Complete genome sequence of a novel negevirus isolated from Culex tritaeniorhynchus in China.

A novel negevirus, tentatively named Manglie virus (MaV), was isolated from Culex tritaeniorhynchus from the village of Manglie, Yunnan, China, in August 2011. It was identified by high-throughput sequencing of cell culture supernatants, and the complete genome was sequenced using an Illumina MiSeq sequencer. The complete MaV genome comprised 9,218 nt encoding three hypothetical proteins and had a poly(A) tail. BLASTn analysis showed that the genome had the greatest similarity to Ngewotan virus strain Nepal22, with query coverage of 100% and 79% identity. Genomic and phylogenetic analyses demonstrated that MaV should be considered a novel negevirus.

Complete genome sequence of Menghai flavivirus, a novel insect-specific flavivirus from China.

Menghai flavivirus (MFV) was isolated from Aedes albopictus in Menghai county of Yunnan Province, China, during an arboviruses screening program in August 2010. Whole genome sequencing of MFV was performed using an Ion PGM™ Sequencer. The complete genome of MFV was 10897 nucleotides in length and encoded a polyprotein and fairly interesting flavivirus orf (FIFO). The polyprotein contained three flavivirus structural proteins (C, prM/M and E) and seven nonstructural proteins. Nucleotide BLAST analysis revealed that the MFV genome showed highest similarity to Xishuangbanna Aedes flavivirus, a novel insect-specific flavivirus recently isolated from the same area. These species shared a query cover of 99%, but only 71% identity, while FIFO showed no similarity with any of the published sequences. Genomic and phylogenetic analyses suggested that MFV was a novel species of the genus Flavivirus. Our findings enrich our understanding of the genetics and prevalence of the family Flaviviridae.

Complete genome sequence of Menghai rhabdovirus, a novel mosquito-borne rhabdovirus from China.

Menghai rhabdovirus (MRV) was isolated from Aedes albopictus in Menghai county of Yunnan Province, China, in August 2010. Whole-genome sequencing of MRV was performed using an Ion PGM™ Sequencer. We found that MRV is a single-stranded, negative-sense RNA virus. The complete genome of MRV has 10,744 nt, with short inverted repeat termini, encoding five typical rhabdovirus proteins (N, P, M, G, and L) and an additional small hypothetical protein. Nucleotide BLAST analysis using the BLASTn method showed that the genome sequence most similar to that of MRV is that of Arboretum virus (NC_025393.1), with a Max score of 322, query coverage of 14%, and 66% identity. Genomic and phylogenetic analyses both demonstrated that MRV should be considered a member of a novel species of the family Rhabdoviridae.

Complete genome sequence of Xishuangbanna flavivirus, a novel mosquito-specific flavivirus from China.

A new flavivirus, Xishuangbanna flavivirus (XFV), infecting Aedes albopictus mosquitoes in Yunnan Province, China, was isolated and sequenced. The single-stranded RNA genome of 10,884 nt contained two open reading frames (ORFs) encoding the polyprotein and FIFO. The genome had a maximum nucleotide sequence identity of 65 % to Parramatta River virus with coverage of only 27 %. Phylogenetic analysis suggested that this virus is most closely related to recognized classical insect-specific flaviviruses (cISF) and most likely has a similar host range. Sequence comparisons and phylogenetic analysis demonstrated that XFV is a new member of the genus Flavivirus.

First isolation of dengue virus from Lao PDR in a Chinese traveler.

BACKGROUND: Epidemic dengue activity has been demonstrated in several southern regions of China, but not in Yunnan province, which borders countries in Southeast Asia where dengue is endemic. Many dengue cases imported from Southeast Asia to Yunnan have been reported, but dengue virus (DENV) has not been isolated from any patients. This study is the first to report the isolation of DENV from a Chinese traveler returning to Yunnan from Lao PDR. FINDINGS: A serum sample was collected from a patient presenting with a febrile illness who returned from Lao PDR in 2009 and was used to inoculate Aedes albopictus C6/36 cells for viral isolation. The viral isolate was identified using reverse transcription-polymerase chain reaction, and phylogenetic analyses based on the full E sequence were performed using Clustalx 1.8 software. The analyses detected DENV genome, and thus, a DENV isolate was obtained from the patient's serum sample. The new DENV isolate was grouped into genotype Asia 1, serotype 2. The viral E protein shared the greatest nucleotide sequence identity (99.6%) with the D2/Thailand/0606aTw strain isolated from Thailand in 2006 and demonstrated 94.3% to 100% identity with the predicted amino acid sequence of other DENV 2 strains. CONCLUSIONS: Our findings indicate that DENV serotype 2 is circulating in Lao PDR, and surveillance of patients suspected of infection with dengue should be conducted not only by a serological test but also by pathogenic detection methods.

Hantaviruses in rodents and humans, Xi'an, PR China.

