Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance.

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/µL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.

[Analysis of infection status and pathogenic features of human metapneumovirus among children in Hangzhou between year 2009 and 2011].

OBJECTIVE: To study the infection status and pathogenic features of human metapneumovirus (hMPV) among children with acute respiratory tract infection in Hangzhou. METHODS: A total of 372 children less than 14 years old with acute respiratory tract infections were recruited as subjects from the pediatric clinic or intensive care unit (ICU) of 3 hospitals in Hangzhou during November 2009 to January 2010, and November 2010 to January 2011. A total of 372 specimens were collected, including 351 respiratory swab, 9 nasopharyngeal aspirate material, 8 endotracheal aspirate material and 4 sputum. The total nucleic acid was then extracted from the specimens, and the nucleoprotein (N) gene of hMPV was amplified by RT-PCR, whose positive products were sequenced and analyzed. Africa green monkey kidney cells (Vero-E6) were applied to culture hMPV among the positive samples; meanwhile fluorescence quantitative RT-PCR was adopted to test other respiratory virus infection. RESULTS: Out of 372 patients, 42 (11.2%) were positive for N gene of hMPV. The positive rate of hMPV among boys was 11.5% (26/226), and correspondingly 10.9% (16/146) among girls. The difference showed no statistical significance (χ(2) = 0.026, P > 0.05). The youngest patient was only 2 month-old and the eldest patient was 14 years old. The median of the patients' age was 24 months. Fifteen positive samples amplified by RT-PCR were sequenced, and all turned out to be subtype B1; whose similarity to GD165 found in Guangdong was 98.1% - 99.5% and similarity to BJ1897 in Beijing was 87.8% - 89.2%. The co-infection rate between hMPV and other respiratory virus was 45.2% (19/42); most of which was between hMPV and respiratory syncytial virus, whose rate at 26.1% (11/42). CONCLUSION: hMPV was the single genotype relevant with the acute respiratory tract infection disease among children in Hangzhou district; however, the co-infection with other respiratory virus did exist.

Two adenovirus serotype 3 outbreaks associated with febrile respiratory disease and pharyngoconjunctival fever in children under 15 years of age in Hangzhou, China, during 2011.

Adenovirus serotype 3 and 7 outbreaks have occurred periodically in northern, eastern, and southern China since 1955, but there has been no report since the adenovirus serotype 7 outbreak first occurred in Hangzhou, China, in 1991. Here we explored the epidemiology and etiology of two adenovirus serotype 3 outbreaks in Hangzhou in 2011. One acute respiratory outbreak was found in Chun'an County, where a total of 371 cases were confirmed in 5 of 23 towns from 4 to 31 May 2011. The outbreak affected 18.57% (13/70) of schools and 14.49% (90/621) of classes. The incidence was 5.18% (371/7,163). The population was distributed among individuals ages 7 to 15 years. No parents or teachers were infected. Another pharyngoconjunctival fever outbreak was discovered in the Chenjinglun Swimming Center located in the Xihu District between 1 and 15 July 2011. A total of 134 cases were confirmed in 900 amateur swimmers, with an incidence of 14.89% (134/900). The ages ranged from 4 to 9 years. The two outbreaks had no severe complications or death. The viruses in 66.67% (10/15) of throat swabs from children with acute respiratory infections and 100% (10/10) of the swabs from children with pharyngoconjunctival fever were confirmed to be adenovirus serotype 3 with 100% homology by PCR. Of these samples, 60.0% (12/20) had a classical characteristic cytopathic effect, presented as grape-like clusters at 72 h after infection in HEp-2 cells. In conclusion, the acute respiratory infection and pharyngoconjunctival fever outbreak in Hangzhou were caused by the completely homologous type 3 adenovirus in subgenus B. Moreover, these outbreaks demonstrated rapid transmission rates, possibly due to close contact and droplet transmission.

