Genotypes and Phylogenetic Analysis of Human Adenovirus in Hospitalized Pneumonia and Influenza-Like Illness Patients in Jiangsu Province, China (2013-2021).

Background: Human adenovirus (HAdV) is common pathogens that cause various respiratory diseases. The genetic diversity of viruses caused by recombination is considered to be the main source of emerging outbreaks. The aim of this study is to explore the evolutionary relationship and recombination events of HAdV genome in respiratory tract infections in Jiangsu Province. Methods: Whole-genome sequencing (WGS) technology was used to sequence 66 patients with HAdV infection (37 patients with influenza-like illness (ILI) and 29 hospitalized patients with pneumonia) from Jiangsu Province. Epidemiological analysis was performed on hospitalized pneumonia and ILI patients infected with HAdV. Subsequently, phylogenetic, recombination, and nucleotide and amino acid identity analyses were performed. Results: Epidemiological analysis of patients undergoing WGS showed that 75.7% of ILI patients were infected with the HAdVB strain and 69.0% of hospitalized pneumonia patients were infected with the HAdVC strain. Moreover, the hospitalized pneumonia and ILI patients infected with HAdV were different in region and time. The strains of HAdVB3 and HAdVB7 genotypes were mainly infected in 2015 and 2017, and the strains of HAdVC1 and HAdVC2 genotypes were mainly infected in 2020. The results of histogram analysis showed that the HAdV strain mainly infected children under 5 years old. In addition, 36 novel recombinant strains were identified. The discovery of these recombinant strains may contribute to understanding the epidemiology of HAdV and research on related vaccines. Furthermore, the percentage of nucleotide and amino acid identities revealed a high level of genetic conservation within isolates from HAdVB3, HAdVB7, HAdVC1, HAdVC2 and HAdVC5 genotypes. Conclusion: The WGS analysis reveals the evolutionary relationships and recombination events of HAdV strains in Jiangsu Province, which is helpful to deepen the understanding of HAdV epidemiology and evolution. In addition, it provides a basis for the formulation of public health strategies in Jiangsu Province.

Peptides derived from viral glycoprotein Gc Inhibit infection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by a novel phlebovirus (SFTSV), characterized by fever, thrombocytopenia and leukocytopenia which lead to multiple organ failure with high mortality in severe cases. The SFTSV has spread rapidly in recent years and posed a serious threat to public health in endemic areas. However, specific antiviral therapeutics for SFTSV infection are rare. In this study, we demonstrated that two peptides, SGc1 and SGc8, derived from a hydrophobic region of the SFTSV glycoprotein Gc, could potently inhibit SFTSV replication in a dose-dependent manner without apparent cytotoxicity in various cell lines and with low immunogenicity and good stability. The IC50 (50% inhibition concentration) values for both peptides to inhibit 2 MOI of SFTSV infection were below 10 µM in L02, Vero and BHK21 cells. Mechanistically, SGc1 and SGc8 mainly inhibited viral entry at the early stage of the viral infection. Inhibition of SFTSV replication was specific by both peptides because no inhibitory effect was shown against other viruses including Zika virus and Enterovirus A71. Taken together, our results suggested that viral glycoprotein-derived SGc1 and SGc8 peptides have antiviral potential and warrant further assessment as an SFTSV-specific therapeutic.

Analysis of Clinical Characteristics and Virus Strains Variation of Patients Infected With SARS-CoV-2 in Jiangsu Province-A Retrospective Study.

