High Risk of Influenza Virus Infection Among Swine Workers: Examining a Dynamic Cohort in China.

BACKGROUND: China is thought to be a hotspot for zoonotic influenza virus emergence, yet there have been few prospective studies examining the occupational risks of such infections. METHODS: We present the first 2 years of data collected from a 5-year, prospective, cohort study of swine-exposed and -unexposed participants at 6 swine farms in China. We conducted serological and virological surveillance to examine evidence for swine influenza A virus infection in humans. RESULTS: Of the 658 participants (521 swine-exposed and 137 swine-unexposed), 207 (31.5%) seroconverted against at least 1 swine influenza virus subtype (swine H1N1 or H3N2). Swine-exposed participants' microneutralization titers, especially those enrolled at confined animal feeding operations (CAFOs), were higher against the swine H1N1 virus than were other participants at 12 and 24 months. Despite elevated titers, among the 187 study subjects for whom we had complete follow-up, participants working at swine CAFOs had significantly greater odds of seroconverting against both the swine H1N1 (odds ratio [OR] 19.16, 95% confidence interval [CI] 3.55-358.65) and swine H3N2 (OR 2.97, 95% CI 1.16-8.01) viruses, compared to unexposed and non-CAFO swine workers with less intense swine exposure. CONCLUSIONS: While some of the observed increased risk against swine viruses may have been explained by exposure to human influenza strains, study data suggest that even with elevated preexisting antibodies, swine-exposed workers were at high risk of infection with enzootic swine influenza A viruses.

Prospective surveillance for influenza. virus in Chinese swine farms.

Pork production in China is rapidly increasing and swine production operations are expanding in size and number. However, the biosecurity measures necessary to prevent swine disease transmission, particularly influenza. viruses (IAV) that can be zoonotic, are often inadequate. Despite this risk, few studies have attempted to comprehensively study IAV ecology in swine production settings. Here, we present environmental and animal sampling data collected in the first year of an ongoing five-year prospective epidemiological study to assess IAV ecology as it relates to swine workers, their pigs, and the farm environment. From March 2015 to February 2016, we collected 396 each of environmental swab, water, bioaerosol, and fecal/slurry samples, as well as 3300 pig oral secretion samples from six farms in China. The specimens were tested with molecular assays for IAV. Of these, 46 (11.6%) environmental swab, 235 (7.1%) pig oral secretion, 23 (5.8%) water, 20 (5.1%) bioaerosol, and 19 (4.8%) fecal/slurry specimens were positive for influenza. by qRT-PCR. Risk factors for IAV detection among collected samples were identified using bivariate logistic regression. Overall, these first year data suggest that IAV is quite ubiquitous in the swine production environment and demonstrate an association between the different types of environmental sampling used. Given the mounting evidence that some of these viruses freely move between pigs and swine workers, and that mixing of these viruses can yield progeny viruses with pandemic potential, it seems imperative that routine surveillance for novel IAVs be conducted in commercial swine farms.

Evidence for Cross-species Influenza A Virus Transmission Within Swine Farms, China: A One Health, Prospective Cohort Study.

Background: Our understanding of influenza A virus transmission between humans and pigs is limited. Methods: Beginning in 2015, we used a One Health approach and serial sampling to prospectively study 299 swine workers and 100 controls, their 9000 pigs, and 6 pig farm environments in China for influenza A viruses (IAVs) using molecular, culture, and immunological techniques. Study participants were closely monitored for influenza-like illness (ILI) events. Results: Upon enrollment, swine workers had higher serum neutralizing antibody titers against swine H1N1 and higher nasal wash total immunoglobulin A (IgA) and specific IgA titers against swine H1N1 and H3N2 viruses. Over a period of 12 months, IAVs were detected by quantitative reverse-transcription polymerase chain reaction in 46 of 396 (11.6%) environmental swabs, 235 of 3300 (7.1%) pig oral secretion, 23 of 396 (5.8%) water, 20 of 396 (5.1%) aerosol, and 19 of 396 (4.8%) fecal-slurry specimens. Five of 32 (15.6%) participants with ILI events had nasopharyngeal swab specimens that were positive for IAV, and 17 (53.1%) demonstrated 4-fold rises in neutralization titers against a swine virus. Reassorted Eurasian avian-lineage H1N1, A(H1N1)pdm09-like, and swine-lineage H3N2 viruses were identified in pig farms. The A(H1N1)pdm09-like H1N1 viruses identified in swine were nearly genetically identical to the human H1N1 viruses isolated from the participants with ILI. Conclusions: There was considerable evidence of A(H1N1)pdm09-like, swine-lineage H1N1, and swine-lineage H3N2 viruses circulating, likely reassorting, and likely crossing species within the pig farms. These data suggest that stronger surveillance for novel influenza virus emergence within swine farms is imperative.

