Dynamic PB2-E627K substitution of influenza H7N9 virus indicates the in vivo genetic tuning and rapid host adaptation.

Avian-origin influenza viruses overcome the bottleneck of the interspecies barrier and infect humans through the evolution of variants toward more efficient replication in mammals. The dynamic adaptation of the genetic substitutions and the correlation with the virulence of avian-origin influenza virus in patients remain largely elusive. Here, based on the one-health approach, we retrieved the original virus-positive samples from patients with H7N9 and their surrounding poultry/environment. The specimens were directly deep sequenced, and the subsequent big data were integrated with the clinical manifestations. Unlike poultry/environment-derived samples with the consistent dominance of avian signature 627E of H7N9 polymerase basic protein 2 (PB2), patient specimens had diverse ratios of mammalian signature 627K, indicating the rapid dynamics of H7N9 adaptation in patients during the infection process. In contrast, both human- and poultry/environment-related viruses had constant dominance of avian signature PB2-701D. The intrahost dynamic adaptation was confirmed by the gradual replacement of 627E by 627K in H7N9 in the longitudinally collected specimens from one patient. These results suggest that host adaptation for better virus replication to new hosts, termed "genetic tuning," actually occurred in H7N9-infected patients in vivo. Notably, our findings also demonstrate the correlation between rapid host adaptation of H7N9 PB2-E627K and the fatal outcome and disease severity in humans. The feature of H7N9 genetic tuning in vivo and its correlation with the disease severity emphasize the importance of testing for the evolution of this avian-origin virus during the course of infection.

Newly Emergent Highly Pathogenic H5N9 Subtype Avian Influenza A Virus.

UNLABELLED: The novel H7N9 avian influenza virus (AIV) was demonstrated to cause severe human respiratory infections in China. Here, we examined poultry specimens from live bird markets linked to human H7N9 infection in Hangzhou, China. Metagenomic sequencing revealed mixed subtypes (H5, H7, H9, N1, N2, and N9). Subsequently, AIV subtypes H5N9, H7N9, and H9N2 were isolated. Evolutionary analysis showed that the hemagglutinin gene of the novel H5N9 virus originated from A/Muscovy duck/Vietnam/LBM227/2012 (H5N1), which belongs to clade 2.3.2.1. The neuraminidase gene of the novel H5N9 virus originated from human-infective A/Hangzhou/1/2013 (H7N9). The six internal genes were similar to those of other H5N1, H7N9, and H9N2 virus strains. The virus harbored the PQRERRRKR/GL motif characteristic of highly pathogenic AIVs at the HA cleavage site. Receptor-binding experiments demonstrated that the virus binds α-2,3 sialic acid but not α-2,6 sialic acid. Identically, pathogenicity experiments also showed that the virus caused low mortality rates in mice. This newly isolated H5N9 virus is a highly pathogenic reassortant virus originating from H5N1, H7N9, and H9N2 subtypes. Live bird markets represent a potential transmission risk to public health and the poultry industry. IMPORTANCE: This investigation confirms that the novel H5N9 subtype avian influenza A virus is a reassortant strain originating from H5N1, H7N9, and H9N2 subtypes and is totally different from the H5N9 viruses reported before. The novel H5N9 virus acquired a highly pathogenic H5 gene and an N9 gene from human-infecting subtype H7N9 but caused low mortality rates in mice. Whether this novel H5N9 virus will cause human infections from its avian host and become a pandemic subtype is not known yet. It is therefore imperative to assess the risk of emergence of this novel reassortant virus with potential transmissibility to public health.

Influenza H7N9 and H9N2 viruses: coexistence in poultry linked to human H7N9 infection and genome characteristics.

