Human infection of avian influenza A H3N8 virus and the viral origins: a descriptive study.

BACKGROUND: The H3N8 avian influenza virus (AIV) has been circulating in wild birds, with occasional interspecies transmission to mammals. The first human infection of H3N8 subtype occurred in Henan Province, China, in April, 2022. We aimed to investigate clinical, epidemiological, and virological data related to a second case identified soon afterwards in Hunan Province, China. METHODS: We analysed clinical, epidemiological, and virological data for a 5-year-old boy diagnosed with H3N8 AIV infection in May, 2022, during influenza-like illness surveillance in Changsha City, Hunan Province, China. H3N8 virus strains from chicken flocks from January, 2021, to April, 2022, were retrospectively investigated in China. The genomes of the viruses were sequenced for phylogenetic analysis of all the eight gene segments. We evaluated the receptor-binding properties of the H3N8 viruses by using a solid-phase binding assay. We used sequence alignment and homology-modelling methods to study the effect of specific mutations on the human receptor-binding properties. We also conducted serological surveillance to detect the H3N8 infections among poultry workers in the two provinces with H3N8 cases. FINDINGS: The clinical symptoms of the patient were mild, including fever, sore throat, chills, and a runny nose. The patient's fever subsided on the same day of hospitalisation, and these symptoms disappeared 7 days later, presenting mild influenza symptoms, with no pneumonia. An H3N8 virus was isolated from the patient's throat swab specimen. The novel H3N8 virus causing human infection was first detected in a chicken farm in Guangdong Province in December, 2021, and subsequently emerged in several provinces. Sequence analyses revealed the novel H3N8 AIVs originated from multiple reassortment events. The haemagglutinin gene could have originated from H3Ny AIVs of duck origin. The neuraminidase gene belongs to North American lineage, and might have originated in Alaska (USA) and been transferred by migratory birds along the east Asian flyway. The six internal genes had originated from G57 genotype H9N2 AIVs that were endemic in chicken flocks. Reassortment events might have occurred in domestic ducks or chickens in the Pearl River Delta area in southern China. The novel H3N8 viruses possess the ability to bind to both avian-type and human-type sialic acid receptors, which pose a threat to human health. No poultry worker in our study was positive for antibodies against the H3N8 virus. INTERPRETATION: The novel H3N8 virus that caused human infection had originated from chickens, a typical spillover. The virus is a triple reassortment strain with the Eurasian avian H3 gene, North American avian N8 gene, and dynamic internal genes of the H9N2 viruses. The virus already possesses binding ability to human-type receptors, though the risk of the H3N8 virus infection in humans was low, and the cases are rare and sporadic at present. Considering the pandemic potential, comprehensive surveillance of the H3N8 virus in poultry flocks and the environment is imperative, and poultry-to-human transmission should be closely monitored. FUNDING: National Natural Science Foundation of China, National Key Research and Development Program of China, Strategic Priority Research Program of the Chinese Academy of Sciences, Hunan Provincial Innovative Construction Special Fund: Emergency response to COVID-19 outbreak, Scientific Research Fund of Hunan Provincial Health Department, and the Hunan Provincial Health Commission Foundation.

Estimation of Avian Influenza Viruses in Water Environments of Live Poultry Markets in Changsha, China, 2014 to 2018.

In routine surveillance for avian influenza viruses (AIVs) in the environments of live poultry markets (LPMs), certain samples were positive for AIVs type A while negative for subtypes (e.g., H5, H7, and H9). However, little attention has been paid to these unsubtyped AIVs samples. To reveal the dynamic distribution and molecular characteristics of AIVs, especially the unsubtyped AIVs, we reported and analyzed 1969 samples collected from the water environments of LPMs in Changsha, China, from January 2014 to November 2018. Our results revealed that 1504 (76.38%) samples were positive for AIV type A. Of these samples, the predominant hemagglutinin (HA) subtype was H9, followed by H5 and H7 (P < 0.05). The positive rate of H5 subtype in water environmental samples exhibited seasonality, which reached a peak in each winter-spring season from January 2014 to March 2017. The positive rates of AIVs (including type A, subtype H9, and mixed subtype H5/H7/H9) in non-central-city regions were higher than that in the central-city regions (P < 0.05). Notably, 161 unsubtyped AIVs samples were detected during the routine surveillance. However, subtyping with the commercial kit further identified eight different HA and seven different neuraminidase subtypes. Analyses unraveled that further subtyped AIVs H1, H6, and H11 had only one basic amino acid (R or K) at the cleavage site and residues Q226 and G228 at the receptor-binding associated sites. Overall, in addition to H5, H7, and H9 subtypes, we should also pay attention to unsubtyped AIVs samples during the routine surveillance for AIVs in the environments of LPMs.

