Importation of a novel Indian Ocean lineage carrying E1-K211E and E2-V264A of Chikungunya Virus in Zhejiang Province, China, in 2019.

The chikungunya virus (CHIKV) is widespread. In Zhejiang province, China, CHIKV infection is often associated with travelers from tropical and subtropical countries. In the present study, three CHIKV isolates from serum samples of travelers in Zhejiang province in 2019 were sequenced, and phylogenetically analyzed to study their molecular characteristics. Sequence analysis showed that the non-structural protein and the structural protein had 37 and 28 amino acid mutations, respectively; no mutation site was found at the E1-A226 residue, which could increase the adaptability of CHIKV to Aedes albopictus. All three samples carried two mutations, namely, E1-K211E and E2-V264A, which were introduced to Bangladesh around late 2015 and Thailand in early 2017. Phylogenetic analysis revealed that these three CHIKVs were Indian Ocean lineage of the East Africa/Central/South Africa genotype (ECSA) and that the MF773566 strain from Bangladesh (Australia/Bangladesh 2017) had the closest evolutionary relationship. The three CHICKs imported into Zhejiang province in 2019 belonged to the ECSA genotype and had multiple amino acid variation sites. The variation in the three samples provides a certain reference for the subsequent research on CHIKV evolution.

Molecular epidemiologic characteristics of hemagglutinin from five waves of avian influenza A (H7N9) virus infection, from 2013 to 2017, in Zhejiang Province, China.

There have been five waves of influenza A (H7N9) epidemics in Zhejiang Province between 2013 and 2017. Although the epidemiological characteristics of the five waves have been reported, the molecular genetics aspects, including the phylogeny, evolution, and mutation of hemagglutinin (HA), have not been systematically investigated. A total of 154 H7N9 samples from Zhejiang Province were collected between 2013 and 2017 and sequenced using an Ion Torrent Personal Genome Machine. The starting dates of the waves were 16 March 2013, 1 July 2013, 1 July 2014, 1 July 2015, and 1 July 2016. Single-nucleotide polymorphisms (SNPs) and amino acid mutations were counted after the HA sequences were aligned. The evolution of H7N9 matched the temporal order of the five waves, among which wave 3 played an important role. The 55 SNPs and 14 amino acid mutations with high frequency identified among the five waves revealed the dynamic occurrence of mutation in the process of viral dissemination. Wave 3 contributed greatly to the subsequent epidemic of waves 4 and 5 of H7N9. Compared with wave 1, wave 5 was characterized by more mutations, including A143V and R148K, two mutations that have been reported to weaken the immune response. In addition, some amino acid mutations were observed in wave 5 that led to more lineages. It is necessary to strengthen the surveillance of subsequent H7N9 influenza outbreaks.

Co-circulation of influenza A(H1N1), A(H3N2), B(Yamagata) and B(Victoria) during the 2017-2018 influenza season in Zhejiang Province, China.

Influenza is a major human respiratory pathogen. Due to the high levels of influenza-like illness (ILI) in Zhejiang, China, the control and prevention of influenza was challenging during the 2017-2018 season. To identify the clinical spectrum of illness related to influenza and characterise the circulating influenza virus strains during this period, the characteristics of ILI were studied. Viral sequencing and phylogenetic analyses were conducted to investigate the virus types, substitutions at the amino acid level and phylogenetic relationships between sequences. This study has shown that the 2017/18 influenza season was characterised by the co-circulation of influenza A (H1N1) pdm09, A (H3N2) and B viruses (both Yamagata and Victoria lineage). From week 36 of 2017 to week 12 of 2018, ILI cases accounted for 5.58% of the total number of outpatient and emergency patient visits at the surveillance sites. Several amino acid substitutions were detected. Vaccination mismatch may be a potential reason for the high percentage of ILI. Furthermore, it is likely that multiple viral introductions played a role in the endemic co-circulation of influenza in Zhejiang, China. More detailed information regarding the molecular epidemiology of influenza should be included in long-term influenza surveillance.

Respiratory virus associated with surgery in children patients.