[Heterozygous genotypes and molecular characteristics of Organophosphorus resistance associated esterase B2 genes of Culex pipiens complex].

OBJECTIVE: To investigate the heterozygous genotype and molecular characteristics of Organophosphorus resistance associated with heterozygous Estbeta2 of esterase B2 gene from natural population of Culex pipiens complex. METHODS: Genomic DNA was extracted from natural populations of Culex pipiens complex in Hangzhou. The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase gene. Estbeta2 of esterase B2 gene was identified by PCR-RFLP, and the genotyping for heterozygous Estbeta2 was carried out after restriction enzyme digesting by Bfm I endonuclease. RESULTS: The DNA was isolated from 207 Culex pipiens respectively, while 156 PCR samples showed positive and the positive rate was 75.36% (156/207). The PCR-RFLP assay of esterase B2 gene revealed that the Estbeta2 was accounted about 28.20% (44/156) in 156 positive samples. There were two genotypes identified, namely homozygous Estbeta2 (90.90%, 30/33) and heterozygous Estbeta2 (9%, 3/33), heterozygous Estbeta2 was in existence of a hybrid form as which combined with Estbeta2 and a subtype (Estbeta2/Estbeta2(1)). CONCLUSION: Heterozygous Estbeta2 of Organophosphorus resistance associated with esterase genotype was determined in natural population of Culex pipiens, and a genotyping method was established.

Vibrio cholerae O139 multiple-drug resistance mediated by Yersinia pestis pIP1202-like conjugative plasmids.

A conjugative plasmid, pMRV150, which mediated multiple-drug resistance (MDR) to at least six antibiotics, including ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole, was identified in a Vibrio cholerae O139 isolate from Hangzhou, eastern China, in 2004. According to partial pMRV150 DNA sequences covering 15 backbone regions, the plasmid is most similar to pIP1202, an IncA/C plasmid in an MDR Yersinia pestis isolate from a Madagascar bubonic plague patient, at an identity of 99.99% (22,180/22,183 nucleotides). pMRV150-like plasmids were found in only 7.69% (1/13) of the O139 isolates tested during the early period of the O139 epidemic in Hangzhou (1994, 1996, and 1997); then the frequency increased gradually from 60.00% (3/5) during 1998 and 1999 to 92.16% (47/51) during 2000 to 2006. Most (42/51) of the O139 isolates bearing pMRV150-like plasmids were resistant to five to six antibiotics, whereas the plasmid-negative isolates were resistant only to one to three antibiotics. In 12 plasmid-bearing O139 isolates tested, the pMRV150-like plasmids ranged from approximately 140 kb to 170 kb and remained at approximately 1 or 2 copies per cell. High (4.50 x 10(-2) and 3.08 x 10(-2)) and low (0.88 x 10(-8) to 3.29 x 10(-5)) plasmid transfer frequencies, as well as no plasmid transfer (under the detection limit), from these O139 isolates to the Escherichia coli recipient were observed. The emergence of pMRV150-like or pIP1202-like plasmids in many bacterial pathogens and nonpathogens occupying diverse niches with global geographical distribution indicates an increasing risk to public health worldwide. Careful tracking of these plasmids in the microbial ecosystem is warranted.

Preparation of armored RNA as a control for multiplex real-time reverse transcription-PCR detection of influenza virus and severe acute respiratory syndrome coronavirus.

The common respiratory viruses, including influenza A, influenza B, and newly emerging severe acute respiratory syndrome (SARS) viruses, may cause similar clinical symptoms. Therefore, differential diagnosis of these virus pathogens is frequently required for single clinical samples. In addition, there is an urgent need for noninfectious and stable RNA standards and controls for multivirus detection. In this study, reverse transcription-PCR (RT-PCR) targeting of the RNAs of influenza A and influenza B viruses and SARS coronavirus was performed, and the resulting products were spliced into a fragment which was packaged into armored RNA for use as a noninfectious, quantifiable synthetic substitute. Furthermore, in the present study we developed a multiplex real-time RT-PCR assay in which the armored RNA was used as an external positive control and the three RNA viruses could be detected simultaneously in a single reaction mix. The detection limit of the multiplex real-time PCR was 10 copies/microl of armored RNA.