Complete genome analysis of coxsackievirus A2 and A5 strains in Changsha / 中国热带医学

@#Abstract: Objective　To investigate the molecular characteristic and evolutionary trends of full-genome sequences of coxsackievirus A2 (CV-A2) and A5 (CV-A5) in Changsha City. Methods　The CV-A2 and CV-A5 strains were isolated and detected from patients with hand, foot and mouth disease (HFMD) cases. The full-genome sequences of CV-A2 and CV-A5 strains were obtained using NGS sequencing. Homology and phylogenetic tree analysis were performed, and the recombination regions of the strains were examined by SimPlot software. Results　The full-genome sequences of CV-A2 and CV-A5 strains were obtained from routine surveillance cases of HFMD in Changsha in 2019. The CV-A2 strain was named S281/Changsha/CHN/2019 with the full-genome sequence of 7 422 bp long; the CV-A5 strain was named S272/Changsha/CHN/2019 with the full-genome sequence of 7 425 bp long. Homology analysis of the isolates by comparison with the nucleic acid sequences of CV-A2 and other CV-A2 strains in China showed that the non-structural protein region shared lower similarity than that of structural protein region. The CV-A2 showed 79.20% similarity with Fleetwood strain (NC038306), showed the highest similarity 95.60% with MN419014 strain from Hubei Province. The non-structural protein 3C and 3D region shared the lowest similarity with MN419014, 90.51 and 92.06%, respectively. Phylogenetic tree analysis showed that 3C and 3D regions were located in the CV-A4 branch. Amino acid mutation sites were found in non-structural protein region, and the amino acid sequence in structural protein region was conserved. SimPlot analysis showed that genetic recombination was found in the 3C and 3D region of CV-A2 strains. The full-genome sequence of CV-A5 showed 80.7% similarity with the Swartz (AY421763) and 97.43% similarity with the strain (MH111030) from Australian. Homology analysis showed that the non-structural protein region shared lower similarity than that of structural protein region, based on full-genome of CV-A5. Phylogenetic tree analysis showed that CV-A5 and MH111030 were in the same branch, indicating that CV-A5 strain not from local. The amino acid sequence of CV-A5 strain was conserved. Conclusions　The CV-A2 strain in Changsha City shared genome sequence information with CV-A4, and the CV-A5 strain was imported from abroad. Our findings are expected to understand the molecular and recombination characteristics of CV-A2 and CV-A5, provided the data of evolution and genetic features of the coxsackievirus, and interrupt disease transmission in a timely and effective manner.

Live poultry feeding and trading network and the transmission of avian influenza A(H5N6) virus in a large city in China, 2014-2015.

OBJECTIVES: To understand the transmission mechanisms of the avian influenza A(H5N6) virus. METHODS: This study explored the live poultry feeding and trading network (LPFTN) around Changsha city, China. Field epidemiological investigations were performed in Changsha to investigate the LPFTN with the environmental samples systematically collected during 2014-2015 to monitor and analyze the spread of the A(H5N6) virus. Two surveillance systems were also applied to find possible human cases of A(H5N6) infection. RESULT: The information of all the 665 live poultry farming sites, five wholesale markets, and 223 retail markets in Changsha was collected to investigate the LPFTN. Moreover, about 840 environmental samples were systematically collected from the LPFTN during 2014-2015 to monitor the spread of the A(H5N6) virus, with 8.45% (71/840) positive for the N6 subtype. Furthermore, the full genome sequences of 10 A(H5N6) viruses detected from the environmental samples were obtained, which were then characterized and phylogenetically analyzed with the corresponding gene segments of the A(H5N6) virus obtained from GenBank, to determine the source of human infection. CONCLUSION: It was demonstrated that the LPFTN provided a platform for the H5N6 transmission, and formed an infectious pool for the spread of the virus to humans.