BACKGROUND: Viral respiratory infection (VRI) is a common contraindication to elective surgery. Asymptomatic shedding among pediatric surgery patients (PSPs) could potentially lead to progression of symptomatic diseases and cause outbreaks of respiratory diseases. The aim of this study is to investigate the incidence of infection among mild symptomatic PSP group and asymptomatic PSP group after surgical procedure. METHODS: We collected the induced sputum from enrolled 1629 children (under 18 years of age) with no respiratory symptom prior to pediatric surgery between March 2017 and February 2019. We tested 16 different respiratory virus infections in post-surgery mild symptomatic PSP group and asymptomatic PSP group using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay panel. We analyzed symptom data and quantitative viral load to investigate the association between viruses, symptoms and viral quantity in qRT-PCR-positive PSPs. RESULTS: Out of 1629 children enrolled, a total of 204 respiratory viruses were present in 171 (10.50%) PSPs including 47 patients with mild symptoms and 124 with no symptoms after surgery. Commonly detected viruses were human rhino/enterovirus (HRV/EV, 42.19%), parainfluenza virus 3 (PIV3, 24.48%), coronavirus (CoV NL63, OC43, HKU1, 11.46%), and respiratory syncytial virus (RSV, 9.9%). PIV3 infection with a higher viral load was frequently found in PSPs presenting with mild symptoms, progressing to pneumonia with radiographic evidence after surgery. HRV/EV were the most commonly detected pathogens in both asymptomatic and mild symptomatic PSPs. CoV (OC43, HKU1) infections with a higher viral load were mostly observed in asymptomatic PSPs progressing to alveolar or interstitial infiltration. CONCLUSIONS: Our study suggested that PIV3 is a new risk factor for VRI in PSPs. Employing a more comprehensive, sensitive and quantitative method should be considered for preoperative testing of respiratory viruses in order to guide optimal surgical timing.

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples.

BACKGROUND: Numerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly. RESULTS: In this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1-26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis. CONCLUSIONS: The evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.

Clinical evaluation of a panel of multiplex quantitative real-time reverse transcription polymerase chain reaction assays for the detection of 16 respiratory viruses associated with community-acquired pneumonia.

We developed a panel of multiplex quantitative real-time reverse transcription polymerase chain reaction (mqRT-PCR) assay consisting of seven internally controlled qRT-PCR assays to detect 16 different respiratory viruses. We compared the new mqRT-PCR with a previously reported two-tube mRT-PCR assay using 363 clinical sputum specimens. The mqRT-PCR assay performed comparably with the two-tube assay for most viruses, offering the advantages of quantitative analysis, easier performance, lower susceptibility to contamination, and shorter turnaround time in laboratories equipped with conventional real-time PCR instrumentation, and it could therefore be a valuable tool for routine surveillance of respiratory virus infections in China.

Transcriptomic analysis reveals the inhibition mechanism of pulsed light on fungal growth and ochratoxin A biosynthesis in Aspergillus carbonarius.

Pulsed light (PL) technology has a good effect on the control of fungi in postharvest fruit. In this present work, PL inhibited the growth of Aspergillus carbonarius in a dose-dependent manner, the mycelial growth decreased by 4.83 %, 13.91 % and 30.01 % at a fluence of 4.5 J·cm-2 (PL5), 9 J·cm-2 (PL10) and 13.5 J·cm2 (PL15), respectively. When inoculated with PL15 treated A. carbonarius, the scab diameter of the pears, ergosterol and OTA content was reduced by 23.2 %, 27.9 % and 80.7 % after 7 days, respectively. The third-generation sequencing technique was applied to study the transcriptome response of A. carbonarius treated with PL. Compared with the blank control, a total number of 268 and 963 differentially expressed genes (DEGs) were discovered in the group of PL10 and PL15, respectively. To be specific, a large amount of DEGs involved in DNA metabolism were up-regulated, while most of DEGs related to cell integrity, energy and glucose metabolism, ochratoxin A (OTA) biosynthesis and transport were down-regulated. In addition, the stress response of A. carbonarius was imbalanced, including up-regulation of Catalase and PEX12 and down-regulation of taurine and subtaurine metabolism, alcohol dehydrogenase and glutathione metabolism. Meanwhile, the results of transmission electron microscopy, mycelium cellular leakage and DNA electrophoresis indicated that PL15 treatment caused mitochondrial swelling, the destroyed cell membrane permeability and imbalance of DNA metabolism. The expression of P450 and Hal involved in OTA biosynthesis pathway were down-regulated in PL treated samples detected by qRT-PCR. In conclusion, this study reveals the molecular mechanism of pulsed light on inhibiting the growth, development and toxin production of A. carbonarius.

Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method.

The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (p = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.

Epidemiology of hand, foot and mouth disease and genomic surveillance of coxsackievirus A10 circulating in Zhejiang Province, China during 2017 to 2022.

BACKGROUND: Coxsackievirus A10 (CA10) is one of the etiological agents associated with hand, foot and mouth disease (HFMD). OBJECTIVES: We aimed to perform a retrospective analysis of the molecular epidemiological characteristics and genetic features of HFMD associated with CA10 infections in Zhejiang Province from 2017 to 2022. STUDY DESIGN: Epidemiologic features were summarized. Throat swab specimens were collected and tested. The VP1 regions were sequenced for genotyping. CA10 positive samples were isolated. Whole genomes of CA10 isolations were sequenced. Nucleotide and amino acid changes were characterized. Phylogenetic trees were constructed. RESULTS: The number of HFMD cases fluctuated from 2017 to 2022. Children aged below 3 years accounted for the majority (66.29%) and boys were more frequently affected than girls. Cases peaked in June. The positivity rate of HEV was 62.69%. A total of 90 strains of CA10 were isolated and 53 genomes were obtained. All CA10 in this study could be assigned to two genogroups, C (C2) and F (F1 and F3). CONCLUSION: The clinical manifestations of HFMD associated with HEV are complex and diverse. CA10 infection may be emerging as a new and major cause of HFMD because an upward trend was observed in the proportion of CA10 cases after the use of EV71 vaccines. Different genogroups of CA10 had different geographic distribution patterns. Surveillance should be strengthened and further comprehensive studies should be continued to provide a scientific basis for HFMD prevention and control.

Effects of lactobacillus plantarum ZJ316 on pig growth and pork quality.

BACKGROUND: Lactobacillus plantarum is a plant-associated bacterial species but it has also been found in human, mouse and porcine gastrointestinal tracts. It can ferment a broad spectrum of plant carbohydrates; it is tolerant of bile salts and low pH, and it has antagonistic potential against intestinal pathogens. However, experiments reporting the use of L. plantarum as a probiotic are limited. In this study, the effects of L. plantarum ZJ316 isolated from infant fecal samples on pig growth and pork quality were investigated. RESULTS: One hundred and fifty newly weaned pigs were selected randomly and divided into five groups. Group 1 was fed a diet supplemented with the antibiotic mequindox; Groups 2, 3 and 4 were fed a diet supplemented with L. plantarum and no antibiotic; and Group 5 was fed a mixture of mequindox and L. plantarum. After a 60 days initial treatment, samples were collected for evaluation. The results showed that, the L. plantarum ZJ316 has probiotic effects on pig growth and that these effects are dose dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of a dose of 5 × 109 CFU/d or 1 × 1010 CFU/d. In Group 2 (1 × 109 CFU/d), the diarrhea (p = 0.000) and mortality rates (p = 0.448) were lower than in antibiotic-treated pigs (Group 1), and the daily weight gain (p = 0.001) and food conversion ratios were better (p = 0.005). Improved pork quality was associated with Lactobacillus treatment. pH (45 min, p = 0.020), hardness (p = 0.000), stickiness (p = 0.044), chewiness (p = 0.000), gumminess (p = 0.000) and restoring force (p = 0.004) were all significantly improved in Lactobacillus-treated pigs (Group 2). Although we found that L. plantarum exerted probiotic effects on pig growth and pork quality, the mechanisms underlying its action require further study. Polymerase chain reaction-denaturing gradient gel electrophoresis results showed that the gut bacterial communities in Lactobacillus- and antibiotic-treated pigs were very similar and the quantity of L. plantarum ZJ316 was below the detection limits of DGGE-band sequencing. The concentration of short-chain fatty acids in Lactobacillus- and antibiotic-treated fecal samples were not significantly different (p = 0.086). However, the villus height of ilea (p = 0.003), jejuna (p = 0.000) and duodena (p = 0.036) were found to be significantly improved by Lactobacillus treatment. CONCLUSION: L. plantarum ZJ316 was found to have probiotic effects, improving pig growth and pork quality. The probiotic mechanism might not involve L. plantarum colonization and alteration of the gut bacterial community. Rather, it might be related to the inhibition of the growth of opportunistic pathogens and promotion of increased villus height.

