Emergence of Eurasian Avian-Like Swine Influenza A (H1N1) virus in a child in Shandong Province, China.

BACKGROUND: Influenza A virus infections can occur in multiple species. Eurasian avian-like swine influenza A (H1N1) viruses (EAS-H1N1) are predominant in swine and occasionally infect humans. A Eurasian avian-like swine influenza A (H1N1) virus was isolated from a boy who was suffering from fever; this strain was designated A/Shandong-binzhou/01/2021 (H1N1). The aims of this study were to investigate the characteristics of this virus and to draw attention to the need for surveillance of influenza virus infection in swine and humans. METHODS: Throat-swab specimens were collected and subjected to real-time fluorescent quantitative polymerase chain reaction (RT‒PCR). Positive clinical specimens were inoculated onto Madin-Darby canine kidney (MDCK) cells to isolate the virus, which was confirmed by a haemagglutination assay. Then, whole-genome sequencing was carried out using an Illumina MiSeq platform, and phylogenetic analysis was performed with MEGA X software. RESULTS: RT‒PCR revealed that the throat-swab specimens were positive for EAS-H1N1, and the virus was subsequently successfully isolated from MDCK cells; this strain was named A/Shandong-binzhou/01/2021 (H1N1). Whole-genome sequencing and phylogenetic analysis revealed that A/Shandong-binzhou/01/2021 (H1N1) is a novel triple-reassortant EAS-H1N1 lineage that contains gene segments from EAS-H1N1 (HA and NA), triple-reassortant swine influenza H1N2 virus (NS) and A(H1N1) pdm09 viruses (PB2, PB1, PA, NP and MP). CONCLUSIONS: The isolation and analysis of the A/Shandong-binzhou/01/2021 (H1N1) virus provide further evidence that EAS-H1N1 poses a threat to human health, and greater attention should be given to the surveillance of influenza virus infections in swine and humans.

Molecular epidemiology and genetic diversity of norovirus among hospitalized patients with acute gastroenteritis in Shandong, China, 2016-2018.

Norovirus (NoV) infection is a leading cause of acute gastroenteritis (AGE) for people of all ages. Here, we reported the molecular epidemiology and genetic diversity of NoVs among hospitalized patients with AGE between 2016 and 2018 in Shandong Province, China. Two thousand sixty-nine AGE patients from sentinel hospitals were enrolled. The stool samples were collected and tested for NoVs by real-time RT-PCR. The RNA-dependent RNA polymerase (RdRp) and capsid gene of 163 strains were amplified and sequenced for genotyping. Phylogenetic analyses and genomic characterization were conducted with the VP1 and RdRp region of the full genome sequences. Four hundred seventy two (21.76%) samples were NoV-positive. The positive rate in 2016 was higher than those of 2017 and 2018. We observed diverse NoV genotypes. GII.2[P16] emerged in January 2017 and became the dominant genotype between May and June 2017. Phylogenetic analyses showed that our GII.2[P16] genomes clustered in the SC1 in VP1 region, while they belonged to the Emerging GⅡ.P16 (2015-2017) clade in RdRp region. Our GⅡ.4 strains displayed two amino acid mutations, positions R297H and D372N, in epitope A of the VP1 region. Our study highlighted that NoV is an important pathogen of viral AGE in Shandong and, therefore, it is necessary to strengthen its surveillance.

Surveillance of avian influenza viruses in live bird markets of Shandong province from 2013 to 2019.

Avian influenza viruses (AIVs) seriously affect the poultry industry and pose a great threat to humans. Timely surveillance of AIVs is the basis for preparedness of the virus. This study reported the long-term surveillance of AIVs in the live bird market (LBM) of 16 cities in Shandong province from 2013 to 2019. A total of 29,895 samples were obtained and the overall positive rate of AIVs was 9.7%. The H9 was found to be the most predominant subtype in most of the time and contributed most to the monthly positve rate of AIVs as supported by the univariate and multivariate analysis, while H5 and H7 only circulated in some short periods. Then, the whole-genome sequences of 62 representative H9N2 viruses including one human isolate from a 7-year-old boy in were determined and they were genetically similar to each other with the median pairwise sequence identities ranging from 0.96 to 0.98 for all segments. The newly sequenced viruses were most similar to viruses isolated in chickens in mainland China, especially the provinces in Eastern China. Phylogenetic analysis showed that these newly sequenced H9N2 viruses belonged to the same clade for all segments except PB1. Nearly all of these viruses belonged to the G57 genotype which has dominated in China since 2010. Finally, several molecular markers associated with human adaptation, mammalian virulence, and drug resistance were identified in the newly sequenced H9N2 viruses. Overall, the study deepens our understanding of the epidemic and evolution of AIVs and provides a basis for effective control of AIVs in China.