Background: At present, the global sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2) situation is still grim, and the risk of local outbreaks caused by imported viruses is high. Therefore, it is necessary to monitor the genomic variation and genetic evolution characteristics of SARS-CoV-2. The main purpose of this study was to detect the entry of different SARS-CoV-2 variants into Jiangsu Province, China. Methods: First, oropharyngeal swabs were collected from 165 patients (55 locally confirmed cases and 110 imported cases with confirmed and asymptomatic infection) diagnosed with SARS-CoV-2 infection in Jiangsu Province, China between January 2020 and June 2021. Then, whole genome sequencing was used to explore the phylogeny and find potential mutations in genes of the SARS-CoV-2. Last, association analysis among clinical characteristics and SARS-CoV-2 Variant of Concern, pedigree surveillance analysis of SARS-COV-2, and single nucleotide polymorphisms (SNPs) detection in SARS-COV-2 samples was performed. Results: More men were infected with the SARS-CoV-2 when compared with women. The onset of the SARS-CoV-2 showed a trend of younger age. Moreover, the number of asymptomatic infected patients was large, similar to the number of common patients. Patients infected with Alpha (50%) and Beta (90%) variants were predominantly asymptomatic, while patients infected with Delta (17%) variant presented severe clinical features. A total of 935 SNPs were detected in 165 SARS-COV-2 samples. Among which, missense mutation (58%) was the dominant mutation type. About 56% of SNPs changes occurred in the open reading frame 1ab (ORF1ab) gene. Approximately, 20% of SNP changes occurred in spike glycoprotein (S) gene, such as p.Asp501Tyr, p.Pro681His, and p.Pro681Arg. In total, nine SNPs loci in S gene were significantly correlated with the severity of patients. It is worth mentioning that amino acid substitution of p.Asp614Gly was significantly positively correlated with the clinical severity of patients. The amino acid replacements of p.Ser316Thr and p.Lu484Lys were significantly negatively correlated with the course of disease. Conclusion: Sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may further undergo a variety of mutations in different hosts, countries, and weather conditions. Detecting the entry of different virus variants of SARS-CoV-2 into Jiangsu Province, China may help to monitor the spread of infection and the diversity of eventual recombination or genomic mutations.

A fatal paediatric case infected with reassortant avian influenza A(H5N6) virus in Eastern China.

Avian influenza A(H5N6) keeps evolving, causing outbreaks in birds and sporadic infections in human. Here, we report a fatal paediatric infection caused by a novel reassortant H5N6 virus. The patient was an obese 9-year-old girl. She initiated with fever and cough, then developed pneumonia, acute respiratory distress syndrome (ARDS) and respiratory failure. Lower respiratory tract aspirates and anal swabs were serially taken till the patient's death. Viral isolation, genome sequencing and phylogenetic analysis were conducted. A novel reassortant H5N6 virus was isolated from the patient. Except the PA gene, all other 7 genes of the virus belonged to H5N6 genotype A (S4-like virus). The PA gene was probably obtained from Eurasian waterfowl influenza viruses. The H5N6 virus was consistently detected from the patient's respiratory samples till the 17th day after symptom onset, but not from anal swabs or urine sample by real-time reverse transcription polymerase chain reaction (RT-PCR). Significantly elevated (32-fold) serum antibodies to H5N6 virus were observed during the patient's course of disease. Aside from the identified novel reassortant H5N6 viral strain, obesity, delayed confirmation of aetiology and specific antiviral treatment, and prolonged virus shedding could have contributed to the poor clinical outcome.

Co-circulation of multiple genotypes of influenza A (H7N9) viruses in eastern China, 2016-2017.

Five epidemic waves of human infection with influenza A (H7N9) virus have emerged in China since spring 2013. We previously described the epidemiological characterization of the fifth wave in Jiangsu province. In this study, 41 H7N9 viruses from patients and live-poultry markets were isolated and sequenced to further elucidate the genetic features of viruses of the fifth wave in Jiangsu province. Phylogenetic analysis revealed substantial genetic diversity in the internal genes, and 18 genotypes were identified from the 41 H7N9 virus strains. Furthermore, our data revealed that 41 isolates from Jiangsu contained the G186V and Q226L/I mutations in their haemagglutinin (HA) protein, which may increase the ability of these viruses to bind the human receptor. Four basic amino acid insertions were not observed in the HA cleavage sites of 167 H7N9 viruses from Jiangsu, which revealed that highly pathogenic avian influenza (HPAI) H7N9 viruses did not spread to Jiangsu province in the fifth wave. These findings revealed that multiple genotypes of H7N9 viruses co-circulated in the fifth wave in Jiangsu province, which indicated that the viruses have undergone ongoing evolution with genetic mutation and reassortment. Our study highlights the need to constantly monitor the evolution of H7N9 viruses and reinforce systematic influenza surveillance of humans, birds, and pigs in China.