UNLABELLED: Avian influenza virus A of the novel H7N9 reassortant subtype was recently found to cause severe human respiratory infections in China. Live poultry markets were suspected locations of the human H7N9 infection sources, based on the cases' exposure histories and sequence similarities between viral isolates. To explore the role of live poultry markets in the origin of the novel H7N9 virus, we systematically examined poultry and environmental specimens from local markets and farms in Hangzhou, using real-time reverse transcription-PCR (RT-PCR) as well as high-throughput next-generation sequencing (NGS). RT-PCR identified specimens positive for the H7 and N9 genomic segments in all of the 12 poultry markets epidemiologically linked to 10 human H7N9 cases. Chickens, ducks, and environmental specimens from the markets contained heavily mixed subtypes, including H7, N9, H9, and N2 and sometimes H5 and N1. The idea of the coexistence of H7N9 and H9N2 subtypes in chickens was further supported by metagenomic sequencing. In contrast, human H7N9 infection cases (n = 31) were all negative for H9N2 virus according to real-time RT-PCR. The six internal segments were indistinguishable for the H7N9 and H9N2 viruses. The H9, N2, and internal-segment sequences were very close to the sequence of the H9N2 virus circulating in chickens in China recently. Our results provide direct evidence that H9N2 strains coexisted with the novel human-pathogenic H7N9 influenza virus in epidemiologically linked live poultry markets. Avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus and continues to do so. IMPORTANCE: Our results suggest that avian influenza A virus of the H9N2 subtype likely made a recent contribution to the evolution of the H7N9 virus, a novel reassortant avian influenza virus A subtype, and continues to do so. The finding helps shed light on how the H7N9 virus emerged, spread, and transmitted to humans. It is of considerable interest for assessing the risk of the possible emergence of novel reassortant viruses with enhanced transmissibility to humans.

Epidemiologic characterization of 30 confirmed cases of human infection with avian influenza A(H7N9) virus in Hangzhou, China.

BACKGROUND: We examined the clinical and epidemiological characteristics of 30 cases of human infection with avian influenza A(H7N9) virus in Hangzhou and investigated their external environments to provide evidence for contact tracing and disease prevention and control. METHODS: The cases confirmed from April 1 through May 1, 2013 were studied. Field epidemiologic surveys were conducted to collect the clinical and epidemiologic data. Case-related and environmental specimens were collected for etiologic detection. RESULTS: Thirty cases of human infection with avian influenza A(H7N9) virus were confirmed in Hangzhou from April 1 through May 1, 2013, including one pregnant woman and three deaths. The median age of the patients was 62 years (range: 38-86 years). Twenty-three of the patients were men (76.67%). The median duration between disease onset and occurrence of respiratory failure and confirmed diagnosis was 5 and 6 days, respectively. Maximum medical observation of 666 close contacts of the patients revealed no irregularity. Of 314 external environmental specimens, the overall positive detection rate of H7N9 nucleic acid was 28.98%. Eight districts of Hangzhou city had positive detections in the external environments, the highest rate being in Yuhang District (78.13%). Statistical analysis of the specimen collection locations indicates a significant difference between the case-linked locations and the non-case locations (χ2 = 16.563, p < 0.05) in terms of H7N9 viral nucleic acid detection rate. No epidemiologic link has been found among the 30 cases. CONCLUSIONS: Most of the infected were retired individuals aged 60 years or older. Men made the majority. The cases are sporadic at present, with no evidence of human-to-human transmission. Exposures to poultry and live poultry markets may be important sources of infection.

Control of RNA stability by NrrF, an iron-regulated small RNA in Neisseria gonorrhoeae.