Full-genome sequences of GII.13[P21] recombinant norovirus strains from an outbreak in Changsha, China.

On 31 March 2019, 68 school students suffered from vomiting, diarrhea, and abdominal pain after participating in a group activity at a commercial park. In this outbreak, multiple norovirus genotypes were observed, including GII.2[P16], GII.17[P17], and GII.13[P21]. Further, we determined the full-genome sequences of two strains of GII.13[P21] recombinant noroviruses, which were 7434 nt long. Phylogenetic analysis based on open reading frames (ORFs) 1 and 2 revealed that these recombinants were related to stains of different genotypes from different countries. The full genome nucleotide sequences of the two isolates were 97.0% and 98.0% identical to those of strains from London and Thailand, respectively. Simplot analysis revealed the presence of a break point at nt 5059 in the ORF1 region. The histo-blood group antigen binding sites were conserved in both recombinant viruses. Our findings not only provide valuable genetic information about a recombinant norovirus but also contribute to our general understanding of the evolution, genetic diversity, and distribution of noroviruses.

Development and evaluation of a real-time RT-PCR assay for detection of a novel avian influenza A (H5N6) virus.

As of Aug 25, 2017, 17 incidences of human infection and 6 deaths due to the novel H5N6 virus have been reported in China. Genetic analysis of the viral genome revealed that this reassortant virus is highly pathogenic to poultry, and that the virus has a risk of transmission to humans. Accordingly, the development of a rapid, sensitive, and specific molecular diagnostic assay is critical for public health. In this study, a real-time reverse-transcription PCR (RT-PCR) assay was developed to specifically detect the novel H5N6 virus, with primer pairs targeting the hemagglutinin and neuraminidase gene sequences of this virus. RNA was extracted from throat swab specimens from patients with influenza-like illness (ILIs), and environmental samples were collected from live poultry markets (LPMs) for H5N6 virus detection by real-time RT-PCR. The method was demonstrated to enable specific detection of the avian H5N6 virus, with no cross-reactivity with seasonal influenza viruses (H1N1, H1N1 pdm09, H3N2 or B); H5N1, H7N9, H9N2 viruses; or other human respiratory viruses. The detection limit of the assay was 1.0 × 101 copies per reaction for N6 and 1.0 × 102 copies per reaction for H5 assays. The assay is a powerful tool for rapid, sensitive, and specific detection of H5N6 virus infection in specimens derived from humans, animals, and the environment.

Epidemiological and Genetic Characteristics of Rabies Virus Transmitted Through Organ Transplantation.

In January 2016, two patients died of rabies after receiving kidney transplants from a common organ donor at a hospital in Changsha, Hunan, China. The medical records, epidemiological data of the organ donor, two kidney and a liver recipients were reviewed. Intravitam saliva samples of the two kidney recipients were tested for rabies virus (RABV) using real-time RT-PCR, and the nucleoprotein (N) gene was amplified and sequenced by Sanger sequencing. Whole genome sequences were analyzed using next-generation sequencing. The N genes of the two kidney recipients showed 100% nucleic acid identity. Phylogenetic analysis of the complete genome, N and glycoprotein (G) genes indicated that the RABV was homologous with dog isolates from the Hunan province and belong to the China I lineage, which is widespread in China. The organ donor was a 22-month-old boy who died from unknown acute progressive encephalitis. After undergoing sub-hypothermia hibernation therapy, rabies-associated symptoms were atypical, and rabies was neglected because serum RABV-specific antibodies were negative. An unknown wound on the forehead of the donor was found 2 months before the onset of symptoms. Based on the clinical, epidemiological, and molecular findings, we speculated that the RABV initially originated in the donor from a dog bite, and was then transmitted to the recipients by organ transplantation. An uncertain exposure history and misdiagnosis played important roles in the spread of the RABV. Rabies should be considered in patients with acute progressive encephalitis of unexplained etiology, especially in potential organ donors.

Primers and probe design and precision assessment of the real time RT-PCR assay in Coxsackievirus A10 and enterovirus detection.