Predictors for fatal human infections with avian H7N9 influenza, evidence from four epidemic waves in Jiangsu Province, Eastern China, 2013-2016.

BACKGROUND: Four epidemic waves of human infection with H7N9 have been recorded in China up to 1 June 2016, including in Jiangsu Province. However, few studies have investigated the differences in patients' characteristics among the four epidemic waves, and the analyses of factors associated with fatal infection lacked statistical power in previous studies due to limited sample size. METHODS: All laboratory-confirmed A(H7N9) patients in Jiangsu province were analysed. Patients' characteristics were compared across four waves and between survivors and those who died. Multivariate analyses were used to identify independent predictors of death. RESULTS: Significant differences were found in the lengths of several time intervals (from onset of disease to laboratory confirmation, to onset of ARDS and respiratory failure, and to death) and in the development of heart failure. The proportions of overweight patients and rural patients increased significantly across the four waves. Administration of glucocorticoids and double-dose neuraminidase inhibitors became the norm. Predictors of death included complications such as ARDS, heart failure and septic shock, administration of glucocorticoids, and disease duration. CONCLUSION: Characteristics of H7N9 patients and clinical treatment options changed over time. Particular complications and the use of particular treatment, along with disease duration, could help clinicians predict the outcome of H7N9 infections.

Molecular epidemiology of genogroup II norovirus infection among hospitalized children with acute gastroenteritis in Suzhou (Jiangsu, China) from 2010 to 2013.

Noroviruses (NoVs) are the most common cause of acute gastroenteritis in both sporadic and outbreak cases. Genotyping and recombination analyses were performed in order to help getting more knowledge of the distribution and genetic diversity of NoVs in Suzhou, located in Jiangsu province of China. All stool samples were collected from hospitalized children younger than 5 years old with acute gastroenteritis. For genotyping, the open reading frame (ORF) 1 and ORF2 were partially amplified and sequenced. 26.9% of stool samples were positive for genogroup II NoVs. The most common genotype was GII.4 and its variants included Den Haag-2006b, New Orleans-2009, and Sydney-2012. The Den Haag-2006b variants predominated during 2010-2012. In 2013, it was replaced by the Sydney-2012 variant. The second most common genotype was GII.12/GII.3. NoVs could be detected throughout the year, with GII.4 and GII.12/GII.3 coexisting during the cold months, and GII.4 was the main genotype during the warm months. The highest prevalence of NoV was detected in young children aged <24 months. Patients infected with GII.4 had a higher chance of getting moderate fever than other NoV-positive patients, while those infected with GII.12/GII.3 tended to have a mild degree of fever. NoV is an important pathogen responsible for viral gastroenteritis among children in Suzhou. Analyses of NoV circulating between 2010 and 2013 revealed a change of predominant variant of NoV GII.4 in each epidemic season and intergenotype recombinant strains represented an important part.

Clinical characteristics and risk factors of an outbreak with scrub typhus in previously unrecognized areas, Jiangsu province, China 2013.

Scrub typhus, caused by Orientia tsutsugamushi, has emerged recently in Jingjiang City, China where the disease had not been known to exist. We analyzed epidemiological data, clinical characteristics and risk factors of scrub typhus outbreak in Jingjiang City, 2013. The 271 clinically diagnosed patients were predominantly farmers 50 to 69 years old and the peak of onset was early to mid-November. For the 187 laboratory-confirmed cases, the major clinical manifestations of the patients were fever (100%), eschar (88.2%), rash (87.7%), chills (87.7%), and headache (66.8%). A community-based case-control study was carried out to investigate the risk factors of the scrub typhus outbreak. Bundling or moving waste straw (OR=9.0, 95%CI 4.6-17.8) and living at the edge of village (OR=0.6, 95%CI 0.4-0.9) posed the highest risks through single- and multi-variable conditional logistic regression. Phylogenetic analysis of the 56-kDa TSA gene showed that the new cluster (GB-C2) and the previously reported cluster (GB-C1) of O. tsutsugamushi were associated with this outbreak. These findings are useful for the establishment of a detailed control strategy for scrub typhus infection in previously unrecognized areas of Jiangsu Province, China.

