An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens.

OBJECTIVE: To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. METHODS: Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. RESULTS: NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events. CONCLUSION: The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

[Detection of respiratory viruses in influenza-like illness in Shijiazhuang, China in 2011].

This study aimed to investigate viral infections and the prevalence of influenza-like illness (ILI) in Shijiazhuang, China, in 2011 and to provide a scientific basis for the diagnosis and control of respiratory tract infections. Throat swab specimens were collected from 483 cases of ILI who were outpatients in the influenza surveillance sentinel hospitals in Shijiazhuang between January and December 2011. All specimens were examined by multiplex RT-PCR for the following 15 respiratory tract viruses: adenovirus (ADV), human rhinovirus (HRV), human parainfluenza virus (PIV types 1-4), influenza virus A (FluA), influenza virus B (FluB), human enterovirus (HEV), respiratory syncytial virus (RSV-A and -B), human metapneumovirus (HMPV), human coronavirus (HCoV-229E/NL63 and -OC43/HKU1), and human bocavirus (HBoV). Among the 483 cases of ILI, 214 (44.31%) were positive for viruses, including ADV (8.7%), HEV (8.7%), RSV-A (8.07%), HRV (7.45%), FluA (5.38%), HCoV-OC43/ HKU1 (2.9%), PIV-3 (2.9%), HMPV (1.86%), PIV-1 (1.24%), HCoV-229E/NL63 (1.04%), PIV-2 (1.04%), HBoV (0.83%), and FluB (0.41%). Twenty-six (5.38%) of all cases were co-infected with two or more viruses, most commonly HEV/HRV with other viruses. Cases of viral infection were detected throughout the year, with peaks in January and February. ADV and HRV were detected throughout almost the whole year without obvious seasonality. HEV was detected between April and November, with a peak of prevalence in summer and autumn. FluA and FluB reached epidemic levels mainly in winter and spring. All cases of RSV were identified to be subtype A. PIV infection was mainly caused by PIV-3. The positive rate of HCoV-OC43/HKU1 infection was significantly higher than that of HCoV-229E/NL63. The leading five viruses that resulted in ILI Shijiazhuang in 2011 were HEV, ADV, RSV-A, HRV, and FluA, and these viruses have different epidemiological features.

Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction for the detection of human enterovirus 71 subgenotype C4 in nasopharyngeal swab specimens.

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID(50)) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China.

Application of an autoregressive integrated moving average model for predicting the incidence of hemorrhagic fever with renal syndrome.

The Box-Jenkins approach was used to fit an autoregressive integrated moving average (ARIMA) model to the incidence of hemorrhagic fever with renal Syndrome (HFRS) in China during 1986-2009. The ARIMA (0, 1, 1) × (2, 1, 0)(12) models fitted exactly with the number of cases during January 1986-December 2009. The fitted model was then used to predict HFRS incidence during 2010, and the number of cases during January-December 2010 fell within the model's confidence interval for the predicted number of cases in 2010. This finding suggests that the ARIMA model fits the fluctuations in HFRS frequency and it can be used for future forecasting when applied to HFRS prevention and control.

Effectiveness of a Chinese hamster ovary cell derived hepatitis B vaccine in Chinese rural communities.

OBJECTIVE: To evaluate the effectiveness of a Chinese hamster ovary cell derived hepatitis B vaccine in a country community in Hebei Province, PR China. METHOD: A cross-sectional investigation was carried out in 4 of 7 randomly selected country communities in Zhengding County in 2005. All of the children who were born between 1997 and 1999 were selected as study objects. Their serum samples were taken to test for HBV markers, and HBsAg prevalence was compared to that of the same age group before hepatitis B vaccination in 1983. In addition, for HBsAg positive children, their mothers were visited and tested for serum HBV markers, in order to distinguish maternal HBV transmissions. RESULTS: Among the 2205 children of the selected birth cohort, 1696 (76.9%) were visited. The prevalence of HBsAg was 0.53%, and by comparing to that of before hepatitis B vaccination, the effectiveness was 95.3%, similar to that of yeast derived hepatitis B vaccines. Among 7 mothers of HBsAg positive children, 5 were HBsAg positive, indicating maternal HBV transmissions; and although one mother was HBsAg negative, her kid was not vaccinated, which indicates a horizontal transmission. As for the other kid, he was adopted and the HBV infection status of his birth mother was unknown. CONCLUSION: The effectiveness of the CHO derived hepatitis B vaccine is comparable to yeast derived ones, and after the hepatitis B vaccination maternal transmission is the most important route of spreading HBV.

[Study on the epidemiological of rotavirus diarrhea in Lulong in 2008-2009].