An external quality assessment for the molecular testing of the SARS-CoV-2 virus genome in Zhejiang Province, China.

The COVID-19 pandemic has necessitated the rapid expansion of laboratories that conduct SARS-CoV-2 tests. A provincial external quality assessment (EQA) scheme on SARS-CoV-2 tests was organized by Zhejiang Provincial CDC to assess the accuracy of the tests in individual CDC municipal and county laboratories in Zhejiang Province, China. Three positive samples in high, medium, and low concentrations, respectively, were prepared using the serial dilutions from the culture with the viral titer concentration of 1×106.3 TCID50/mL, and one negative sample were included. A total of 93 laboratories participated, contributing results from 36 distinct combinations of nucleic acid extraction methods and PCR reagents. There was 100% concordance among all laboratories for all EQA samples, and no false-positive or false-negative results were observed. The EQA survey provides confidence in the identification of infected individuals or asymptomatic populations and assurance for clinical and public health decision-making based on test results.

Comparison and Analysis of Neutralizing Antibody Levels in Serum after Inoculating with SARS-CoV-2, MERS-CoV, or SARS-CoV Vaccines in Humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus (SARS-CoV) pose a great threat to humanity. Every pandemic involving these coronaviruses has seriously affected human health and economic development. Currently, there are no approved therapeutic drugs against their infections. Therefore, the development of vaccines is particularly important to combat these coronaviruses. In this review, we summarized and analyzed the progress of vaccines against SARS-CoV, MERS-CoV, and SARS-CoV-2, including inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines, and viral vector vaccines. In addition, we compared the levels of neutralizing antibodies in the serum of patients with these three kinds of coronaviruses at different stages, and their ability and effects against SARS-CoV-2, MERS-CoV, and SARS-CoV. This review provides useful information for vaccine evaluation and analysis.

Simultaneous Detection of Ebola Virus and Pathogens Associated With Hemorrhagic Fever by an Oligonucleotide Microarray.

Ebola virus infection causes severe hemorrhagic fever, and its mortality rates varied from 25 to 90% in the previous outbreaks. The highly infectious and lethal nature of this virus highlights the need for reliable and sensitive diagnostic methods to distinguish it from other diseases present with similar clinical symptoms. Based on multiplex polymerase chain reaction (PCR) and oligonucleotide microarray technology, a cost-effective, multipathogen and high-throughput method was developed for simultaneous detection of Ebola virus and other pathogens associated with hemorrhagic fever, including Marburg virus, Lassa fever virus, Junin virus, Machupo virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, malaria parasite, hantavirus, severe fever with thrombocytopenia syndrome virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A virus, and influenza B virus. This assay had an excellent specificity for target pathogens, without overlap signal between the probes. The limit of detection was approximately 103 pathogen copies/µl. A total of 60 positive nucleic acid samples for different pathogens were detected, a concordance of 100% was observed between microarray assay and real-time PCR analysis. Consequently, the described oligonucleotide microarray may be specific and sensitive assay for diagnosis and surveillance of infections caused by Ebola virus and other species of hemorrhagic fever pathogens.

Molecular Characterization of Dengue Virus Type 1 in Zhejiang in 2019.

Dengue fever (DF) is a mosquito-borne viral disease caused by the dengue virus (DENV), which is considered one of the most important arboviruses in the world. This study aimed to determine the molecular, epidemiological, and phylogenetic characterization of 174 DENV-1 (132 indigenous cases and 42 imported cases) isolated from nine municipalities of Zhejiang province in 2019. The analyses of phylogenetics, haplotypes, and amino acid substitutions were conducted based on the full envelope (E) gene sequences. Sixty-four haplotypes were clustered into two main clades, with isolates from Wenzhou and Taizhou mainly clustered into clade I and Hangzhou and Ningbo cases clustered into clade II. Six sites of amino acid substitutions including A88T, F96L, M297V, T339S, I378L, and V436I were only observed in strains isolated from Ningbo and Hangzhou, while two sites of amino acid substitutions including V312L and V380I were observed in strains from Taizhou and Wenzhou. In our study, strains were in high homology with the strains from Southeast Asian countries, thus cases in Zhejiang were probably imported from Southeast Asian countries. The strains from different regions in Zhejiang were clustered in the same branch which may be caused by the continuous import of cases in the same country at different time periods. After the continuous outbreak in Zhejiang province, some sites of the dengue gene have mutated, and the effects need further study.