Incidence, circulation, and spatiotemporal analysis of seasonal influenza in Shandong, China, 2008-2019: A retrospective study.

BACKGROUND: Understanding the influenza-like illness (ILI) incidence, circulation pattern of virus strains and spatiotemporal pattern of influenza transmission are important for designing control interventions. Based on the 10 years' surveillance data, we aimed to provide a baseline characterization and the epidemiology and dynamics of influenza virus in Shandong. METHODS: We extracted surveillance and laboratory testing data. We estimated the ILI incidence and analyzed the predominant virus. A wavelet power analysis was used to illustrate the periodicity. In addition, we applied a linear regression model to characterize the correlation of influenza seasonality with longitude. RESULTS: The average ILI incidence was estimated to be 3744.79 per 1 million (95% confidence interval [CI]: 2558.09-4931.45) during 2009-2018. Influenza A/H1N1 and A/H3N2 strains predominated in the most influenza seasons in Shandong. The annual amplitude of influenza epidemics decreased with longitude (P < 0.05). In contrast, the epidemic peak of influenza emerged earlier in the western region and increased with longitude in influenza A (P < 0.05). The annual peak of the influenza B epidemic lagged a median of 4.2 weeks compared with that of influenza A. CONCLUSIONS: The development or modification of seasonal influenza vaccination strategies requires the recognition that the incidence is higher in preschool- and school-aged children. Although seasonal influenza circulates annually in Shandong, the predominant virus strain circulation pattern is extremely unpredictable and strengthening surveillance for the predominant virus strain is necessary. Lower longitude inland regions need to take nonpharmaceutical or pharmaceutical interventions in advance during influenza high-occurrence seasons.

Effects of ambient temperature on influenza-like illness: A multicity analysis in Shandong Province, China, 2014-2017.

Background: The associations between ambient temperature and influenza-like illness (ILI) have been investigated in previous studies. However, they have inconsistent results. The purpose of this study was to estimate the effect of ambient temperature on ILI in Shandong Province, China. Methods: Weekly ILI surveillance and meteorological data over 2014-2017 of the Shandong Province were collected from the Shandong Center for Disease Control and Prevention and the China Meteorological Data Service Center, respectively. A distributed lag non-linear model was adopted to estimate the city-specific temperature-ILI relationships, which were used to pool the regional-level and provincial-level estimates through a multivariate meta-analysis. Results: There were 911,743 ILI cases reported in the study area between 2014 and 2017. The risk of ILI increased with decreasing weekly ambient temperature at the provincial level, and the effect was statistically significant when the temperature was <-1.5°C (RR = 1.24, 95% CI: 1.00-1.54). We found that the relationship between temperature and ILI showed an L-shaped curve at the regional level, except for Southern Shandong (S-shaped). The risk of ILI was influenced by cold, with significant lags from 2.5 to 3 weeks, and no significant effect of heat on ILI was found. Conclusion: Our findings confirm that low temperatures significantly increased the risk of ILI in the study area. In addition, the cold effect of ambient temperature may cause more risk of ILI than the hot effect. The findings have significant implications for developing strategies to control ILI and respond to climate change.

Long-term clinical prognosis of human infections with avian influenza A(H7N9) viruses in China after hospitalization.