Regulation of gene expression by small noncoding RNAs (sRNAs) plays a critical role in bacterial response to physiological stresses. NrrF, a trans-acting sRNA in Neisseria meningitidis and Neisseria gonorrhoeae, has been shown in the meningococcus to control indirectly, in response to iron (Fe) availability, the transcription of genes encoding subunits of succinate dehydrogenase, a Fe-requiring enzyme. Given that in other organisms, sRNAs target multiple mRNAs to control gene expression, we used a global approach to examine the role of NrrF in controlling gonococcal transcription. Three strains, including N. gonorrhoeae FA1090, an nrrF deletion mutant, and a complemented derivative, were examined using a custom CombiMatrix microarray to assess the role of this sRNA in controlling gene expression in response to Fe availability. In the absence of NrrF, the mRNA half-lives for 12 genes under Fe-depleted growth conditions were longer than those in FA1090. The 12 genes controlled by NrrF encoded proteins with biological functions including energy metabolism, oxidative stress, antibiotic resistance, and amino acid synthesis, as well as hypothetical proteins and a regulatory protein whose functions are not fully understood.

Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress.

The incidence of fluoroquinolone-resistant Shigella strains has risen rapidly, presumably in response to ciprofloxacin antibiotic stress. Understanding the molecular mechanisms underlying this resistance phenotype is critical to developing novel and effective therapeutic strategies. In this study, the frequency of ciprofloxacin-induced mutation was measured in antibiotic resistance genes (gyrA, gyrB, parC, parE, marOR, and marA) of Shigella flexneri. The S. flexneri 2a strain 301 was cultured on Luria-Bertani agar plates containing one of seven different ciprofloxacin concentrations (range: 0.03125-2 µg mL(-1)). Resistant colonies were selected for gene-targeted sequencing analysis; the identified point mutations were subsequently confirmed by insertion into antibiotic cassette plasmids and growth under ciprofloxacin stress. The results demonstrated that the seven different ciprofloxacin concentrations produced dose-dependent frequencies of spontaneous mutations: 10(-8) (0.03125 and 0.0625 µg mL(-1)), 10(-9) (0.125 µg mL(-1)), and <10(-9) (0.25, 0.5, 1, 2 µg mL(-1)). PCR sequencing of the ten randomly selected resistant colonies (minimum inhibitory concentrations (MICs) of 0.125 µg mL(-1), n = 5 and 0.25 µg mL(-1), n = 5) revealed that all colonies had mutations in the gyrA gene at either codon 83 (Ser83 → Leu) or 87 (Asp87 → Tyr or → Gly), both of which were confirmed at MIC of 0.125 µg mL(-1). None of the spontaneous mutation colonies exhibited gyrB, parC, parE, marOR, or marA mutations. In conclusion, S. flexneri is normomutable under ciprofloxacin antibiotic stress and fluoroquinolone resistance by spontaneous mutation occurs at a low rate. Codon mutations gyrA 83 and/or gyrA 87 cause a 4-fold increase in the ciprofloxacin MIC, and may represent the natural mechanism of fluoroquinolone resistance.

Population genomics implies potential public health risk of two non-toxigenic Vibrio cholerae lineages.

Diarrheal cases caused by non-toxigenic Vibrio cholerae have been reported globally. Lineages L3b and L9, characterized as ctxAB-negative and tcpA-positive (CNTP), pose the highest risk and have caused long-term epidemics in different regions worldwide. From 2001 to 2018, two waves (2001-2012 and 2013-2018) of epidemic caused by non-toxigenic V. cholerae occurred in the developed city of Hangzhou, China. In this study, through the integrated analysis of 207 genomes of Hangzhou isolates from these two waves (119 and 88) and 1573 publicly available genomes, we showed that L3b and L9 lineages together caused the second wave as had happened in the first wave, but the dominant lineage shifted from L3b (first wave: 69%) to L9 (second wave: 50%). We further found that the genotype of a key virulence gene, tcpF, in the L9 lineage during the second wave shifted to type I, which may have enhanced bacterial colonization in humans and potentially promoted the pathogenic lineage shift. Moreover, we found that 21% of L3b and L9 isolates had changed to predicted cholera toxin producers, providing evidence that gain of complete CTXφ-carrying ctxAB genes, rather than ctxAB gain in pre-CTXφ-carrying isolates, led to the transition. Taken together, our findings highlight the possible public health risk associated with L3b and L9 lineages due to their potential to cause long-term epidemics and turn into high-virulent cholera toxin producers, which necessitates a more comprehensive and unbiased sampling in further disease control efforts.