OBJECTIVE: To analyze the feature of epidemiological of rotavirus diarrhea in Lulong county, Hebei province. METHODS: 426 stool specimens were collected from inpatant with acute diarrhea from children less than 5 years old. Rotavirus-positive specimens were identified by ELISA kit. G/P typing assays were confirmed with multiplex seminested RT-PCR. RESULTS: Rotavirus was detected in 202 of 426 (47.42%) specimens. Genotyping of rotavirus showed that G3 was predominant (57.9%), followed by Gmix (16.3%), G9 (14.9% ), G1 (7.9%), G4 (1%), G2 (0.5%), P-genotyping showed that P [8], Pmix, P [4], P [9], type were found in 58.4%, 28.7%, 6.9% and 1% respectively. The most common G/P combination identified was G3P [8]. CONCLUSION: Group A rotaviruses was a major pathogen of diarrhea in Children in Lulong. G3P [8] was the predominant type in 2009, Gmix and Pmix abound, and G9 serotypes has become the second predominant after G3 strain in the region.

[12 years immunological effects of hepatitis B vaccine made by recombinant deoxyribonucleic acid techniques in Chinese hamster ovary cell].

OBJECTIVE: To understand the long-term immunological effects of a recombined hepatitis B vaccine [HepB made by recombinant deoxyribonucleic acid (DNA) techniques in Chinese hamster ovary (CHO) cell, HepB (CHO)]. METHOD: from 1997 to 1999 all newborns in Zhengding County were administered to HepB (CHO)at birth. Children in 3 of all 7 towns in the county were selected as study objects to check their hepatitis B vaccination history and test for HBV markers. RESULTS: Children who aged 10, 11 and 12 years after immunization, the anti-HBs positive rates were 71.0%, 72.1% and 68.2% respectively, and the geometric mean concentration (GMCs)were 197.9 mIU/ml, 296.0 mIU/ml and 158.0 mIU/ml respectively; Anti-HBs GMCs were 197.9 mIU/ml, 296.0 mIU/ml and 158.0 mIU/ml respectively. The Average GMC was 207.9 mIU/ml. HBsAg and anti-HBc positive rates were 0.50% and 2.26% respectively. CONCLUSION: The long-term immunological effects of HepB (CHO) are excellent.

[Virological surveillance and molecular characteristics of influenza B viruses in Hebei Province during 2004-2008].

OBJECTIVE: To understand influenza B viruses circulating and antigenic variation and distributing in Hebei province during 2004-2008. METHODS: Viruses were isolated with Madin-Darby canine kidney (MDCK) cells. Viral RNA was extracted from isolates after serological identification and transcribed into cDNA by reverse transcript and ampified by PCR. The PCR product was purified and nucleotide sequence of HA1 gene of the influenza B viruses was analyzed. RESULTS: 131 influenza B viruses were obtained from October 2004 to March 2008. Among them, 48 strains were Victoria-lineage and 83 strains were Yamagata-lineage viruses. Yamagata-lineage viruses were detected in the 2004-2005, replaced by Victoria-lineage viruses in the 2005-2006. Both Victoria-lineage and Yamagata-lineage viruses were isolated in 2006-2007 and 2007-2008. There were several amino acids substitution in both Victoria and Yamagata lineages according to the result of sequence analysis of the hemagglutinin HA1 genes, and Yamagata lineages viruses took more frequently substitution in isolates. Influenza B viruses in 2008 were differed by 8 amino acids from virus stains, in 2005 with homology of 97.4%-98.0%. CONCLUSION: The antigenicity of Victoria-lineage virus didn't take place varidation apparently in Hebei provinece during 2004-2008, but Yamagata-lineage virus took drift and caused outbreak of influenza in the epidemic season of 2007-2008.

[Investigation on the source of the first human of avian influenza A (H5N1) case in Beijing].

OBJECTIVE: To investigate the source of the first human case of avian influenza A (H5N1) infection in Beijing. METHODS: Interviewing the relatives of the case and other key persons, collecting and detecting samples of related biological, epidemiological and environmental data of the case were conducted. Later, the infection source was thoroughly investigated. RESULTS: The case ever contacted a slaughtered duck 5 days prior to the onset of illness, and the duck was bought from a stall of a wet market in Yanjiao area of Hebei province. Ten environmental samples were collected in this stall and the neighboring stall of the market. Another 6 samples were tested positive for H5N1 virus by PCR method, with 5 virus strains isolated. The whole-genome sequencing indicated that the amino acid homology between the H5N1 virus strains from the environment and the virus isolated from the case reached 99.8% - 100%. CONCLUSION: From both epidemiological and virological evidence, it was proved that the first human case of avian influenza A (H5N1) infection in Beijing was infected by a duck that carrying H5N1 virus the case contacted 5 days proceeding the onset of illness.

[Evaluation of 5 kinds of China-made enzyme immuoassay kits for antibody to hepatitis B virus core antigen detection].