BACKGROUND: Mainland China has experienced five epidemics of human cases of avian influenza A(H7N9) virus infection since 2013. We conducted a prospective study to assess long-term clinical, pulmonary function testing, and chest computed tomography (CT) imaging findings after patients were discharged from hospital. METHODS: A(H7N9) survivors in five provinces and one municipality underwent follow-up visits from August 2013 to September 2018, at three, six, and 12 months after illness onset, and a subset was also assessed at 18 and 64 months after onset. Thirteen patients were enrolled from the first A(H7N9) epidemic in 2013, 36 from the 2013-2014 second epidemic, and 12 from the 2016-2017 fifth epidemic. At each visit, A(H7N9) survivors received a medical examination, including the mMRC (modified Medical Research Council) dyspnea scale assessment, chest auscultation, pulmonary function testing and chest CT scans. FINDINGS: The median age of 61 A(H7N9) survivors was 50 years. The cumulative rate of pulmonary dysfunction was 38·5% and 78·2% for chest CT scan abnormalities at the end of follow-up. Restrictive ventilation dysfunction was common during follow-up. Mild dyspnea was documented at three to 12-month follow-up visits. INTERPRETATION: Patients who survived severe illness from A(H7N9) virus infection had evidence of persistent lung damage and long-term pulmonary dysfunction. FUNDING: National Science Fund for Distinguished Young Scholars (grant number 81525023); Program of Shanghai Academic/Technology Research Leader (grant number 18XD1400300); National Science and Technology Major Project of China (grant numbers 2017ZX10103009-005, 2018ZX10201001-010).

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.

Dynamic patterns of circulating influenza virus from 2005 to 2012 in Shandong Province, China.

To identify circulating emerging/reemerging viral strains and epidemiological trends, an influenza sentinel surveillance network was established in Shandong Province, China, in 2005. Nasal and/or throat swabs from patients with influenza-like-illness were collected at sentinel hospitals. Influenza viruses were detected by reverse transcription polymerase chain reaction (RT-PCR) or virus isolation. From October 2005 to March 2012, 7763 (21.44 %) of 36,209 swab samples were positive for influenza viruses, including 5221 (67.25 %) influenza A and 2542 (32.75 %) influenza B. While the influenza viruses were detected year-round, their type/subtype distribution varied significantly. Peak influenza activity was observed from November to February. The proportion of laboratory-confirmed influenza cases was highest among participants aged 0-4 years (14.97 %) in the 2005-2009 and 2010-2012 influenza seasons and the positivity rate of influenza A(H1N1)pdm09 was highest in the 15 to 24 year age group during the 2009-2010 influenza season. Genetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes revealed that the viruses matched seasonal influenza vaccine strains in general, with some amino acid mutations. Influenza A(H1N1)pdm09 strains isolated in Shandong Province were characterized by an S203T mutation that is specific to clade 7 isolates. This report illustrates that the Shandong Provincial influenza surveillance system was sensitive in detecting influenza virus variability by season and by genetic composition. This system will help official public health target interventions such as education programs and vaccines.

Viral Etiology of acute respiratory tract infections in hospitalized children and adults in Shandong Province, China.

BACKGROUND: The dominant viral etiologies responsible for acute respiratory infections (ARIs) are poorly understood, particularly among hospitalized patients. Improved etiological insight is needed to improve clinical management and prevention of ARIs. METHODS: Clinical and demographic information and throat swabs were collected from 607 patients from 2011 to 2013 in Shandong Province, China. Multiplex RT-PCR (SeeplexTM RV detection, Seegene) was performed to detected 12 respiratory viral pathogens. RESULTS: A total of 607 hospitalized patients were enrolled from 2011 to 2013. Viruses were identified in 35.75 % (217/607) of cases, including 78 influenza virus A and B (IVA and IVB), 47 para-influenza viruses (PIVs), 41 respiratory syncytial virus (RSV) and 38 adenovirus (ADV). For the children under 15 year old, the common detected viruses were influenza viruses, RSV, PIVS and ADV, while the principal respiratory viruses were human coronaviruses (HCoV), PIVs, influenza viruses for the old adults. Co-infections with multiple viruses were detected in 15.67 % of patients. Children under 5 years were more likely to have one or more detectable virus associated with their ARI. The peak of ARI caused by the respiratory viruses occurred in winter. CONCLUSION: This study demonstrated respiratory viruses were the major cause of hospitalized ARI patients in Shandong Province, influenza virus was the most common detected, RSV was the highest incidence among the young children (≤5 years). These findings also gave a better understand of virus distribution among different age and seasons, which help to consider potential therapeutic approaches and develop effective prevention strategies for respiratory virus infection.