Estimating daily salt intake based on 24†h urinary sodium excretion in adults aged 18-69†years in Shandong, China.

OBJECTIVE: 24 h urinary sodium extretion was used to estimate the daily salt intake of shandong residents aged from 18 to 69 years in China. SETTING: 20 selected counties/districts in Shandong stratified by geographic region (Eastern, Central Southern and North Western) and residence type (urban vs rural). PARTICIPANTS: Among 2184 randomly selected adults, 2061 provided usable 24 h urine samples. Urine volume <500 mL or male creatinine <3.81 (female creatinine <4.57) are not included in the analysis. RESULTS: The mean sodium level excreted over 24 h was 237.61 mmol (95% CI 224.77 to 250.44) mmol. Overall, the estimated mean salt intake was 13.90 g/day (95% CI 13.15 to 14.65). The mean salt intake among rural residents was higher than that among urban residents (14.00 vs 13.68 g; p<0.01). Salt intake in men was higher than that in women (14.40 vs 13.37 g; p<0.01). Approximately 96% of the survey participants had a dietary salt intake of ≥6 g/day. CONCLUSIONS: The salt intake in Shandong is alarmingly higher than the current recommended amount (6 g/day). Thus, effective interventions to reduce salt intake levels to combat the increasing burden of non-communicable diseases need to be developed and implemented.

Risk factors associated with severe manifestations of 2009 pandemic influenza A (H1N1) infection in China: a case-control study.

BACKGROUND: No studies on the risk factors of 2009 pandemic influenza A (H1N1) in China have been reported. We aimed to investigate the risk factors for severe manifestations of 2009 pandemic H1N1 influenza in China METHODS: A case-control study with 343 severe hospitalized patients and 343 randomly selected mild controls was conducted. The diagnosis was established by assessment of clinical symptoms and confirmed by the real-time reverse-transcriptase-polymerase chain reaction assay. Severe or mild patients were classified by uniform criteria issued by the Ministry of Health in China. RESULTS: The multivariable logistic regression analysis showed that the overweight or obese subjects admitted to hospital with H1N1 influenza were more likely to experience severe manifestations. The ORs were 3.70 (95% CI: 2.04-6.72) and 35.61 (95% CI: 7.96-159.21) respectively. Subjects at age less than 5 years or older than 60 years had an increased risk of severe manifestations (OR = 21.14, 95% CI: 7.79-57.33). We also observed increased risk among subjects with longer time interval from symptom onset to hospital admission (OR = 3.26, 95% CI: 2.08-5.11) or peasants (OR = 9.79, 95% CI: 5.11-18.78). Those with chronic disorders had increased risk of severe manifestations of H1N1 influenza. CONCLUSION: We provide evidence on the risk factors associated with severe manifestations of 2009 pandemic H1N1 influenza in a study of hospitalized subjects in China.

[The status of awareness, treatment and control of hypertension in adult population in Shandong province in 2011].

OBJECTIVE: To identify the status of awareness, treatment and control of hypertension in adult population in Shandong province in China. METHODS: A total of 15 350 representative subjects aged 18 to 69 in Shandong province were selected with multistage stratified and clustered sampling design. Questionnaire investigation and physical examination including measurement of blood pressure, height and weight, were taken for all of them. The prevalence was estimated by weighted SURVEYFREQ model. RESULTS: In Shandong province, 34.5% of the hypertensive patients were aware of their high blood pressure (31.1% in male, 38.5% in female), 27.5% of them were taking antihypertensive medications (24.1% in male, 31.7% in female), and 14.9% of them (13.7% in male, 16.4% in female) were under control for their blood pressure (<140/90 mm Hg). CONCLUSION: The rates of awareness, treatment and control of hypertension in adult hypertensive population in Shandong province, China were low, and it is urgently needed to take steps for intervention and control for hypertension prevention, particularly in rural areas.

