The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes.

BACKGROUND: Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. METHODS: A GeXP-based multiplex RT-PCR assay (GeXP assay) was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay. RESULTS: The GeXP assay achieved a sensitivity of 20-200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51%) of the specimens. GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for the detection of 12 samples was completed within 2.5 hours. CONCLUSIONS: In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.

Current status of human adenovirus infection in China.

BACKGROUND: Outbreaks of severe, acute hepatitis among children have recently attracted global attention. The pathogen causing the outbreak remains unknown, but there is growing evidence that it may be associated with human adenovirus (HAdV). DATA SOURCES: A review of adenovirus-related clinical studies, epidemiological studies, etiological studies, and case reports was conducted by reviewers independently. RESULTS: HAdV can cause a wide variety of clinical symptoms. In the Mainland of China, HAdV infection accounts for 5.8%-13% of patients with acute respiratory infections, and these infections are mainly caused by species B, C, and E of HAdV. For acute conjunctivitis, 39.8%-74.9% of sporadic cases were infected by B and D species of HAdV. Outbreaks of keratoconjunctivitis and pharyngoconjunctival fever related to HAdV infection could be found throughout the country. In pediatric patients with acute gastroenteritis, HAdV-41 was the predominant HAdV type, followed by HAdV species B and C. Several types of HAdV, including HAdV-5, HAdV-7, HAdV-1, and HAdV-2, have previously been reported as potential pathogens associated with HAdV hepatitis in immunocompromised patients. However, few HAdV-related hepatitis cases have been reported in China to date. CONCLUSIONS: There are no systematic surveillance and clinical studies on HAdV hepatitis in China. Therefore, it is imperative to establish a nationwide HAdV virological surveillance system to collect relevant clinical, epidemiological and virological surveillance data and risk factor information as soon as possible to assess the potential risk of HAdV hepatitis among children.

Measles Virus IgG Avidity Assay for Use in Identification of Measles Vaccine Failures in Tianjin, China.

OBJECTIVE: To identify measles vaccine failures in Tianjin, China using a measles virus IgG avidity assay. METHODS: The China Information System for Disease Control and Prevention (CISDCP) was used to collect information about measles cases and blood specimens in Tianjin from 2013 to 2015. Measlesspecific IgM and IgG antibodies were detected using Enzyme-Linked Immunosorbent Assay (ELISA). Avidity testing for measles IgG was performed using a commercial enzyme immunoassay (EIA). RESULTS: A total of 284 confirmed measles cases were identified. Of this total, 262 (92.25%) were in patients aged ⪖ 20 years. High avidity was exhibited in 172 (60.56%) cases, while 80 (28.17%) cases demonstrated low avidity. High avidity was detected in only 21.43% of cases in patients aged < 1 year. The proportion of high avidity increased with age, and was significantly higher in patients aged 30-39 years at 70.07% (χ2 = 17.27, P = 0.002). Of the 52 measles cases in patients with a history of vaccinations, 41 (78.85%) cases showed high avidity, indicating secondary vaccine failures (SVF). In these vaccinations, there was no significant difference (P > 0.05) in clinical severity between high avidity and low avidity cases. However, regardless of vaccination status, clinical severity was significantly lower in high avidity cases (P < 0.001) than in low avidity cases. The percentages of positive measles IgM results in high avidity and low avidity cases were 66.28% and 91.25%, respectively. Geometric Mean Concentration (GMC) was significantly lower in high avidity cases at 33.73 U/mL, compared to 166.07 U/mL in low avidity cases. CONCLUSION: Low clinical severity and inconclusive IgM antibody results are more likely in high avidity measles cases. Measles cases were more common in adults. Therefore, a further dose of vaccines should be recommended for 30-39 years in Tianjin..

Human bocavirus in children suffering from acute lower respiratory tract infection in Beijing Children's Hospital.