[Status of acute upper respiratory infection, influenza-like illness, and influenza vaccination coverage among community residents in Jinan].

OBJECTIVE: To analyze the status of acute upper respiratory infection and influenza-like illness (ILI) among community residents in Jinan in 2015, and to make a understand of the patient's medical treatment behavior and influenza vaccination coverage status in 2014. METHODS: Balloting method and convenient sampling method were used to launch a household survey. The residents who had been in Jinan for more than 3 months were selected, to investigate the residents' attack ratio of acute upper respiratory and influenza-like from Jan. 8 to Feb. 7, 2015. Totally, 1 300 persons from 410 families were involved in this survey which recovered 1 241 valid questionnaires with the efficiency of 95.5%. Based on the national age-urban demographic statistics in 2010, the attack rates of acute respiratory infections, influenza-like illness were estimated by the direct standardization method, and the influenza vaccination rates were also calculated in this study. χ(2)-test method was used to compare the different status of incidence and vaccination among residents with different features. RESULTS: The attack rate of acute upper respiratory infection and influenza-like illness in Jinan from January 8, 2015 to February 7, 2015 were 30.2% (375 cases), and 6.1% (76 cases), respectively, with a standardized rate of 29.1% and 5.4%. 5.3% (66 cases) of the residents have vaccinated with the influenza vaccine inoculation, with an adjusted rate of 3.8%. The attack rate difference of acute upper respiratory tract infections was statistically significant between each age group (χ(2)=17.121, P= 0.002). The 0-4 age group had a highest attack rate (45.4%) of acute respiratory infection, while the 15-24 age group got the lowest (26.5%). 38.9% (146 cases) of patients went for a treatment in hospital. Among them, 37.7% (55 cases) of them selected the county level hospitals for treatment, 37.7% (55 cases) selected the community level hospitals, and 24.6% (36 cases) selected the individual clinic. Significant differences of influenza-like illness attack rate between each age group were also found in this study (χ(2)=76.79, P<0.001). 0-4 age group had the highest attack rate (22.7%). 81.6% (62 cases) of the ILI sought treatment in the hospital, of which 53.2% (33 cases) selected county level hospital or above, and 33.9% (21 cases) selected community hospital, and 12.9% (8 cases) selected the individual clinic. CONCLUSION: The attack rate of acute upper respiratory infections among the residents of Jinan was high, whereas that of influenza-like illness was relatively low. 0-4 age group had a higher risk of ILI than other age groups. Most of the cases were likely to take the treatment in large hospitals. In general, the coverage rate of influenza vaccination was relatively low.

One family cluster of avian influenza A(H7N9) virus infection in Shandong, China.

BACKGROUND: The first case of human infection with avian influenza A (H7N9) virus was identified in March, 2013 and the new H7N9 virus infected 134 patients and killed 45 people in China as of September 30, 2013. Family clusters with confirmed or suspected the new H7N9 virus infection were previously reported, but the family cluster of H7N9 virus infection in Shandong Province was first reported. CASE PRESENTATION: A 36-year-old man was admitted to Zaozhuang City Hospital with progressive respiratory distress and suspicion of impending acute respiratory distress syndrome on April 21. The chest radiography revealed bilateral ground-glass opacities and pulmonary lesions. The second case, the first case's 4 year old son, was admitted to the same hospital on April 28 with fever and multiple patchy shadows in the bilateral lungs. Both of the two cases were confirmed to infect with H7N9 virus by the results of real-time reverse transcriptase-polymerase-chain reaction (rRT-PCR), virus isolation and serum antibody titer. At the same time, one environment samples was detected positive for H7N9 virus in the living poultry market in Zaozhuang. The homologous analysis of the full genome sequence indicated that both viruses from the patients were almost genetically identical. The field epidemiology investigation showed that the two cases had no recognized exposure to poultry, but had the exposure to the environment. The second case had substantial unprotected close exposure to his ill father and developed symptoms seven days after his last contact with his father. After surgery, the index case and his son were discharged on May 16 and May 6, respectively. 11 close contacts of both patients were identified and tested negative both the throat swabs and the serum antibody. CONCLUSION: The infection of the index case probably resulted from contact with environmentally contaminated material. For the son, the probable infection source was from the index case during unprotected exposure, but the possibility from the environment or other sources could not be completely ruled out.