Clinical progress and risk factors for death in severe fever with thrombocytopenia syndrome patients.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV) with an average fatality rate of 12%. The clinical factors for death in SFTS patients remain unclear. METHODS: Clinical features and laboratory parameters were dynamically collected for 11 fatal and 48 non-fatal SFTS cases. Univariate logistic regression was used to evaluate the risk factors associated with death. RESULTS: Dynamic tracking of laboratory parameters revealed that during the initial fever stage, the viral load was comparable for the patients who survived as well as the ones that died. Then in the second stage when multi-organ dysfunction occurred, from 7-13 days after disease onset, the viral load decreased in survivors but it remained high in the patients that died. The key risk factors that contributed to patient death were elevated serum aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and creatine kinase fraction, as well as the appearance of CNS (central nervous system) symptoms, hemorrhagic manifestation, disseminated intravascular coagulation, and multi-organ failure. All clinical markers reverted to normal in the convalescent stage for SFTS patients who survived. CONCLUSIONS: We identified a period of 7-13 days after the onset of illness as the critical stage in SFTS progression. A sustained serum viral load may indicate that disease conditions will worsen and lead to death.

[Establishment of minireplicon system for severe fever with thrombocytopenia syndrome bunyavirus].

Severe fever with thrombocytopenia syndrome bunyavirus is a newly emerging virus in China, enveloped with a tripartite, single-stranded RNA genome of negative polarity. The regulatory elements for viral transcription and replication, as well as encapsidation and packaging signals, are thought to be located within these noncoding regions (NCRs). The terminal nucleotides are genus specific and highly conserved. The function of the remaining nucleotides of the NCRs is still not well understood. In this study, we developed the plasmid-driven RNA polymerase I minireplicon system for SFTSV firstly, using reporter genes GFP and luciferase. The function of the noncoding regions of the three Bunyaviridae RNA segments (L, M, S) in transcription was analyzed. Reporter genes are successfully expressed in SFTSV minireplicon system. Our results suggest that the NCRs of SFTSV from all three segments contain the necessary signals to initiate transcription. Quantitative detection of the luciferase expression level shows that promoter activity in the three segments is different.

[Isolation, identification and characterization of SFTS bunyavirus from ticks collected on the surface of domestic animals].

To understand the maintenance and transmission of SFTS virus, the potential vector ticks were collected from sheep, cattle and dogs in the endemic areas of SFTSV in Shandong Province. Among the collected ticks, the dominant species was H. longicornis ticks. Real-time PCR for RNA detection, virus isolation and characterization, genomic sequencing, phylogenetic and antigenic analysis were performed in this investigation. The results showed that the SFTS viral RNA was detected in 2.14% H. longicornis, and a SFTS virus was isolated from one of viral RNA positive ticks collected from sheep. Whole genome analysis of the SFTSV isolates with 11 human-origin SFTS virus revealed a highly pairwise similarity, and the growth curve analysis showed nearly identical in virus yield and the dynamic of virus reproduction compared to human derived viral isolates. Immunofluorescence and neutralization test showed identical serological reaction character of the two different origin viral strains. In this study, the characters of a SFTSV isolate was firstly described, which suggested that the tick species H. longicornis acting important vector role in the transmission of SFTS virus.

[Study of molecular subtypes of biotype 1A Yersinia enterocolitica in Shandong province from 2008 to 2009].

OBJECTIVE: To investigate the molecular subtypes of 73 strains of Yersinia enterocolitica biotype 1A isolated in Shandong province by PFGE, and thereby to analyze the relationship between PFGE typing and biological characteristics. METHODS: Seventy-three strains of Yersinia enterocolitica biotype 1A were isolated from animal feces and meat products in Gaomi city and Wulian county in Shandong province from 2008 to 2009. Motility test, serum agglutination and virulent genes detection by PCR were used to learn the biological characteristics of the isolated strains. The molecular subtypes were determined by PFGE, whose relationships with motility, serotypes and virulent genotypes were also analyzed. RESULTS: Out of the 73 strains of Yersinia enterocolitica, 5 showed medium-active motility while the other 68 showed well-active motility. The dominated serotypes were O:5(17/73) and O:8(14/73), followed by O:9(5/73) and O:7, 8(1/73), and there was no O:3 serotype found. Meanwhile, 36 strains couldn't be serotyped. All the strains were negative with the gene ail, ystA, yadA and virF, yet the positive rate of ystB gene was 72.6% (53/73). The 73 strains of Yersinia enterocolitica isolated could be subtyped into 54 PFGE patterns (K6GN11SD0001-K6GN11SD0054), most of which only had 1 or 2 isolated strains, and no pattern was dominant. The strains in the same or similar cluster were from different hosts; each serotype and toxic genotype scattered in the clustering trees, without specific correlation with PFGE subtypes. 4 out of 5 strains, which showed medium-active motility, belonged to one branch, with the similarity coefficient at 80.9% - 100.0%; while all the toxic genotype belonged to type B. CONCLUSION: Biotype 1A Yersinia enterocolitica has many clones, whose PFGE types had relations with motility, but no relations with virulent genotype and host.