Seroprevalence of antibodies against SFTS virus infection in farmers and animals, Jiangsu, China.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a newly identified viral zoonosis caused by a phlebovirus. Most reported SFTS cases are farmers living in rural areas. The seroprevalence of SFTS virus in farmers has not been investigated. The current knowledge of SFTS virus seroprevalence in animals, especially in wild animals, is still poor. OBJECTIVES: To investigate SFTS virus seroprevalence among farmers and a variety of animal species. STUDY DESIGN: SFTS virus antibodies in sera were determined using a double-antigen sandwich ELISA. Serum samples were collected from 2547 farmers and 2741 animals in 6 SFTS-endemic counties from March 2012 to February 2013 in Jiangsu province. The farmer participants aged from 15 to 90 years. All of them were interviewed using a structured questionnaire. The animals sampled included 6 domesticated animal species and 2 wild animal species. RESULTS: SFTSV antibodies were found in a total of 33 farmers (1.30%) and was more prevalent in males than in females (respectively 1.87% and 0.71%, P<0.01). The mean age of seropositive farmers was 56.5 years and seroprevalence increased gradually with age. Seroprevalence in animal species were: goats (66.8%), cattle (28.2%), dogs (7.4%), pigs (4.7%), chickens (1.2%), geese (1.7%), rodents (4.4%) and hedgehogs (2.7%). Multiple variable logistic regression analysis showed that grazing, grass mowing, raising cattle, age, farm work time and tick bites were risk factors for SFTS virus infection among farmers. CONCLUSIONS: SFTSV readily infects humans with farming-related exposures as well as numerous domestic and wild animals. Serological results further suggest that the virus circulates widely in Jiangsu province.

A two-tube multiplex real-time RT-PCR assay for the detection of four hemorrhagic fever viruses: severe fever with thrombocytopenia syndrome virus, Hantaan virus, Seoul virus, and dengue virus.

The aim of this study was to develop and evaluate a two-tube multiplex real-time RT-PCR assay for the detection and identification of four viral hemorrhagic fever (VHF) pathogens, severe fever with thrombocytopenia syndrome virus (SFTSV), Hantaan virus (HTNV), Seoul virus (SEOV), and dengue virus (DENV), from human clinical samples. The two-tube multiplex real-time RT-PCR assay we developed has a sensitivity of 10 copies/µL for each of the targets, and the performance was linear within the range of at least 10(7) transcript copies. Moreover, we evaluated the specificity of the assay using other virus RNA as template, and found no cross-reactivity. This new assay is able to detect SFTSV, HTNV, SEOV and DENV in two reactions and brings a cost of 40 % compared to separate reactions. Evaluation of this assay with clinical serum samples from laboratory-confirmed patients and healthy donors showed 100 % clinical diagnostic sensitivity and over 99 % specificity. The assay was applied for scanning 346 clinical samples collected from patients admitted to the hospital with suspected VHF and compared with virus isolation and immunofluorescence assay (IFA). The assay indentified 59 SFTSV-, 12 HTNV-, 11 SEOV- and 9 DENV-positive samples and showed higher sensitivity. This assay thus provides a reliable and cost-effective screening tool for early clinical diagnosis of SFTSV, HTNV, SEOV and DENV in the acute phase.

Development and application of a one-step real-time RT-PCR using a minor-groove-binding probe for the detection of a novel bunyavirus in clinical specimens.

A highly sensitive one-step real-time RT-PCR method using a minor-groove-binding (MGB) probe was developed for detection and quantitation of severe febrile with thrombocytopenia syndrome virus (SFTSV). The assay could discriminate SFTSV infection from other related viral diseases in human with a minimum detection limit of 10 viral RNA copies/µl and was 1,000 times more sensitive than the conventional PCR. Strong linear correlations (r(2) > 0.99) between the C(t) values and viral RNA standards over a linear range were obtained. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The RT-PCR was also shown to be highly specific, as no positive signals were detected for other related viruses. Evaluation of this assay with serum samples from laboratory confirmed cases and healthy donors showed 100% clinical diagnostic sensitivity and over 99% specificity. Clinical application with samples from 287 patients admitted to the hospital with suspected SFTSV infection showed that 15% were infected by SFTSV. This assay was rapid, requiring just over 2 hr, including the nucleic acid extraction step.