Seroepidemiological study of influenza A (H1N1) pdm09 virus following the 2009-2010 wave in Shandong Province, China.

INTRODUCTION: The aims of this study were to understand the geographic extent, risk factors, and attack rate of influenza A (H1N1) pdm09 virus (pH1N1) infection in Shandong Province China and to elucidate influencing factors. METHODS: In January and March 2010, a cross-sectional seroprevalence survey of pH1N1 was carried out. Serum samples from 9022 participants in the survey were subjected to the hemagglutination inhibition assay. RESULTS: Among the 9022 participants, the overall rate of seropositivity against pH1N1 was 22.6%. The weighted rate, adjusted for gender, age, and region, was estimated to be 18.7%. Among 8340 subjects who did not report previous vaccination, the rate was 16.7%, as compared to 50.9% among 682 subjects reporting previous vaccination (p < 0.001). Within the unvaccinated population, the rate of seropositivity among 16-24 and 6-15 y-old subjects was 31.8% and 29.9%, respectively, as compared to 9.6% among subjects aged ≥ 60 y (p < 0.001) and 23% in the 0-5 y age group (p < 0.001). Those aged 6-15 y and aged 16-24 y had higher odds of seropositivity than those aged 0-5 y (odds ratio 1.53 and 1.48, respectively). CONCLUSIONS: These study findings help enhance our understanding of pH1N1 epidemiology and provide valuable information for the vaccination strategy for the influenza season.

Post-pandemic seroprevalence of 2009 pandemic influenza A (H1N1) virus in Shandong Province, China.

The purpose of this study was to understand the geographic extent, risk factors, and attack rate of the 2009 pandemic influenza A (H1N1) virus [A(H1N1)pdm09] infection in Shandong Province, China and to identify the influencing factors. A randomized serological survey of A(H1N1)pdm09 infection was carried out in August and September 2010. A total of 4,549 participants involved in the survey had their antibody levels tested by hemagglutination-inhibition assay. The overall seropositive rate for A(H1N1)pdm09 antibodies was 25.85%. The seropositive rate was 25.89% for the unvaccinated group, with statistically significant differences among individuals of different age groups, occupations, and cities. The highest seropositive rate was observed in young children aged 0-5 years and elderly people aged ≥60 years. Multivariate logistic regression revealed that subjects in rural areas had significantly higher odds ratio of A(H1N1)pdm09 seropositivity than those in the capital city. Individuals belonging to all professions, except for teachers, had significantly lower odds ratio of A(H1N1)pdm09 seropositivity compared with children in family care. Our data indicated that almost 26% of the residents in Shandong Province had appropriate antibody titers against A(H1N1)pdm09. This seroepidemiology study provides valuable data for understanding the epidemiology of the 2009 pandemic influenza and for planning future intervention strategies; moreover, it highlights the significance of seroprevelance studies.

[Advances in the structure and function of pandemic A/H1N1/2009 influenza virus HA protein].

Since March 2009, pandemic A/H1N1/2009 influenza virus has been spreading throughout many countries including China. The emerged virus caused great harm to human health and social economy. Hemagglutinin (HA) is the most important viral surface glycoprotein, mainly possessing three kinds of functions: (1) binding to host cell receptor, (2) triggering the fusion between viral envelop and target cell membrane, (3) stimulating the body to generate the neutralizing antibody. Advances in the structure, primary function, evolution and antigenicity of pandemic A/H1N1/2009 influenza virus HA protein are reviewed in this paper.

Using geographic information system-based ecologic niche models to forecast the risk of hantavirus infection in Shandong Province, China.

Hemorrhagic fever with renal syndrome (HFRS) is an important public health problem in Shandong Province, China. In this study, we combined ecologic niche modeling with geographic information systems (GIS) and remote sensing techniques to identify the risk factors and affected areas of hantavirus infections in rodent hosts. Land cover and elevation were found to be closely associated with the presence of hantavirus-infected rodent hosts. The averaged area under the receiver operating characteristic curve was 0.864, implying good performance. The predicted risk maps based on the model were validated both by the hantavirus-infected rodents' distribution and HFRS human case localities with a good fit. These findings have the applications for targeting control and prevention efforts.