BACKGROUND: Human bocavirus (HBoV) is a parvovirus recently found to possibly cause respiratory tract disease in children and adults. This study investigated HBoV infection and its clinical characteristics in children younger than five years of age suffering from acute lower respiratory tract infection in Beijing Children's Hospital. METHODS: Nasopharyngeal aspirates were collected from children suffering from acute lower respiratory tract infection during the winters of 2004 to 2006 (from November through the following February). HBoV was detected by polymerase chain reaction amplification and virus isolation and the amplification products were sequenced for identification. RESULTS: HBoV infection was detected in 16 of 333 study subjects. Coinfections with respiratory syncytial virus were detected in 3 of 16 HBoV positive patients with acute lower respiratory tract infection. The median age for HBoV positive children was 8 months (mean age, 17 months; range, 3 to 57 months). Among the HBoV positive children, 14 were younger than 3 years old, 9 were younger than 1 year old and 7 were younger than 6 months. These 16 positive HBoV children exhibited coughing and abnormal chest radiography findings and more than 60% of these children had wheezing and fever. Ten children were clinically diagnosed with pneumonia, 2 bronchiolitis, 2 acute bronchitis and 2 asthma. One child died. CONCLUSIONS: HBoV was detected in about 5% of children with acute lower respiratory infection seen in Beijing Children's Hospital. Further investigations regarding clinical and epidemiologic characteristics of HBoV infection are needed.

[Protective effects of gastrodin on the cellular model of Alzheimer's disease induced by Abeta25-35].

OBJECTIVE: To assess the effects of gastrodin (GAS) on the induced neurons of Alzheimer's disease (AD). METHODS: The cellular model of AD was established with primary cultured cerebral cortical and hippocampal cells induced by Abeta325-35. The total saponin ginseng was taken as positive control. The effects of gastrodin on the cellular model of AD were studied by morphological observation and biochemical methods. RESULTS: Compared with the control, the cellular model induced by Abeta25-35 showed remarkable morphological changes, significant increase in LDH release, and significant decrease in cell survival. When different doses of gastrodin, total saponin of ginseng were added beforehand, significant decrease in LDH release and significant increase in cell survival were noted by comparison with the experimental control, which indicated that the two medicines could protect the cells from the damage induced by Abeta25-35 to some extent. CONCLUSION: The results suggested that gastrodin has potential effects on the prevention and therapy of AD.

[Analysis the viral etiology of fever and respiratory tract infection syndrome in Shaanxi province].

OBJECTIVE: Analysis the viral pathogenic spectrum for patients with fever and respiratory tract infection syndrome in Shaanxi province during 2010 and investigate the molecular epidemiology characteristics of respiratory syncytial virus. METHODS: A total of 208 patients' pharyngeal swabs were collected based on surveillance definition from January 2010 to January 2011 and screened for sixteen human respiratory virus types/subtypes by Qiaxcel-based multiplex reverse transcription-PCR assay, including HRV,HCoV, Flu, HPIV, ADV, HRSV, HMPV and HBoV and investigate molecular epidemiology of HRSV by sequencing and phylogenetic analysis of the C-terminal second hypervariable region of the G gene. RESULTS: 109 out of 208 specimens (53%) were positive for one or more viruses. HRSV(42. 2%) was the dominant pathogen detected, followed by Flu(24. 5%), PIV(20%), HRV(13.6%) and ADV( 10.9%),there were also 8 strains of HCoV, 5 strains of HMPV and 3 strains of HBoV detected. The results showed that 22 specimens were positive for two or more viruses, PIV (14/22) was the most frequently detected viral agent among co-infection specimens, and the highest incidence of mixed infection is aged 15-39 years group (P < 0.05). The overall viral detection rate was no related to age. In addition to Flu, HMPV and PIV, other viruses (HRV, HBoV, HCoV, ADV, RSV) mainly infected 0 to 4 years old children. Among 46 HRSV positive specimens, 42 HRSV-A strains clustered into NA1 genotype and two HRSV-B strains clustered into two genotypes, BA9 and GB2. CONCLUSION: HRSV is the dominate pathogen collected from patients with fever and respiratory tract infection syndrome in Shaanxi and HRSV A is the predominant subtype. For most viruses, infection was most prevalent among children aged <4 years. PIV was the most common pathogen in co-infection.

[Characterization of human rhinovirus in children with acute respiratory infections in Gansu Province during 2011].