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.

[Molecular characteristics and antibiotic resistances of Vibrio cholerae O1 isolates in Hangzhou in 2009].

OBJECTIVE: To study the molecular characteristics and antibiotic resistances of Vibrio cholerae (V. cholerae) O1 isolates in Hangzhou in 2009. METHODS: The virulence genes ctxA and tcpA of the thirty V. cholerae O1 isolates from 7 counties and districts of Hangzhou were detected by PCR. Pulsed-field gel electrophoresis (PFGE) was performed for molecular typing and similarity analysis. Antibiotic resistances of these isolates were measured by the Kirby-Bauer method. RESULTS: Virulence gene analysis showed that 80.00% (24/30) of the genotype in V. cholerae isolates was ctxA- and tcpA+, 13.33% (4/30) was ctxA- and tcpA-, and 6.67% (2/30) was ctxA+ and tcpA+. Twenty-seven isolates tested were typed into 11 PFGE patterns (P1-P11). Twenty-three isolates with genotype ctxA- and tcpA+ were clustered into 7 PFGE patterns (P1-P7, termed P1-like cluster) with the similarity to be equal or greater than 91.4%, and 56.52%(13/23) of them belong to P1. 7 isolates with very high similarity (97.6%), belonging to P1 (6 isolates), and P2 (1 isolate), respectively, were collected from one foodborne disease outbreak. The resistant rates of the 24 isolates with genotype ctxA- and tcpA+ to ampicillin, tobramycin and amikacin were 20.83% (5/24), 4.17% (1/24) and 4.17% (1/24), respectively. CONCLUSION: The genotype of the epidemic strains of V. cholerae O1 isolates in Hangzhou in 2009 with high similarity was ctxA- and tcpA+; The level of drug resistances of this kind of V. cholerae O1 isolates were not high.

Composition and Dynamics of H1N1 and H7N9 Influenza A Virus Quasispecies in a Co-infected Patient Analyzed by Single Molecule Sequencing Technology.

A human co-infected with H1N1 and H7N9 subtypes influenza A virus (IAV) causes a complex infectious disease. The identification of molecular-level variations in composition and dynamics of IAV quasispecies will help to understand the pathogenesis and provide guidance for precision medicine treatment. In this study, using single-molecule real-time sequencing (SMRT) technology, we successfully acquired full-length IAV genomic sequences and quantified their genotypes abundance in serial samples from an 81-year-old male co-infected with H1N1 and H7N9 subtypes IAV. A total of 26 high diversity nucleotide loci was detected, in which the A-G base transversion was the most abundant substitution type (67 and 64%, in H1N1 and H7N9, respectively). Seven significant amino acid variations were detected, such as NA:H275Y and HA: R222K in H1N1 as well as PB2:E627K and NA: K432E in H7N9, which are related to viral drug-resistance or mammalian adaptation. Furtherly, we retrieved 25 H1N1 and 22 H7N9 genomic segment haplotypes from the eight samples based on combining high-diversity nucleotide loci, which provided a more concise overview of viral quasispecies composition and dynamics. Our approach promotes the popularization of viral quasispecies analysis in a complex infectious disease, which will boost the understanding of viral infections, pathogenesis, evolution, and precision medicine.

Genomic epidemiology of Vibrio cholerae reveals the regional and global spread of two epidemic non-toxigenic lineages.