Development of an inactivated vaccine candidate for SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale production of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6 micrograms per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without observable antibody-dependent enhancement of infection. These data support the clinical development and testing of PiCoVacc for use in humans.

Epidemiological Characterization of the 2017 Dengue Outbreak in Zhejiang, China and Molecular Characterization of the Viruses.

Dengue, a mosquito-borne disease caused by the dengue virus (DV), has been recognized as a global public health threat. In 2017, an unexpected dengue outbreak occurred in Zhejiang, China. To clarify and characterize the causative agent of this outbreak, data on dengue fever cases were collected from the China Information System for Disease Control and Prevention in Zhejiang province for subsequent epidemiological analysis. A total of 1,229 cases were reported, including 1,149 indigenous and 80 imported cases. Most indigenous cases (1,128 cases) were in Hangzhou. The epidemic peak occurred in late August and early September, and the incidence rate of elderly people (4.34 per 100,000) was relatively high. Imported cases were reported all year round, and most were from South-East Asia and Western Pacific regions. Young people and men accounted for a large fraction of the cases. Acute phase serums of patients were collected for virus isolation. And 35 isolates (including 25 DV-2, 8 DV-1, 1 DV-3, and 1 DV-4) were obtained after inoculation and culture in mosquito C6/36 cells. The E genes of the 35 new DV isolates and the complete genome of a DV-2 isolate (Zhejiang/HZ33/2017), and the E gene of a DV-2 isolate from Ae. albopictus (Zhejiang/Aedes-1/2017) were determined. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. Phylogenetically, DVs of all four serotypes with multiple genotypes (mainly including 21 Cosmopolitan genotype DV-2, 4 Asian I genotype DV-2, 6 genotype I DV-1, and 2 genotype V DV-1) were present in the indigenous and imported cases in Zhejiang during the same period. Most of the isolates probably originated from South-East Asia and Western Pacific countries. The imported cases, high density of mosquito vector, and missed diagnosis might contribute to the 2017 outbreak in Zhejiang.

Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles.

OBJECTIVE: In previous studies, Lactobacillus plantarum ZJ316 showed probiotic properties, such as antimicrobial activity against various pathogens and the capacity to significantly improve pig growth and pork quality. The purpose of this study was to reveal the genes potentially related to its genetic adaptation and probiotic profiles based on comparative genomic analysis. METHODS: The genome sequence of L. plantarum ZJ316 was compared with those of eight L. plantarum strains deposited in GenBank. BLASTN, Mauve, and MUMmer programs were used for genome alignment and comparison. CRISPRFinder was applied for searching the clustered regularly interspaced short palindromic repeats (CRISPRs). RESULTS: We identified genes that encode proteins related to genetic adaptation and probiotic profiles, including carbohydrate transport and metabolism, proteolytic enzyme systems and amino acid biosynthesis, CRISPR adaptive immunity, stress responses, bile salt resistance, ability to adhere to the host intestinal wall, exopolysaccharide (EPS) biosynthesis, and bacteriocin biosynthesis. CONCLUSIONS: Comparative characterization of the L. plantarum ZJ316 genome provided the genetic basis for further elucidating the functional mechanisms of its probiotic properties. ZJ316 could be considered a potential probiotic candidate.

Living poultry markets in rural area: Human infection with H7N9 virus re-emerges in Zhejiang Province, China, in winter 2014.