To study the epidemic characteristics of human rhinovirus (HRV) in children with acute respiratory infections in Gansu Province. 286 throat swabs were collected from children with acute respiratory in fections in Gansu Province during 2011. Multiplex reverse transcription-PCR (multiplex RT-PCR) assay was used to screen those specimens for detection of common respiratory tract pathogens. For HRV-positive samples, nested reverse transcription polymerase chain reaction (nested RT-PCR) was performed to amplify VP1 and VP4/VP2 gene fragments of HRV. The VP4/VP2 and VP1 regions of HRV-positive samples were sequenced and performed genotype analysis. Of 286 specimens fested, 27 were positive for HRV by multiplex RT-PCR and nested RT-PCR, of which 16 children were made (16/185), 8.64%) and 11 female (11/101,10.89%). The positive rate was 9.44% (27/286). The mean age of HRV-positive children was 3 years in this study, children less than one year old had the highest proportion 44.4% (12/ 27, 44.4%). The highest HRV positive rate fell on May, 2011 (6/27, 22.2%). Common cold accounted for the highest proportion, 12.24% (12/98) followed by pneumonia, 8.50% (13/153). The remaining 2 cases were bronchitis. Sequence analysis showed HRV A was the predominant genotype in Gansu Province in 2011, accounting for 84.62% (22/26) of positive cases, followed by HRV C (11.54%, 3/26) and only one HRV B was detected (3.85%, 1/26). HRV could be detected throughout the year in Gansu Province and primarily infected children under one year old. The group A was the epidemic genotype of HRV and move than one genotype existed in Gansu Province during 2011.

[Simultaneous detection of human parainfluenza viruses 1, 2, 3 by multiplex real-time reverse transcription-polymerase chain reaction with LNA probes].

OBJECTIVE: Human parainfluenza virus (HPIV) types 1, 2 and 3 are major viral pathogens responsible for upper and lower respiratory tract infections. In this study, a real-time RT-PCR was developed using multiplex primers-probe (HPIV-1, 2, 3) for the simultaneous detection of both HPIV1, HPIV2 and HPIV3 genomes. METHODS: Optimal primers and probes were designed using specialized software. The conditions for multiplex real-time RT-PCR had been optimized. The synthesis of RNA standards of HPIV1, 2, 3 were used a T7 RNA polymerase. Check the specificity sensitivities and stability of one step RT-PCR assay. RESULTS: Obtained in a 10-fold dilution series assay demonstrate a high sensitivity of the assay with a lowest detection limit of 10 copies for HPIV1, 100 copies for HPIV2 and 100 copies for HPIV3. CONCLUSION: The assays demonstrates an improved sensitivity and scope of detecting HPIV1, 2, 3 viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the pre-diagnosis and respiratory virus pathogenesis.

[Transmission and prevalence patterns of C4a evolutionary lineage of human enterovirus 71 circulating in mainland China, 2008-2010].

OBJECTIVE: To understand the evolutionary relationship between the C4a evolutionary lineage of human enterovirus 71 (HEV71) strains circulating in mainland of China during 2008-2010 and 2008 Fuyang strains and study the prevalence and transmission patterns of 2008 Fuyang strains. METHODS: Download all the complete VP1 ( > or = 891 bp) or approximate complete VP1 (> or = 876 bp) gene nucleotide sequences from GenBank of HEV71 strains circulating in Mainland of China during 2008-2010. And analyze the phylogenetic relationship between Fuyang strains and other provinces' strains using the MEGA software, version 5.0. RESULTS: All of the HEV71 isolates circulating in Mainland of China during 2008-2010 were clustered into evolutionary lineage C4a except for eight strains grouped in the genotype A and one isolate belongs to evolutionary lineage C4b; the homology analysis showed there were 96.5%-100% identity between C4a viruses circulating in mainland China during 2008-2010 and 2008 Fuyang strains, and they were evolved from C4b viruses of 1998. The transmission chains of Fuyang strains were mainly transmitted in Guangdong, Jiangsu, Shanghai, Hunan, Shandong provinces. CONCLUSION: The predominant viruses circulating in Mainland of China during 2008-2010 were evolutionary lineage C4a of human Enterovirus 71; Fuyang transmission chains mainly distributed in southern of China and the Central China around Anhui provinces.

[The study of human rhinovirus in infants with lower respiratory tract infections].