Non-toxigenic Vibrio cholerae isolates have been found associated with diarrheal disease globally, however, the global picture of non-toxigenic infections is largely unknown. Among non-toxigenic V. cholerae, ctxAB negative, tcpA positive (CNTP) isolates have the highest risk of disease. From 2001 to 2012, 71 infectious diarrhea cases were reported in Hangzhou, China, caused by CNTP serogroup O1 isolates. We sequenced 119 V. cholerae genomes isolated from patients, carriers and the environment in Hangzhou between 2001 and 2012, and compared them with 850 publicly available global isolates. We found that CNTP isolates from Hangzhou belonged to two distinctive lineages, named L3b and L9. Both lineages caused disease over a long time period with usually mild or moderate clinical symptoms. Within Hangzhou, the spread route of the L3b lineage was apparently from rural to urban areas, with aquatic food products being the most likely medium. Both lineages had been previously reported as causing local endemic disease in Latin America, but here we show that global spread of them has occurred, with the most likely origin of L3b lineage being in Central Asia. The L3b lineage has spread to China on at least three occasions. Other spread events, including from China to Thailand and to Latin America were also observed. We fill the missing links in the global spread of the two non-toxigenic serogroup O1 V. cholerae lineages that can cause human infection. The results are important for the design of future disease control strategies: surveillance of V. cholerae should not be limited to ctxAB positive strains.

Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing.

The novel SARS-CoV-2 outbreak has swiftly spread worldwide. The rapid genome sequencing of SARS-CoV-2 strains has become a helpful tool for better understanding the genomic characteristics and origin of the virus. To obtain virus whole-genome sequences directly from clinical specimens, we performed nanopore sequencing using a modified ARTIC protocol in a portable nanopore sequencer and validated a routine 8-h workflow and a 5-h rapid pipeline. We conducted some optimization to improve the genome sequencing workflow. The sensitivity of the workflow was also tested by serially diluting RNA from clinical samples. The optimized pipeline was finally applied to obtain the whole genomes of 29 clinical specimens collected in Hangzhou from January to March 2020. In the 29 obtained complete genomes of SARS-CoV-2, 33 variations were identified and analyzed. The genomic variations and phylogenetic analysis hinted at multiple sources and different transmission patterns during the COVID-19 epidemic in Hangzhou, China. In conclusion, the genomic characteristics and origin of the virus can be quickly determined by nanopore sequencing following our workflows.

Characterization of fluoroquinolone-resistant Shigella flexneri in Hangzhou area of China.

OBJECTIVES: The aim of this study was to characterize fluoroquinolone-resistant Shigella and determine whether the qnr and aac(6')-Ib-cr genes could contribute to sporadic shigellosis at the clinic in the Hangzhou area of China. METHODS: A total of 202 strains of Shigella (79 Shigella sonnei and 123 Shigella flexneri ) isolated from sporadic cases of shigellosis from 1998 to 2007 were analysed for their antimicrobial susceptibility. The gyrA, gyrB, parC, parE, qnr and aac(6')-Ib-cr genes and the profiles and incompatibility of plasmids were characterized. Chromosomal DNA fingerprinting was determined by XbaI-based digestion and PFGE. RESULTS: All strains of S. sonnei were susceptible to fluoroquinolones (ciprofloxacin and levofloxacin) while 15 out of 123 strains of S. flexneri were resistant. All of the 15 resistant strains displayed common mutations in the gyrA and parC genes and formed eight distinct groups with unique molecular characteristics. Notably, 10 isolates showed mutations at codon 87 of gyrA, and the other 5 were qnrS-positive. Two strains were positive for the aac(6')-Ib-cr gene. Importantly, this is the first report of qnrS- and aac(6')-Ib-cr-positive Shigella in China, the qnrS-positive S. flexneri serotypes 1a, 2a and 4c and the aac(6')-Ib-cr-positive S. flexneri serotypes 2a and 4c worldwide. CONCLUSIONS: The common mutations at position 83 of gyrA and position 80 of parC were crucial for resistance to nalidixic acid in S. flexneri. The mutation at position 87 of gyrA or the presence of the qnrS gene is necessary for high-level resistance to fluoroquinolones in Shigella isolates from China.

Multiple Lineages of Dengue Virus Serotype 2 Cosmopolitan Genotype Caused a Local Dengue Outbreak in Hangzhou, Zhejiang Province, China, in 2017.