[Multiplex real-time PCR detecting Salmonella, Shigella and diarrheagenic Escherichia coli].

OBJECTIVE: To develop a multiplex real-time PCR for the detection of Salmonella invasion protein A gene (invA), enterotoxigenic Escherichia coli (ETEC) heat-labile I enterotoxin gene (elt), and Shigella or enteroinvasive E. coli (EIEC) invasive plasmid antigen H gene (ipaH). METHODS: Under the optimized reaction conditions of the multiplex real-time PCR, invA, elt, and ipaH were determined in 10-fold series of dilution of DNA extracted from Salmonella enterica serovar Typhimurium, ETEC 44815 strain and Shigella F301 strain. The three genes were examined in 90 fecal samples from diarrhea patients using the multiplex real-time PCR. When PCR-positive samples were found, the target strains were isolated and identified. RESULTS: The detectable concentration for this multiplex real-time PCR was 10 CFU/microl for Shigella F301 strain, 10(2) CFU/microl for S. enterica serovar Typhimurium and ETEC 44815 strain, respectively. Out of 90 fecal samples from diarrhea patients, thirteen were found positive for elt gene (14.4%), and five were found positive for ipaH gene (5.6%). Three E. coli strains positive for elt gene and four E. coli strains positive for ipaH gene were isolated successfully from the PCR-positive samples mentioned above. The detection of invA, elt and ipaH genes was completed in 10 h, which included an enrichment period of 6 h. CONCLUSION: The multiplex real-time PCR assay can detect invA, elt, ipaH simultaneously in a single reaction, moreover, it can detect for virulence genes in strains of Salmonella, ETEC, and Shigella or EIEC and screen these pathogens in fecal specimens from patients with diarrhea with a high specificity.

[Genotypes, allele frequencies and dynamic distribution on resistance-associated esterase genes of Culex pipiens complex in Hangzhou].

OBJECTIVE: To investigate the genotypes , allele frequencies and dynamic distribution on resistance associated esterase genes of Culex pipiens complex in Hangzhou. METHODS: The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase genes, and dynamic surveillance on frequencies of the resistance associated esterase gene of natural population of Culex pipiens complex in Hangzhou during 2003-2005, and phenotype of the resistance associated esterase genes were detected by esterase starch gel electrophoresis technique. RESULTS: The PCR-RFLP assay of esterase allele genes for three consecutive years disclosed four esterase genotypes, namely, the world-wide highly active homozygous Est beta 1(1) (50%-54%), homozygous Est beta 2 (29%-34%), heterozygous Est beta 1(1)/beta 2 (5%-10%) and Est beta N (3.13%) of a new homozygous genotype. The research of the resistance associated esterase genes phenotype in natural population of Culex pipiens complex in Hangzhou in 2005 with esterase starch gel electrophoresis technique revealed four major types, namely, Est beta 1(1) (61%), Est alpha 2/beta 2 (12%), Est alpha 8/beta 8 (7%) and sensitive phenotype (29%). CONCLUSION: There should be various resistance associated esterase genotypes in natural population of Culex pipiens complex in Hangzhou. During the period of 2003-2005, Est beta 1(1) was the major type; Est alpha 2/beta 2 was the second. Est beta N was a new esterase genotype detected in 2005 only with a mere percentage of 3.13%. As for its resistance to the new insecticide, a follow-up study should be needed. The molecular typing of the amplified esterase gene should be consistent with the resistance associated esterase genes phenotype.

Interim estimates of divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance (2010-2015) in Hangzhou, southeast of China.