Spatiotemporal trends and climatic factors of hemorrhagic fever with renal syndrome epidemic in Shandong Province, China.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease caused by Hantaviruses. It is endemic in all 31 provinces, autonomous regions, and metropolitan areas in mainland China where human cases account for 90% of the total global cases. Shandong Province is among the most serious endemic areas. HFRS cases in Shandong Province were first reported in Yutai County in 1968. Since then, the disease has spread across the province, and as of 2005, all 111 counties were reported to have local human infections. However, causes underlying such rapid spread and wide distribution remain less well understood. METHODS AND FINDINGS: Here we report a spatiotemporal analysis of human HFRS cases in Shandong using data spanning 1973 to 2005. Seasonal incidence maps and velocity vector maps were produced to analyze the spread of HFRS over time in Shandong Province, and a panel data analysis was conducted to explore the association between HFRS incidence and climatic factors. Results show a rapid spread of HFRS from its epicenter in Rizhao, Linyi, Weifang Regions in southern Shandong to north, east, and west parts of the province. Based on seasonal shifts of epidemics, three epidemic phases were identified over the 33-year period. The first phase occurred between 1973 and 1982 during which the foci of HFRS was located in the south Shandong and the epidemic peak occurred in the fall and winter, presenting a seasonal characteristic of Hantaan virus (HTNV) transmission. The second phase between 1983 and 1985 was characterized by northward and westward spread of HFRS foci, and increases in incidence of HFRS in both fall-winter and spring seasons. The human infections in the spring reflected a characteristic pattern of Seoul virus (SEOV) transmission. The third phase between 1986 and 2005 was characterized by the northeast spread of the HFRS foci until it covered all counties, and the HFRS incidence in the fall-winter season decreased while it remained high in the spring. In addition, our findings suggest that precipitation, humidity, and temperature are major environmental variables that are associated with the seasonal variation of HFRS incidence in Shandong Province. CONCLUSIONS: The spread of HFRS in Shandong Province may have been accompanied by seasonal shifts of HTNV-dominated transmission to SEOV-dominated transmission over the past three decades. The variations in HFRS incidence were significantly associated with local precipitation, humidity, and temperature.

[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.

Genetic properties of medium (M) and small (S) genomic RNA segments of Seoul hantavirus isolated from Rattus norvegicus and antigenicity analysis of recombinant nucleocapsid protein.

A novel isolate of Seoul (SEO) hantaviruses was detected and identified in Rattus norvegicus in Shandong Province, China and designated as JUN5-14. The partial M segment and the coding region of nucleocapsid protein (NP) in the S segment of JUN5-14 were PCR-amplified and sequenced. Nucleotide sequence analysis of the partial M segment (300 bp) revealed that JUN5-14 isolate was closely related to former SEO isolates in Shandong (97.3-99.0% homology) and non-Shandong SEO viruses (84.1-97.7% homology) but distantly related to other hantaviruses (61.5-75.1% homology). Consistent with the M segment, the coding region of the NP showed 87.5-97.8% and 97.9-99.8% identity with SEO viruses and 55.2-75.8% and 47.2-84.4% homology with other hantaviruses, at nucleotide and amino acid level, respectively. The virus isolate was identified as a member of the subtype 3 (S3) of SEO viruses by phylogenetic trees generated from the nucleotide sequences of the S and M segments. In order to develop a diagnostic assay for hantavirus infection in human, the full-length NP gene of JUN5-14 was expressed in BHK21 cells using the T7 RNA polymerase expression system. The NP expression was confirmed by indirect immunofluorescence assay (IFA) and Western blotting. The expressed NP protein was used as antigen to detect antibody response against NP in patients with hemorrhagic fever with renal syndrome (HFRS) in an IgG-IFA. Sixteen out of seventeen serum samples showed positive for the presence of anti-NP antibodies, indicating that the recombinant NP (rNP) protein of JUN5-14 was a good antigen for detecting hantavirus infection in human.