[Etiological and molecular characteristics of pandemic influenza A (H1N1) virus during 2009-2010 in Shandong Province].

To isolate and identify the influenza virus in Shandong Province in 2009-2010 and analyze the genetic characteristics of hemagglutinin and neuraminidase gene, further study the variation of gene. A total of 17 126 nasopharyngeal swabs from fever patients were collected and detected by real time quantitative RT-PCR method. The results showed 4004 samples were pandemic influenza A (H1N1) virus positive, with an overall positive rate as 23.38%. The positive samples were incubated and cultured in MDCK cells. The HA and NA genes of isolated pandemic influenza A(H1N1) virus were sequenced, the homology analysis of the HA and NA genes showed an average of 96.9%-99.3% and 99.1%-99.6% sequence identity, respectively, compared with WHO-recommended vaccine strain. The genetic evolution and amino acid substitutions were performed with Mega 4.0 Software. Twenty one amino acids were changed in HA protein, of which 11 were located in the antigenic site; Sixteen amino acids were changed in NA protein, which didn't lead to the changes of enzyme sites. Furthermore, one glycosylation site of HA protein and NA protein were changed respectively. No H275Y mutation in NA protein was found. The results showed that the HA and NA genes of the epidemic strains were highly homologous, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process. All the strains were sensitive to oseltamivir.

Fever with thrombocytopenia associated with a novel bunyavirus in China.

BACKGROUND: Heightened surveillance of acute febrile illness in China since 2009 has led to the identification of a severe fever with thrombocytopenia syndrome (SFTS) with an unknown cause. Infection with Anaplasma phagocytophilum has been suggested as a cause, but the pathogen has not been detected in most patients on laboratory testing. METHODS: We obtained blood samples from patients with the case definition of SFTS in six provinces in China. The blood samples were used to isolate the causal pathogen by inoculation of cell culture and for detection of viral RNA on polymerase-chain-reaction assay. The pathogen was characterized on electron microscopy and nucleic acid sequencing. We used enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and neutralization testing to analyze the level of virus-specific antibody in patients' serum samples. RESULTS: We isolated a novel virus, designated SFTS bunyavirus, from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. Electron-microscopical examination revealed virions with the morphologic characteristics of a bunyavirus. The presence of the virus was confirmed in 171 patients with SFTS from six provinces by detection of viral RNA, specific antibodies to the virus in blood, or both. Serologic assays showed a virus-specific immune response in all 35 pairs of serum samples collected from patients during the acute and convalescent phases of the illness. CONCLUSIONS: A novel phlebovirus was identified in patients with a life-threatening illness associated with fever and thrombocytopenia in China. (Funded by the China Mega-Project for Infectious Diseases and others.).

[Antibiotic resistance and molecular typing of Listeria monocytogenes from foods in Shandong province from 2009 to 2010].

OBJECTIVE: To know the antibiotic resistance and molecular characteristics of Listeria monocytogenes in Shandong province and to study the relationship between antibiotic resistance phenotypes and genome types. METHODS: From 2009 to 2010, a total of 80 Listeria monocytogenes isolates were collected from raw meat, cooked meat, aquatic products and other foods in 6 cities of Shandong province. The antibiotic susceptibility was measured by broth microdilution method, PFGE was performed for molecular typing and the relationship between antimicrobial resistance and PFGE patterns was analyzed. RESULTS: 16.25% (13/80) of the isolates were drug-resistant. Imipenem resistance was the most prevalent (12.50%, 10/80), followed by tetracycline and doxycycline (3.75%, 3/80 and 2.50%, 2/80). A total of the 80 isolates were subtyped into 9 antibiotic resistance patterns and 34 PFGE types which were largely dominated by the type 17 and 29. Antibiotic resistance pattern A corresponded to 79.41% (27/34) of PFGE types. CONCLUSION: The antibiotic resistance of Listeria monocytogenes in Shandong province is serious from 2009 to 2010 and there is no correlation between PFGE types and antibiotic resistance patterns.

[Analysis of etiology of four pandemic influenza A (H1N1) virus outbreaks in Shandong province, in 2009].