This data article contains data related to the research article entitled "Rapid detection of enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay" (Chen at al., 2017) [1]. Primers and probe sequence design are among the most critical factors in real-time polymerase chain reaction (PCR) assay optimization. Linearity, sensitivity, specificity and precision are the crucial criteria which are used to evaluate the performance of a new method. This data article report the primers and probe design and precision assessment of the new assay. VP1 gene of Coxsackievirus A10 (CV-A10) and 5'-NCR of different enterovirus (EV) serotypes were retrieved from GenBank database and aligned. The intra- and inter-assay variation were assessed using high, medium and low concentration of control plasmid DNA and viral RNA samples.

Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay.

A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found.

Clinical, epidemiological and virological characteristics of the first detected human case of avian influenza A(H5N6) virus.

A human infection with novel avian influenza A H5N6 virus emerged in Changsha city, China in February, 2014. This is the first detected human case among all human cases identified from 2014 to early 2016. We obtained and summarized clinical, epidemiological, and virological data from this patient. Complete genome of the virus was determined and compared to other avian influenza viruses via the construction of phylogenetic trees using the neighbor-joining approach. A girl aged five and half years developed fever and mild respiratory symptoms on Feb. 16, 2014 and visited hospital on Feb. 17. Throat swab specimens were obtained from the patient and a novel reassortant avian influenza A H5N6 virus was detected. All eight viral gene segments were of avian origin. The hemagglutinin (HA) and neuraminidase (NA) gene segments were closely related to A/duck/Sichuan/NCXN11/2014(H5N1) and A/chicken/Jiangxi/12782/2014(H10N6) viruses, respectively. The six internal genes were homologous to avian influenza A (H5N2) viruses isolated in duck from Jiangxi in China. This H5N6 virus has not gained genetic mutations necessary for human infection and was suggested to be sensitive to neuraminidase inhibitors, but resistant to adamantanes. Epidemiological investigation of the exposure history of the patient found that a live poultry market could be the source place of infection and the incubation period was 2-5days. This novel reassortant Avian influenza A(H5N6) virus could be low pathogenic in humans. The prevalence and genetic evolution of this virus should be closely monitored.

Isolation and characteristic analysis of a novel strain H7N9 of avian influenza virus A from a patient with influenza-like symptoms in China.

A novel H7N9 virus (A/Changsha/1/2013(H7N9)) identified through routine examination in the influenza network laboratory was analyzed retrospectively. The gene sequences of A/Changsha/1/2013(H7N9) were highly homologous to other viruses isolated in mainland China. Mutations of Q226L and G186V were found in the hemagglutinin protein (HA). Amino acid deletions were found at positions 69-73 of the neuraminidase protein (NA) and 218-230 of the non-structural protein (NS1). All viral genes except PB1 were essentially identical to the sequences of other Chinese influenza A H7N9 isolates. Overall, A/Changsha/1/2013(H7N9) is highly homologous to other H7N9 avian influenza viruses isolated in mainland China.

The role of enterovirus 71 and coxsackievirus A strains in a large outbreak of hand, foot, and mouth disease in 2012 in Changsha, China.

BACKGROUND: During 2012, Changsha experienced a large outbreak of hand, foot, and mouth disease (HFMD), resulting in 25,438 cases, including 42 severe cases and eight deaths. METHODS: Seven hundred and forty-six clinical specimens were collected from hospital-based surveillance for HFMD in 2012. The detection and genotyping of enterovirus were performed by real-time RT-PCR and sequencing of the VP1 regions; phylogenetic analysis was performed based on the VP1 sequences. RESULTS: A total of 545 (73.1%) enterovirus-positive samples were identified, with the most frequently presenting serotype being enterovirus 71 (EV-71; n=364, 66.8%), followed by coxsackievirus A16 (CV-A16; n=84, 15.4%), CV-A6 (n=22, 4.0%), and CV-A10 (n=19, 3.5%). Most of the affected patients were children aged ≤5 years (n=524, 96.1%). EV-71 was the major pathogen in the severe and fatal cases (n=22, 78.6%). Phylogenetic analysis of VP1 gene sequences showed the EV-71 isolates to belong to subgenotype C4a, and the CV-A16 isolates to belong to subgenotype B1. The Changsha CV-A6 and CV-A10 circulating strains were homologous to strains circulating in other areas of mainland China. CONCLUSIONS: Our results demonstrate that EV-71 was the primary causative agent responsible for the HFMD outbreak in Changsha in 2012, and the co-circulation of other coxsackievirus A strains posed a potential risk to public health.