OBJECTIVES: In the post-pandemic period 2010-2015, seasonal influenza A(H3N2) virus predominated in Hangzhou, southeast of China, with an increased activity and semi-annual seasons. This study utilized HA virus gene segment sequences to analyze the divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance in Hangzhou. METHODS: Virological and serological analyses of 124 representative A(H3N2) viruses from prospective studies of systematic surveillance samples were conducted to quantify the genetic and antigenic characteristics and their vaccine strain match. RESULTS: Bayesian phylogenetic inference showed that two separate subgroups 3C.3 and 3C.2 probably diverged from group 3C in early 2012 and then evolved into groups 3C.3a and 3C.2a, respectively, in the 2014/15 influenza season. Furthermore, high amino acid substitution rates of the HA1 subunit were found in A(H3N2) group 3C.2a variants, indicating that increased antigenic drift of A(H3N2) group 3C.2a virus is associated with a vaccine mismatch to the 2015/16 vaccine reference strain Switzerland/9715293/2013 (group 3C.3a). CONCLUSIONS: A portion of the group 3C.2a isolates are not covered by the current A(H3N2) vaccine strain. These findings offer insights into the emergence of group 3C.2a variants with epidemic potential in the imminent influenza seasons.

Progressive antigenic drift and phylogeny of human influenza A(H3N2) virus over five consecutive seasons (2009-2013) in Hangzhou, China.

Vaccine efficacy (VE) can be affected by progressive antigenic drift or any new reassortment of influenza viruses. To effectively track the evolution of human influenza A(H3N2) virus circulating in Hangzhou, China, a total of 65 clinical specimens were selected randomly from outpatients infected by A(H3N2) viruses during the study period from November 2009 to December 2013. The results of reduced VE and antigenic drift of the correspondent epitopes (C-D-E to A-B) suggest that the current vaccine provides suboptimal protection against the A(H3N2) strains circulating recently. Phylogenetic analysis of the entire HA and NA sequences demonstrated that these two genes underwent independent evolutionary pathways during recent seasons. The H3-based phylogenetic tree showed that a special strain A/Hangzhou/A289/2012 fell in a cluster among viruses with reduced VE predominantly circulating in 2013. Our findings underscore a possible early warning for the circulation of A(H3N2) variants with antigenic drift during the previous seasons.

Molecular evolution of HA gene of the influenza A H1N1 pdm09 strain during the consecutive seasons 2009-2011 in Hangzhou, China: several immune-escape variants without positively selected sites.

BACKGROUND: Even under immune pressure, the highly active influenza A H1N1 pdm09 variants emerged again in December 2010. Did the variability lead to poor vaccine effectiveness? OBJECTIVES: To study the genetic distance and antigenic drift of the influenza A H1N1 pdm09 strains based on the sequence analysis of HA virus gene segments during consecutive seasons 2009-2011 in Hangzhou, China. STUDY DESIGN: 39 Clinical samples from influenza-like-illness patients with culture-confirmed influenza A H1N1 pdm09 infections were collected over seasons in routine influenza surveillance. The HA gene was amplified and sequenced. A perspective analysis of genetic distance, antigenic drift and positively selected sites were conducted. RESULTS: Analyses of human influenza A H1N1 pdm09 strains isolated in Hangzhou revealed that during the seasons 2009-2011, the antigenic drift had occurred, away from the northern hemisphere 2010/2011 influenza vaccine strain A/California/07/2009. The 2010/2011 viruses cluster in two main branches with a significant genetic distance, characterized by either S202T and S468N, or K180T/I, V216A, P288S, I312V and I389F. Interestingly, the epitopes corresponding to the immune-escape characteristic have altered much, but none of the amino acid substitutions in 2010/2011 variants were positively selected. CONCLUSIONS: The results of genetic surveillance in this study might account for frequent outbreaks of the influenza A H1N1 pdm09 strains since December 2010 and the disappearance after three months circulation. It facilitates early detection of antigenic sites for the virus to escape immunological restraint in 2010/2011 season. Continuous monitoring of antigenic changes is recommended.