Seroprevalence of pandemic (H1N1) 2009 in pregnant women in China: an observational study.

BACKGROUND: We investigated the seropositive rates and persistence of antibody against pandemic (H1N1) 2009 virus (pH1N1) in pregnant women and voluntary blood donors after the second wave of the pandemic in Nanjing, China. METHODOLOGY/PRINCIPAL FINDINGS: Serum samples of unvaccinated pregnant women (n = 720) and voluntary blood donors (n = 320) were collected after the second wave of 2009 pandemic in Nanjing. All samples were tested against pH1N1 strain (A/California/7/2009) with hemagglutination inhibition assay. A significant decline in seropositive rates, from above 50% to about 20%, was observed in pregnant women and voluntary blood donors fifteen weeks after the second wave of the pandemic. A quarter of the samples were tested against a seasonal H1N1 strain (A/Brisbane/59/2007). The antibody titers against pH1N1 strain were found to correlate positively with those against seasonal H1N1 strain. The correlation was modest but statistically significant. CONCLUSIONS AND SIGNIFICANCE: The high seropositive rates in both pregnant women and voluntary blood donors suggested that the pH1N1 virus had widely spread in these two populations. Immunity derived from natural infection seemed not to be persistent well.

[Determination of biphenyl ether herbicides in water using HPLC with cloud-point extraction].

OBJECTIVE: To determine residues of multiple biphenyl ether herbicides simultaneously in water using high performance liquid chromatography (HPLC) with cloud-point extraction. METHODS: The residues of eight biphenyl ether herbicides (including bentazone, fomesafen, acifluorfen, aclonifen, bifenox, fluoroglycofenethy, nitrofen, oxyfluorfen) in water samples were extracted with cloud-point extraction of Triton X-114. The analytes were separated and determined using reverse phase HPLC with ultraviolet detector at 300 nm. Optimized conditions for the pretreatment of water samples and the parameters of chromatographic separation applied. RESULTS: There was a good linear correlation between the concentration and the peak area of the analytes in the range of 0.05-2.00 mg/L (r = 0.9991-0.9998). Except bentazone, the spiked recoveries of the biphenyl ether herbicides in the water samples ranged from 80.1% to 100.9%, with relative standard deviations ranging from 2.70% to 6.40%. The detection limit of the method ranged from 0.10 microg/L to 0.50 microg/L. CONCLUSION: The proposed method is simple, rapid and sensitive, and can meet the requirements of determination of multiple biphenyl ether herbicides simultaneously in natural waters.

[Analysis on the epidemiological and genetic characteristics of enterovirus type 71 and Coxsackie A16 virus infection in Jiangsu, China].

OBJECTIVE: To determine the epidemiological features of hand-foot-and-mouth disease (HFMD) outbreaks and the genetic characteristics of enterovirus type 71(EV71) isolates from patients in Lianyungang, Jiangsu province in May, 2008. METHODS: Epidemiological, microbiological, cellular and molecular methods were performed to investigate pathogens and to describe the homogeneity of isolated strains. RESULTS: 21 cases were reported in this HFMD outbreak with the attack rate as 20.0%. 3 EV71 virus strains were isolated from 10 stool samples. The nucleotide and amino acid homogeneity of these 3 Jiangsu strain with Anhui Fuyang strains were 97.9%-100.0% and 99.7%-100.0%, respectively. These 4 Jiangsu strains were within genotype C sub-geno group C4 in phylogenetic tree. Data from the follow-up study showed that shedding of EV71 and Coxsackie A16 virus (CA16) in the latent period appeared in the outbreak of HFMD. Human beings could be infected by both EV71 virus and CA16 at the same time and could also carry the two viruses. We also discovered that EV71 virus could be expelled out of the human body through stool in the first week and last for 10 weeks. CONCLUSION: The recently identified EV71 isolates from this HFMD outbreak belonged to sub-geno group C4. Facts as: the release of viruses in the latent period, co-infection or coexisting of two viruses at the same time and super long period of expulsion of toxin exist in EV71 and CA16 did exist.