OBJECTIVE: We want to explore the harm degree of human rhinovirus in infants in Beijing area. METHODS: From May 2008 to September 2009, 240 nasopharyngeal aspirates were collected from the children and infants who were hospitalized and with lower respiratory tract infections. These specimens were screened for HRV by real-time reverse transcription PCR (RT-PCR) and statistically analysised. RESULT: In all of 240 hospitalized children, 208 cases were admission diagnosis of pneumonia, accounting for 86.67% (208/240), no deaths, the ratio of male and female patients was 1.93 : 1, and the collected samples reached to a maximum number in February 2009. Real-time PCR used to detect human rhinovirus, positive samples number is 71, positive rate is 29.58% (71/240), and the main symptoms and clinical diagnosis was pneumonia. Most cases were less than 2 years old, making up 81.69% (58/71), amony them, 13 months-18 months age and > or = 24 months groups have the highest incidence rates, the incidence rate is 33.33%. CONCLUSION: Human rhinovirus happened in spring and winter seasons, especially the infants who were under 2 years are the main infection groups, the important symptoms are lower respiratory infections such as pneumonia, bronchitis and bronchiolitis et al. Human rhinovirus is seasonal and contagious, spreads fast, so protective measures in hospitals should be prepared to avoid cross-infection.

[A GeXP based multiplex RT-PCR assay for simultaneous detection of twelve human respiratory viruses].

A GeXP based multiplex RT-PCR assay was developed to simultaneously detect twelve different respiratory viruses types/subtypes including influenza A virus, influenza B virus, influenza A virus sH1N1, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus, human metapneumovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B and human bocavirus. Twelve sets of specific primers were designed based on the conserved sequences of available respiratory-virus sequence database. The specificity of the multiplex system was examined by positive specimens confirmed previously. The sensitivity to detect twelve respiratory viruses simultaneously was 10(3) copies/microL. Twenty four clinical specimens were further detected by this novel assay and the results were compared with that of the real-time RT-PCR. These results showed that this novel assay based on GeXP is a fast, sensitive, and high throughput test for the detection of respiratory virus infections.

[Genetic characterization of measles virus caused the measles outbreak in Xinjiang].

OBJECTIVE: To analyze the genetic characterizations of wild type measles viruses caused the measles outbreak in Xinjiang. METHODS: Vero/Slam cell were used for measles viruses isolation from the specimens collected from measles outbreaks patients. Fragment of 676 nucleotide acids of the carboxylend of nucleoprotein gene were amplified using reverse transcription-polymerase chain reaction (RT-PCR) method and then the PCR products were directly sequenced and analyzed. Phylogenetic tree was constructed based on 450 nucleotide acids of the N-terminus of nucleoprotein gene, and homological analysis was performed at nucleotide acid level. RESULTS: 11 measles viruses were sequenced and all belonged to H1a subgenotype. The nucleotide difference was 0-0.2% between 11 Xinjiang isolates. And the nucleotide difference was 2.2%-2.4% between Xinjiang isolates and H1 genotype reference strain. CONCLUSION: The Measles viruses causing the measles outbreak in Xinjiang were H1a subgenotype.

[Comparison and evaluation of enzyme-linked immunization assay kits with plague reduction neutralization test for detection of measles IgG antibody].

OBJECTIVE: To evaluate and comparison of 2 commercial ELISA kits (Vrion/Serion kit and IBL kit) which ELISA kit will be used in sero-epidemiological survey in China in 2006 by Plaque Reduction Neutralization Test (PRNT). METHODS: 52 serum panel which contain different levels of measles antibody were used. RESULTS: The results showed that the sensitivity, PPV and accurate of Virion/Serion kit achieved highest score when taking PRNT as "gold standard". Virion/Serion kit showed good relationship with PRNT titer: the correlation coefficients are 0.878 (P<0.01). IBL kit get lower correlation coefficients 0.850 (P<0.01). The test value and mean of unit of serum antibody increased along with neutralizing titers increased. CONCLUSION: The Virion/Serion kit is good in sensitivity and specificity. It has quantitative for single well. The kit is suitable for sera epidemiology survey to detection of automatic workstation in China.

[The genetic characterization of enteric cytopathic human orphanviruses type 13 isolated in Shandong province].

OBJECTIVE: To analysis the genetic characteristics of Echo 13 isolated from stool specimens of AFP patient in Shandong. METHODS: The complete VP1 sequences of the 6 isolates were successfully amplified by RT-PCR and were compared with other Echovirus 13 isolates available from GenBank. RESULTS: 6 Shandong isolates were clearly identified as Echo 13 and were grouped into B cluster that agreed with neutralization result. The homogeneity analysis showed that the Shandong isolates share at least 80% similarity in nucleotide level and 94.4% in amino acid level. Phylogenetic tree base on complete VP1 sequence indicated there were many transmitting chains of Echovirus 13 prevailed in China. CONCLUSION: All Echo 13 isolates were divided into three genomic clusters A, B and C. It is suggested that enterovirus surveillance should be strengthened to prevent imported of Echo 13.