OBJECTIVE: To Screen an EIA kit that fits best for using in serological detection of anti-HBc in the national investigation of hepatitis B virus infection. METHODS: Top 5 kinds of kits on the best seller list of China-made anti-HBc EIA kit were selected and each detect the anti-HBc panel (95 positive and 57 negative) 5 times. The co/s ratio of each test was recorded and transformed by Ig(co/s+1). Reliability indices ICC and CV, and validity indices AUC, pAUC and Se(FPR=e) were calculated and compared between each kit. RESULTS: The indices of ICC and CV of the 5 kinds of kits arranged in the same order of A, B, D, C and E, from the best to the worst. And the differences between them were significantly important (Bootstrap method, P < 0.05), except for between kits B and D in CV index; The AUCs ranged from 0.991 to 0.997; For index pAUCs, differences were significant between kits B, D, C ,which had the biggest values, kits A and E which had the lowest ones. For index Se(FPR=e), significant differences were only seen between kits B and A, and B and E (Bootstrap method, P < 0.05). CONCLUSION: The diagnostic ability of all the 5 kinds of kits arrange in an order of B, C, D, A and E, from the best to the worst.

[A nested case control study for risk factors impacting hepatitis B vaccination in Hebei, China].

OBJECTIVE: Study the risk factors that impact the effectiveness of mass hepatitis B vaccination, and try to amend them in the future. METHOD: Based on the national surveillance of hepatitis B, all the 1734 of 1-15 years old children from Hebei Province were enrolled in the present study and they were divided into case and control group according to their sera HBsAg were positive or not. RESULTS: Mother sera HBsAg positive and the hospital the children were born and earlier year of birth were risk factors. CONCLUSION: The effectiveness of mass neonate hepatitis B vaccination has greatly improved and the future focus should be on finding pregnant HBsAg positive women, and encourage them to give birth in better hospitals, and at the mean time, try to make the neonate hepatitis B vaccination perfect, especially in country areas.

[Genotype and sequence analysis on G2 segments of hantavirus from HFRS patients in Hebei Province].

OBJECTIVE: To know the genotype and subtype of hantavirus (HV) which infected persons in Hebei province. METHODS: According to G2 coding region of 76-118 and R22 strains, specific type primers were designed to detect and identity the types of HV in HFRS patients' sera with RT-nested PCR. Nucleotides were assayed from partial products after purification and reclaim. Then, gene analysis was done with DNAStar package. RESULTS: 17 out of 69 positive serum specimens were successfully detected by RT-PCR and the detection rate was 24.64%, among which, or= 14 days were 0. 17 positive specimens were all belonged to SEO. The nucleotide homology of 9 typical specimens was 92.0%-100%. Between HeB7 and other 8 specimens was 92%-95%, and they belonged to different subtypes. When HeB7 compared with R22 strain, it was 97.7%. HeB7 and R22 belonged to S1 subtype. The 8 specimens except HeB7 was 95.7%-100% and they all belonged to S3 subtype. When compared with 76-118 strain, 9 specimens' nucleotide homology was only 70.3%-72.7%, belonged to different type. CONCLUSION: SEO was the major type of HV from HFRS patients in Hebei province, S3 was the major subtype and S1 was also existed. In a certain area, the HV which belonged to the same type was correspondingly conservative, and had the characteristic of regional stability.

H2 strain attenuated live hepatitis A vaccines:protective efficacy in a hepatitis A outbreak.

AIM:To investigate the protective efficacy of H2 strain attenuated live hepatitis A vaccines (H2-strain vaccines) in hepatitis A (HA) outbreaks.METHODS:With the permission of their parents, 5551 pre-school and grade 1-3 primary school children were inoculated with 1 dose (10(6.5) TCID(50)) of H2 strain vaccines in a nonrandomized, controlled trial conducted in Fucheng County, Hebei Province in May 1997.Another 6485 children in the same grades and compatible in gender and age were enrolled as controls. Epidemiological and serological survey was conducted to evaluate the protective efficacy of the vaccines. ELISA was used to detect serum IgM anti-HAV.RESULTS:HA outbreak started in early May 1998, peaked in the middle of the same month, and lasted about 80 days. Overall 302 HA cases were found, 192(63.58%) were 5-9 years old. One vaccinee and 25 control cases were found to have hepatitis A, which account for 0.28% (1/356) and 5.92% (25/422) of all vaccinees and controls in the 14 villages, respectively. The protective efficacy of vaccines was 95.27% (95% CI: 85.83%-104.72%). In subjects tested for anti-HAV IgM from 13 villages, 1(0.40%) overt and 11(4.06%) asymptomatic HAV cases were found in 271 vaccinees but 21(6.69%) of overt and asymptomatic ones were found in 314 controls.CONCLUSION:H2 strain vaccines were excellent in preventing overt hepatitis A,but not so effective in preventing asymptomatic hepatitis A virus infection.A booster dose might be needed to get permanent reliable immunity.