BACKGROUND: Avian influenza A H7N9 virus, previously undetected in humans, has caused infections in many areas in China since February 2013. Here we report the re-emergence of a case of H7N9 in rural Jiaxing city, Zhejiang Province, in the winter of 2014. OBJECTIVES: To understand (1) the clinical syndrome, epidemiological and virological characteristics of this case; (2) the importance of controlling live poultry markets (LPMs) in rural areas. STUDY DESIGN: There is one patient and 16 contacts, including 4 family members living in the same household, and 12 medical personnel. Pharyngeal swabs and serum samples were collected from the patient and her contacts. Environment samples were also obtained from the local LPMs. We conducted detailed clinical and epidemiological investigations and laboratory work, including viral RNA extraction, RT-PCR detection and sequencing. Characteristic and phylogenetic analyses were performed using the obtained sequences. RESULTS: H7N9s were detected in environmental samples collected in LPMs in Jiaxing, Zhejiang. Unknown mutations were discovered in amino acids in the sample from the patient. The strain from the patient was in a clade different from isolates obtained in 2013 in phylogenetic trees of HA, NA and PB2. CONCLUSIONS: A severe case of H7N9 was identified in early winter, 2014. Epidemiological and clinical tests were consistent with patterns reported previously, while laboratory findings showed the virus to be different. Live poultry markets in rural Zhejiang Province are in need of closer supervision and enhanced management.

A study of family clustering in two young girls with novel avian influenza A (H7N9) in Dongyang, Zhejiang Province, in 2014.

BACKGROUND: The avian influenza A H7N9 virus, previously unknown in humans, has infected humans in many areas of China since February 2013. Here we report on a clustering case of H7N9 in two little girls in one family in Dongyang city, Jinhua area, Zhejiang Province. OBJECTIVES: To determine (1) whether the infections were due to person-to-person transmission or to co-exposure to poultry and (2) the prevalence of this novel H7N9 virus in Dongyang inferred by this family clustering case. STUDY DESIGN: Samples were collected from patients and environment. We undertook detailed epidemiological investigations and laboratory work. Phylogenetic analyses were done based on the sequenced genomes. The concentration of cytokines and chemokines in the serum was detected by cytometric bead array analyses. RESULTS: A mixture of H7 and H9 was detected from the environmental sample. The three H7N9 viruses shared one infection source. The index patient who had significantly higher levels of IL-4, IL-8 and IL-10 suffered severe infection. CONCLUSIONS: Based on a comparison with previous isolations of the virus in 2013, H7N9 has evolved different lineages through recombination with local H9N2 viruses. Determining whether it was human-to-human transmission or exposure to the same live poultry, since both patients had identical exposure histories, was ambiguous. The results from the cytokine analyses agreed with the conclusion that H7N9 severity is associated with a higher level of cytokines/chemokines. Long term influenza surveillance remains essential to allow for early warning of potential transmission events.

Characterization of severe fever with thrombocytopenia syndrome in rural regions of Zhejiang, China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) infections have recently been found in rural regions of Zhejiang. A severe fever with thrombocytopenia syndrome (SFTS) surveillance and sero-epidemiological investigation was conducted in the districts with outbreaks. During the study period of 2011-2014, a total of 51 SFTSV infection cases were identified and the case fatality rate was 12% (6/51). Ninety two percent of the patients (47/51) were over 50 years of age, and 63% (32/51) of laboratory confirmed cases occurred from May to July. Nine percent (11/120) of the serum samples from local healthy people without symptoms were found to be positive for antibodies to the SFTS virus. SFTSV strains were isolated by culture using Vero, and the whole genomic sequences of two SFTSV strains (01 and Zhao) were sequenced and submitted to the GenBank. Homology analysis showed that the similarity of the target nucleocapsid gene from the SFTSV strains from different geographic areas was 94.2-100%. From the constructed phylogenetic tree, it was found that all the SFTSV strains diverged into two main clusters. Only the SFTSV strains from the Zhejiang (Daishan) region of China and the Yamaguchi, Miyazakj regions of Japan, were clustered into lineage II, consistent with both of these regions being isolated areas with similar geographic features. Two out of eight predicted linear B cell epitopes from the nucleocapsid protein showed mutations between the SFTSV strains of different clusters, but did not contribute to the binding ability of the specific SFTSV antibodies. This study confirmed that SFTSV has been circulating naturally and can cause a seasonal prevalence in Daishan, China. The results also suggest that the molecular characteristics of SFTSV are associated with the geographic region and all SFTSV strains can be divided into two genotypes.