[Seroepidemical study of Coxsackievirus A 16, in four provinces, China, 2005].

OBJECTIVE: To study the situation of 1- 5-years-old children's antibody against Coxsackievirus A group 16 strain (CVA16) in Guangdong, Heilongjiang,Yunnan Province and Xinjiang Uygur Autonomous Regions, China, 2005, it can offer scientific evidences for preventing and controlling CVA16 causative hand-food and mouth disease. METHODS: Using microneutrilization test, to study 503 serum samples randomly selected from sera collected in 2005. RESULTS: Positive rate of anti-CVA16 antibody were 41.90%, 9.40%, 40.00% and 34.40% in Guangdong, Heilongjiang,Yunnan and Xinjiang, respectively. Antibody titer was relative low (average, 1: 6.1) and there was no statistical difference of geometry mean of antibody titer (GMT) among Guangdong, Heilongjiang, Yunnan (F = 0.97, 0.40, 1.06, respectively; P > 0.05), while there had statistical difference of GMT between Heilongjiang and other three regions( F = 10.61, P < 0.00). CONCLUSIONS: There had probably existed local epidemic in some regions of Guangdong, Heilongjiang, Yunnan Province and Xinjiang Uygur Autonomous Regions, China, 2005 or even before, but the area and degree of transmission and epidemic had difference. Children aged from 1- 5-years-old were relatively susceptible population of CVA16 infection.

[A serological survey of Epstein-Barr virus infection in children in Beijing].

OBJECTIVE: To understand the prevalence of Epstein-Barr virus (EBV) infection in urban and rural areas of Beijing using the serological method. METHODS: Totally 589 serum samples were collected from children in Beijing urban and rural areas who were 0--14 years old and tested with Viron-Seron ELISA classic EBV virus capsid antigen IgG antibody (EBV VCA IgG) kit. Optical density of serum samples was obtained at the wavelength of 405 nanometers. Sero-positive or negative samples were determined according to standard curve and cut-off attached in ELISA classic EBV VCA IgG kits. The activity of EBV VCA IgG was calculated by using special formula. The percentage and activity of EBV VCA IgG from Beijing children were compared with SPSS 13.0 between the urban and rural areas. RESULTS: The percentage of EBV VCA IgG seropositive samples was 83.6%, and 80.8% in those from urban and 86.2% in those from rural areas. The peak value of EBV infection was 71% seen among children under the age of 3 years, and in urban area the rate was 67.7%, which was lower than that in the rural area (75.3%), and was 82.5% by the age of 6, which was lower than the data (up to 90%) reported 30 years ago. There was a significant difference in EBV infection rate and VCA IgG activities in children at different ages between urban and rural areas (P < 0.05). CONCLUSION: The rate of EBV infection in children living in urban area was lower by the age of 6 years. The primary infection of EBV occurred late in part of children lived in urban area.

[Genetic characterization of Chinese rubella virus isolates from 2003 to 2007].

57 rubella virus strains were isolated using Vero cell line or Vero/SLAM cell line from patients' throat swabs during rubella outbreaks and sporadics in 10 provinces of China from 2003 to 2007. Fragments of 1107 nucleotides of E1 genes of the isolates were amplified by RT-PCR, the PCR products were directly sequenced and analyzed. The phylogenetic analysis based on 739 nucleotides showed that out of 57 Chinese rubella virus strains, 55 belong to a distinguish branch of 1E genotype when comparing with 1E genotype rubella strains from other countries, and the other 2 Chinese rubella virus strains belong to 2B genotype. Most of the nucleotide mutations of 57 rubella viruses were silent mutations, and the amino acid sequences were highly conserved. Except one amino acid change (Thr212 --> Ser212) in two rubella viruses at the hemagglutination inhibition and neutralization epitopes, there had no change found at the important antigenic epitope sites of the other rubella viruses. 1E genotype rubella viruses isolated from 10 provinces of China from 2003 to 2007, and two imported 2B genotype rubella viruses from Vietnam suggested that 1E genotype was the predominant genotype in this period of time. The rubella virus genotypes circulated during 2003 to 2007 were different from that circulating during 1979 to 1984 and 1999 to 2002, the rubella prevailed in recent years was mainly caused by 1E genotype rubella viruses with multi-transmission routes.