During July to November 2017, a large dengue outbreak involving 1,138 indigenous cases occurred in Hangzhou, Zhejiang Province, China. All patients were clinically diagnosed as mild dengue. Epidemiology investigation and phylogenetic analysis of circulating viruses revealed that at least three lineages of dengue virus serotype 2 (DENV-2) Cosmopolitan genotype initiated the outbreak during a short time. The analysis of the time to most recent common ancestor estimated that the putative ancestor of these DENV-2 lineages might rise no later than March, 2017, suggesting independent introductions of these lineages into Hangzhou. We presumed that group travelers visiting dengue-endemic areas gave rise to multiple introductions of these lineages during so short a time. Co-circulating of multiple DENV-2 lineages, emerging of disease in urban areas, hot and humid weather in Hangzhou adequate for mosquito breeding, and limited dengue diagnosis abilities of local hospitals, were the reasons causing the large local outbreak in Hangzhou.

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.

[Establishing a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NQO1 gene].

OBJECTIVE: To develop a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene. METHODS: Two primers of NQO1 gene C609T locus were designed. Using these primers, a SYBR Green I fluorescent bidirectional PCR, combined with melting curve analysis of the PCR products, were optimized to differentiate the 609C/T polymorphisms in 191 samples of human genomic DNA. The accuracy of the fluorescent bidirectional PCR was validated by the classical method of PCR-restriction fragment length polymorphism(RFLP) in 62 of these 191 samples. RESULTS: In the 62 samples, the genotypes determined by the fluorescent bidirectional PCR were 100% consistent with the ones by the PCR-RFLP. The frequencies of genotypes of homozygous wild-type (CC), heterozygous (CT), and homozygous mutant (TT) were 28%, 50%, and 22%, respectively, in the 191 samples. CONCLUSION: The single-tube fluorescent bidirectional PCR method established here provides a simple, rapid, accurate and inexpensive assay to determine the 609C/T polymorphism of NQO1 gene. The assay is suitable to detect the single nucleotide polymorphism in large-scale samples.

Complete Sequences and Characterization of Two Novel Plasmids Carrying aac(6')-Ib-cr and qnrS Gene in Shigella flexneri.

The complete sequences of two previously reported plasmids carrying plasmid-mediated quinolone resistance genes from Shigella flexneri in China have not been available. The present study using the p5-C3 assembly method revealed that (1) the plasmid pSF07201 with aac(6')-Ib-cr had 75,335 bp with antibiotic resistance genes CTX-M-3, TEM-1, and FosA3; (2) seven fragments of pSF07201 had more than 99% homology with the seven corresponding plasmids; (3) the other plasmid pSF07202 with qnrS had 47,669 bp with antibiotic resistance gene TEM-1 and 99.95% homology with a segment of pKF362122, which has the qnrS gene from location 162,490 to 163,146. A conjugation and electrotransformation experiment suggested that these two plasmids might horizontally transfer between and coexist in Escherichia coli J53 and S. flexneri 2a 301. Either the aac(6')-Ib-cr or qnrS gene contributed to, but only the coexistence of the two genes conferred to the resistance to ciprofloxacin in these two strains. To the best of our knowledge, this is the first report of the complete sequences of the aac(6')-Ib-cr- and qnrS-positive plasmids in Shigella isolates. Our findings indicate that two genes probably evolve through horizontal plasmid transfer between the different bacterial types.

Outbreak of coinfection with human metapneumovirus and measles virus resulting in the death of a child at a hospital in China.

Two children with different digestive diseases were admitted to the gastroenterology department of a children's hospital in Hangzhou, Zhejiang Province, China, in May 2010. They manifested successively acute lower respiratory tract infection symptoms during their stay in the hospital. The epidemiologic and experimental evidence supports that one child acquired nosocomial coinfection with measles virus and human metapneumovirus from another child while they shared the same ward.