization for 15 minutes as the optimal fixation-permeabilization and removing the blocking step. The overall coefficient of variation (CV) for the reproducibility validation of this method was <15%, showing a good linear relationship with the conventional PRNT. The Spearman correlation coefficient r s was 0.983. This method was used to detect neutralizing antibody titers in mouse sera against HRSV subtype A strain long and subtype B strain 9320, and the fusion proteins combined with AlOH and CpG adjuvant induced the highest neutralizing antibody titers in mice. Conclusion:The HRSV neutralizing antibody assay established in this study is rapid, reproducible, high-throughput, and can be used to detect neutralizing antibodies to HRSV subtypes A and B.

Objective:To understand the genetic characteristics of subgenus C human adenovirus (HAdV-C) strain isolated in Hunan in 2014.Methods:An HAdV-C strain named Hunan2014-s024 was isolated from throat swab collected from a child with severe acute respiratory infection in Changsha city of Hunan province in 2014. The whole genome sequence (WGS) was obtained by segmented amplification and sequence splicing. Database of HAdV-C was constructed with the strain from this study and the HAdV-C representative strains from GenBank. The whole genome characteristics was analyzed by using bioinformatics software.Results:Strain Hunan2014-s024 has the highest homology with the HAdV-C strain (MK041244-CHN-2006) isolated from stool sample of a healthy child in Shanxi province in 2006, and the genome of strain MK041244-CHN-2006 was used as the backbone, and penton based gene was replaced by gene fragment from MK041246-chn-2012 strain isolated from a child with acute lower respiratory tract infection in Hunan province in 2012.Conclusions:Our result indicated that 2014 Hunan strain(Hunan2014-s024)is a recombinant virus of 2006 Shanxi strain(MK041244-CHN-2006)and 2012 Hunan strain(MK041246-CHN-2012). Due to the change of pathogenicity, it is necessary to strengthen the molecular epidemiological surveillance of HAdV-C to understand its potential disease burden and public health significance.

Objective:To evaluate the effect of synthetic CpG oligodeoxynucleotide (CpG-ODN) as adjuvant on immune response induced by inactivated human adenovirus (HAdV)-55 antigen in BALB/c mice.Methods:HAdV-55 virus QS prototype strain was purified by plaque to construct a seed bank of vaccine candidate strain. The amplified product of vaccine candidate strain was inactivated by 0.05%β-propiolactone, and purified to prepare perfect virus particle antigen. The purified HAdV-55 antigen was mixed with the same volume CPG-ODN and aluminum hydroxide adjuvant in low-dose group (0.2 mg/ml) and high-dose group (1 mg/ml), respectively, and inoculated BALB/c mice after emulsification. Meanwhile, the control group was set with PBS, and the immunization was enhanced once every 21 days. Respectively on primary immune 21 and 35 days after collecting venous blood in mice and separation of serum, serum was collected at the end of the time of separating spleen lymphocytes in mice. The levels of HAdV-55 specific IgG antibody and neutralization antibody in serum of immunized mice were observed by ELISA and micro-neutralization test, and the levels of lymphocytes secreting IL-4 and IFN-γ cytokines were detected by ELISpot.Results:No matter with or without adjuvant, along with the increase of the number of immunization and vaccination dose of inactivated HAdV-55 antigen induced BALB/c mice virus specific IgG antibody was also significantly increased. However, neutralizing antibody can reach detectable level only after enhanced immunity, and the geometric mean titer (GMT) of neutralizing antibody is between 1: 11 and 1: 23. Different adjuvants have significant effects on the immune response of mice. Low dose antigen combined with CPG-ODN and aluminum hydroxide mixed adjuvant can induce higher humoral and cellular immune responses in mice, and the levels of specific IgG antibody and neutralizing antibody are 2.2 and 1.8 times higher than those in the aluminum hydroxide adjuvant group, respectively. The number of lymphocytes secreting IFN-γ was 2.3 times that of the group immunized with aluminum hydroxide adjuvant.Conclusions:The novel CPG-ODN adjuvant significantly increased the immunogenicity of the inactivated HAdV-55 whole virus antigen in BALB/c mice and directed the cellular immune response toward Th1 type.

COVID-19 is an emerging infectious disease caused by SARS-CoV-2. After the infection of the virus, the host immune system is stimulated to produce multifarious specific antibodies to decrease or eliminate effects of the pathogen. Study of the specific antibodies dynamic characteristics in patients with COVID-19 is very important for the understanding and diagnosis of the disease, research and development of vaccine, and planning of prevention and control strategy. This paper reviews and summarizes the domestic and oversea research on dynamic characteristics of specific antibodies of COVID-19 patients, including the antibody producing, duration and level, and its possible influencing factors in order to improve the understanding of the immunological characteristics of COVID-19.