OBJECTIVE: To isolate and identify the influenza virus that caused four influenza-like-illness outbreaks in Jining city of Shandong Province in 2009 and analyze the genetic characteristics of hemagglutinin (HA) and neuraminidase (NA) gene, the variation of these genes were studied. METHODS: 34 nasopharyngeal swabs from fever patients of four influenza-like-illness outbreaks were collected and diagnosed by real time quantitative RT-PCR method. The positive samples were incubated and cultured for virus. HA and NA genes of isolated pandemic influenza A (H1N1) virus were sequenced, the homology analysis was done with DNAStar software and the genetic evolution and amino acid substitutions were performed with Mega 4.0 software. The sequences were compared with WHO recommended vaccine virus, native reference virus. RESULTS: Seventeen of 34 nasopharyngeal swabs were positive, 11 pandemic influenza A (H1N1) viruses were isolated and HA and NA genes of 7 strains were sequenced. Phylogenetic analysis for hemagglutinin and neuraminidase gene of Shandong outbreak strains showed that there were 98.4% - 99.6% and 99.2% - 100.0% sequence identity. Compared with WHO-recommended vaccine strain, the reference virus in mainland China strain, eleven amino acids were changed for HA protein, including position 38, 40, 56, 90, 100, 145, 172, 173, 220, 303 and 338, and 38, 40, 303 of HA protein were located in the antigenic determination C cluster, 172, 173 in the D cluster, 56 in the E cluster, site 40 of HA protein were glycosylated. In NA protein, seven amino acids were changed, including position 80, 106, 241, 248, 351, 369 and 386, site 40 of NA protein were glycosylated. No mutations of 275 in NA protein were found. CONCLUSION: The HA and NA genes of the epidemic strains showed high homology, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process.

[Development of TaqMan real-time PCR in detection of Aeromonas hydrophila].

OBJECTIVE: To develop a TaqMan real-time PCR for the detection of Aeromonas hydrophila. METHODS: The conserved region of major adhesion gene of Aeromonas hydrophila (aha) was used to design primers and TaqMan probe. A total of six concentration gradients for forward and reverse primers ranging from 200 -700 nmol/L were chosen, and four concentration gradients for probe ranging from 100 - 400 nmol/L were chosen. And then the concentration of primers and probe were optimized by ANOVA of completely randomized design respectively. The specificity of the established method was evaluated by using bacteria as contrast, including 45 strains Vibrio cholerae, 20 strains Vibrio parahaemolyticus, 10 strains Vibrio fluvialis, 4 strains Vibrio mimicus, 5 strains Vibrio vulnificus, 1 strain Vibrio alginolyticus, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Plesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by Aeromonas hydrophila artificially, and the ability of the established TaqMan real-time PCR system for detection of Aeromonas hydrophila was also evaluated. RESULTS: The cycle threshold (Ct) value deserved from 6 groups of primers concentration gradient was (x +/- s):20.69 +/- 0.33, 20.72 +/- 0.21, 20.81 +/- 0.12, 20.74 +/- 0.12, 20.51 +/- 0.16 and 20.69 +/- 0.11, respectively, and the concentration of forward primer and reverse primer was determined to be 200 nmol/L (F = 1.33, P = 0.28). The Ct value deserved from 4 groups of probe concentration gradient was (x +/- s):20.56 +/- 0.08, 20.82 +/- 0.05, 20.82 +/- 0.11 and 20.93 +/- 0.09, respectively, and the concentration of probe was determined to be 100 nmol/L (F = 5.26, P = 0.01). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/microl respectively, and the sensitivity to detect Aeromonas hydrophila from stool was 8 x 10(3) CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial. CONCLUSION: The TaqMan real-time PCR method targeting the aha gene of Aeromonas hydrophila had a high sensitivity and specificity and might be used to detect Aeromonas hydrophila from pure bacterial and stool rapidly.

[Cloning and sequence analysis of envelope glycoprotein G2 gene of hantavirus in Shandong province].

OBJECTIVE: To construct the cloning vector of glycoprotein G2 gene of hantavirus (HV), to analyze the sequence of G2 gene by the phylogenetic tree, and to study the differences among glycoprotein G2 genes from the world around. METHODS: Envelope glycoprotein G2 gene was amplified from four specimens of Shandong province by RT-PCR, and the product recombined into the PMD-18T vector. The clones that carry the G2 gene were identified. After sequencing, the gene sequence was handled with the software DNASTAR, compared with 24 strains worldwide and the phylogenetic tree was drawn. RESULTS: HV G2 gene was amplified by RT-PCR from 4 specimens, named GM04-38.G2, ZB8.G2, JUN5-14.G2, RCH5.G2, respectively. The map of the phylogenetic tree showed that all the 4 strains belonged to SEO-type hantavirus. The analysis of the sequence showed that all the four HV strains had the highest rates of homology with Z37 strain. The sequence homology of SEO-type HV strains was from 82.3% to 99.8%. CONCLUSION: The four cloning vectors containing the glycoprotein G2 genes were successfully constructed. Envelope glycoprotein G2 gene of four specimens from Shandong province had high homology rates.