Surveillance of influenza virus during 2010-2012 in Changsha, China.

The objective of this study was to examine the circulating influenza viruses in Changsha, China, during 2010-2012. Nasopharyngeal specimens were collected from persons with influenza-like illness (ILI) who presented for care at two hospitals. Of 2,955 patients tested, 278/(9.4%) were positive for influenza virus: 116/(41.7%) were influenza type A(H3N2), 79/(28.4%) were type A(H1N1) pandemic 2009 (pdm09) and 83/(29.9%) were influenza type B. The rates of virus detection varied by age and sex. The highest rate was in the 5-14 year old age group and females were infected more than males. After the initial 2009 A(H1N1) pdm09 outbreak, the number of cases of this virus declined and the season become shorter. Influenza A(H3N2) and B viruses occurred mainly during the spring and summer, while influenza A(H1N1)pdm09 occurred mainly during the winter and spring. Influenza A(H1N1)pdm09 replaced the usual seasonal H1N1 virus during 2010-2012. Continuing epidemiological surveillance of influenza virus is important to monitor trends in influenza infections and to develop prevention and control measures.

Analysis of the full-length genome of a novel strain of the H7N9 avian influenza virus.

The aim of the present study was to analyze the evolution and variation of a novel strain of the avian influenza virus. The virus-positive specimens [A/Changsha/2/2013 (H7N9)] from a patient infected with the novel avian influenza A (H7N9) virus was amplified by reverse transcription-PCR and the full genome was sequenced. The sequencing results were submitted to GenBank and then analyzed by phylogenetic tree analysis using BioEdit and Mega5 software. The phylogenetic tree of the hemagglutinin (HA) and neuraminidase genes revealed that A/Changsha/2/2013 (H7N9) and all the new H7N9 viruses in 2013 were in a large cluster, and their nucleotide evolutionary distances were closely associated. Phylogenetic tree analyses of the nucleoprotein and nonstructural genes demonstrated two main branches. One branch contained novel H7N9 viruses isolated from avian, human and environmental sources in different regions. The other branch contained three novel H7N9 virus strains isolated from environmental sources in Shanghai. All the phylogenetic trees of the matrix protein, polymerase acidic, polymerase basic protein 1 and polymerase basic protein 2 genes also showed two branches, with each branch including the novel H7N9 virus strains isolated from avian, human and environmental sources in different regions. Molecular characterization demonstrated that 52 novel H7N9 viruses sequenced to date contain the G228S and G186V mutations in the receptor binding site of the HA protein. The full-genome sequences of A/Changsha/2/2013 and analyses of its molecular characteristics suggest that the A/Changsha/2/2013 H7N9 virus strain has molecular characteristics that may facilitate adaptation of the virus to mammalian hosts and may even bind to human receptors.

[Risk related to the transmission of H5N1 subtype avian influenza virus in the environment of poultry markets in Changsha, China].