Antioxidant and antibacterial activity of six edible wild plants (Sonchus spp.) in China.

The total phenolic and flavonoid, antioxidant and antibacterial activities of six Sonchus wild vegetables (Sonchus oleraceus L., Sonchus arvensis L., Sonchus asper (L.) Hill., Sonchus uliginosus M.B., Sonchus brachyotus DC. and Sonchus lingianus Shih) in China were investigated. The results revealed that S. arvensis extract and S. oleraceus extract contained the highest amount of phenolic and flavonoid, respectively. Among the methanol extracts of six Sonchus species, S. arvensis extract exhibited the highest radical (DPPH and ABTS+ scavenging power and lipid peroxidation inhibitory power. It also exhibited the highest reducing power at 500 µg mL⁻¹ by A (700) = 0.80. The results of antibacterial test indicated that the S. oleraceus extract showed higher activity than the other five Sonchus wild vegetables extracts, both in Gram-negative bacteria (Escherichia coli, Salmonella enterica and Vibrio parahaemolyticus) and in a Gram-positive bacterium (Staphylococcus aureus). These results indicate that Sonchus wild food plants might be applicable in natural medicine and healthy food.

Anti-obesity and hypolipidemic effects of ethanolic extract from Alpinia officinarum Hance (Zingiberaceae) in rats fed high-fat diet.

To investigate the anti-obesity and hypolipidemic effects of Alpinia officinarum ethanolic extract (AOE) for the first time, anti-obesity models in vivo were used. Ten male Sprague-Dawley rats were fed normal control diet (NC); other groups of rats were fed a high-fat diet (HFD) with or without different proportions of AOE (AOE-1, 3%; AOE-2, 5%) for 6 weeks to examine feed intake, body and adipose tissue weight, serum total cholesterol (Total-C), triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and leptin levels, alanine aminotransferase (ALT) and aspartate aminotranferase activities, hepatic Total-C and TG levels, and the pathological changes in liver and epididymal adipose tissues. Interestingly, feed intakes among the experimental groups were not significantly different. Body weight gains were significantly lowered in the AOE-1 and AOE-2 groups compared with the HFD group (P < .05) and near to the level of the NC group. AOE also improved the lipid profile in serum and the pathological changes in liver and adipose tissue and decreased the relative weights of epididymal and perirenal white adipose tissues. They improved lipid profile by lowering serum Total-C, TG, and LDL-C concentrations, leptin content, and the atherogenic index compared with the HFD group. The HDL-C concentration and the ratio of HDL-C/Total-C significantly increased compared with those of the HFD group. The serum ALT activity of the AOE-2 group was notably lower than that of the HFD group. Our data suggest that AOE can be considered as an anti-obesity agent that is effective for suppressing body weight gain and decreasing lipid profile.

[Preparation of armored RNA containing M gene of influenza H3N2].

OBJECTIVE: To prepare the armored RNA containing M gene of influenza H3N2. METHODS: The vector pAR-1 was constructed from expression vector pET30b in which the bacteriophage MS2 DNA fragment, containing the genes for maturase and coat protein and the pac site, was inserted. The M gene fragment of influenza A was inserted into the HindIII site downstream of the pac site on the pAR-1, which formed a new recombinant plasmid pAR-2. After the prokaryotic expression was carried out, armored RNA AR-2 containing M gene was obtained. AR-2 was purified, and then was quantified by real time RT-PCR. Moreover, the stability of AR-2 was checked. RESULTS: AR-2 was expressed successfully. AR-2 remained stable under various storage environments. Approximately 8.9 x 10(11) copies of AR-2 particles can be purified from one milliliter of culture. CONCLUSION: It showed that AR-2 was stable and RNase-resistant, which, as a virus surrogate, would be used as RT-PCR standards, controls and training or proficiency samples.