OBJECTIVE: To investigate the risk of H5N1 subtype avian influenza virus (AIV) transmission in the poultry market environment in Changsha city. H5N1 antibody levels among the groups related occupational exposure and AIV nucleic acid in the environment of poultry markets were detected. The characteristics of haemagglutinin (HA) genes of H5N1 AIV in the environment were analyzed. METHODS: One district and one county from Changsha city were selected randomly and two poultry markets at inner city or township levels were selected in the same district or county respectively. H5N1 antibody of the occupational exposure groups in the poultry market was tested and AIV nucleic acid in the poultry market environment monitored. One hundred and two blood samples of the occupational exposure groups were tested for H5N1 antibody with single radioimmunoassay diffusion hemolysis (SRH) while 160 environment samples (from sewage, birds stools, feathers and smearing samples of poultry cages) in the poultry market were also detected for AIV nucleic acid with real-time PCR method. Four sewage samples of H5N1 subtype AIV were collected from poultry markets in Changsha, and the HA genes of H5N1 subtype AIV amplified by RT-PCR and then sequenced with TA cloning. Amino acid sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega 5.0 software. RESULTS: The results through H5N1 antibody monitoring program showed that H5N1 antibody positive rates from workers were 25.5% (26/102), 50.0% (9/18) and 25.4% (17/67) respectively in the poultry markets of township and inner cities. H5N1 antibody positive rate in the township poultry markets was higher than in the inner cities poultry markets. RESULTS: from the surveillance on AIV nucleic acid showed that the overall H5 subtype positive rate in Changsha poultry markets was 31.3% (50/160), and the positive rate of townships poultry markets was 37.3% (31/83), which were both higher than those from the inner cities poultry markets (24.7%, 19/77). H5 subtype AIV positive rate was different in the tested specimens, with ranking of positive rates were sewage (50.0%, 24/48), feathers (44.5%, 4/9), birds stools (29.8%, 14/47) and smearing samples of poultry cages (14.3%, 8/56), with statistically significant differences (P < 0.01). Four H5N1 HA genes TA cloning were successfully constructed and identified as Eurasian branch, similar to viruses isolated in mainland China and Hong Kong in the same group, according to genetic analysis. Sequence data of the four HA genes showed the same feature of high pathogenicity, compared to the H5N1 AIV from mainland China of human origin. The receptor specificities were still with avian influenza origin (QSG) and the connecting peptide between HA1 and HA2 possessing the polybasic motif (RERRRKK or RERRGKK). CONCLUSION: One of the reasons for H5N1 antibody positive rate of 25.5% among poultry markets workers was that there were large numbers of H5N1 subtype AIV detected in the environment of poultry markets and HA genes of H5N1 subtype AIV in the poultry markets environment carried molecular characteristics of highly pathogenic which could increase the risk for H5N1 subtype AIV transmission in the environment of poultry markets.

[Genetic analysis of the NS genes of H5N1 avian influenza viruses isolated from sewage in poultry markets].

In order to investigate the transmission risk of H5N1 avian influenza viruses (AIV) from sewage in Changsha poultry markets, the evolution relationship and molecular characteristics of non-structural (NS) genes of H5N1 AIV from sewage were analyzed. Nine H5N1 AIV environmental sewage specimens were collected from Changsha poultry markets. The NS genes were amplifyed by PCR and then sequenced with TA cloning. Amino acid(aa) sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega5 software. Eight NS genes TA cloning were constructed successfully. Phylogenetic tree indicated that they were belonged to the allele A subgroup. Aa homology analysis showed 90.1% 92.5% identity in NS1 proteins and 91.0% - 92.6% identity in NS2 proteins compared with reference viruses of the allele A (A/chicken/ Hubei/ w h/ 1999). The homologies of the amino sequences of NS1 and NS2 in this study were 93.8%-100.0% and 98.4%-100.0%, respectively. The C terminal of all eight H5N1 NS1 proteins from sewage in poultry markets carried a ESEV of PL motif and the 92 amino acids were E, furthermore, the 80 to 84aa were missed which were the characteristics of highly pathogenic AIV. The NS genes of H5N1 AIV from sewage in poultry markets have molecular characteristics of highly pathogenic and have the potential risk of H5N1 virus spreading.

[Rapid detection of norwalk GII with reverse transcription loop-mediated isothermal amplification].

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.

[Pathogenic diagnosis of an unexplained epidemic event of infectious diarrhea].

OBJECTIVE: To determine the pathogen of an unexplained epidemic event of infectious diarrhea by laboratory diagnosis of suspected cases samples. METHODS: 28 samples from 28 suspected cases (22 fecal samples, 3 vomitus samples, 3 anus swab samples) were tested for Norovirus by RT-PCR. Sequencing and phylogenetic analysis were acomplished of 5 positive samples. RESULTS: 160 of 5694 population were ill with an attack rate of 2.81%. The peak period was 7-9, March. 14 of 28 samples were tested Norovirus positive.Sequencing and phylogenetic analysis showed Norovirus type GII/4 was the causative agent and it had highest identity (97. 9%) with epidemic strain 2006b. CONCLUSION: The epidemic event ofinfectious diarrhea were caused by GII/4 Norovirus strains.