Xi'an, the capital of Shaanxi province, located in north-west China, is one of the major endemic areas for haemorrhagic fever with renal syndrome (HFRS). In this study, the epidemiological data of HFRS in Xi'an from 1959 to 2010, especially in the past ten years (2001-2010), were surveyed. The features of hantavirus (HV) host carriers, the molecular characteristics of the HV S gene from hosts and patients, and the genome of the viral isolate were also investigated. Data showed that there might be a ten-year cycle of HFRS in Xi'an. Although the main population group infected over the past ten years was still the 16-59-year-old male farmers, the composition of the population and geographical distribution of HFRS cases have changed slowly, accompanied by the development of environmental and socio-economic situations. Apodemus agrarius remains the dominant host of HV. The HV strains from host rodents and patients in Xi'an belonged to the Hantaan virus (HTNV); no Seoul virus strains were found. Phylogenetic analysis of the small segments of strains taken from hosts and patients, and the whole genome of a viral isolate showed that the virus circulating in Xi'an had high similarity to Guizhou strains. The study also indicated that the vaccine candidate strain A16 isolated during the past century in Xi'an might be a recombinant strain of HTNV and the Amur virus, thus it may not be an optimal vaccine strain.

Encephalitis temporally associated with live attenuated Japanese encephalitis vaccine: four case reports.

BACKGROUND: Japanese encephalitis (JE) vaccination is the most effective measure for preventing JE disease. The live attenuated JE vaccine, which has shown good efficacy and safety, has been widely used in China. CASE PRESENTATIONS: We report four laboratory-confirmed JE cases detected in JE-endemic areas during the JE virus (JEV) transmission season, who all received a first dose of live attenuated JE vaccine within 2 weeks prior to the onset of illness. All cases presented with acute encephalitis and rapidly reduced consciousness. All cerebrospinal fluid (CSF) samples from the patients were positive for JEV-specific immunoglobulin M (IgM) antibodies, but viral isolation and polymerase chain reaction (PCR) detection of JEV were both negative. CONCLUSIONS: It is difficult to identify a causal link between the disease and the vaccination, as the source of positive CSF JEV IgM antibodies might be natural JEV infection or possibly due to a traumatic lumbar puncture. Our observations highlight the need for public health officers and doctors to consider reasonable vaccination policies during the JE season. In addition, continued surveillance as well as thorough investigation of any events that occur after JE vaccination is necessary.

Clinical characteristics of the autumn-winter type scrub typhus cases in south of Shandong province, northern China.

BACKGROUND: Before 1986, scrub typhus was only found endemic in southern China. Because human infections typically occur in the summer, it is called "summer type". During the autumn-winter period of 1986, a new type of scrub typhus was identified in Shandong and northern Jiangsu province of northern China. This newly recognized scrub typhus was subsequently reported in many areas of northern China and was then called "autumn-winter type". However, clinical characteristics of associated cases have not been reported. METHODS: From 1995 to 2006, all suspected scrub typhus cases in five township hospitals of Feixian county, Shandong province were enrolled. Indirect immunofluorescent assay (IFA) was used as confirmatory serodiagnosis test. Polymerase chain reaction (PCR) connected with restriction fragment length polymorphism (RFLP) and sequence analyses were used for genotyping of O. tsutsugamushi DNAs. Clinical symptoms and demography of confirmed cases were analyzed. RESULTS: A total of 480 scrub typhus cases were confirmed. The cases occurred every year exclusively between September and December with a peak occurrence in October. The case numbers were relatively higher in 1995, 1996, 1997, and 2000 than in other years. 57.9% of cases were in the group aged 21-50. More cases occurred in male (56%) than in female (44%). The predominant occupational group of the cases was farmers (85.0%). Farm work was reported the primary exposure to infection in 67.7% of cases. Fever, rash, and eschar were observed in 100.0%, 90.4%, and 88.5% of cases, respectively. Eschars formed frequently on or around umbilicus, abdomen areas, and front and back of waist (34.1%) in both genders. Normal results were observed in 88.7% (WBC counts), 84.5% (PLT counts), and 89.7% (RBC counts) of cases, respectively. Observations from the five hospitals were compared and no significant differences were found. CONCLUSION: The autumn-winter type scrub typhus in northern China occurred exclusively from September to December with a peak occurrence in October, which was different from the summer type in southern China. In comparison with the summer type, complications associated with autumn-winter type scrub typhus were less severe, and abnormalities of routine hematological parameters were less obvious.

Anaplasma phagocytophilum and Borrelia burgdorferi in rabbits from southeastern China.

A total of 54 wild rabbits captured from southeastern China were examined for Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato by polymerase chain reaction (PCR) assays. One and three samples were positive for A. phagocytophilum and B. burgdorferi, respectively. Sequence analyses of PCR products identified a variant of A. phagocytophilum and a B. garinii genotype. This is the first detection of the two tick-borne agents in Chinese rabbits, the role of which in the maintenance of the agents deserve further investigations.

The characteristics of current natural foci of hemorrhagic fever with renal syndrome in Shandong Province, China, 2012-2015.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS), an infectious disease caused by hantaviruses, is endemic in China and remains a serious public health problem. Historically, Shandong Province has had the largest HFRS burden in China. However, we do not have a comprehensive and clear understanding of the current epidemic foci of HFRS in Shandong Province. METHODOLOGY/PRINCIPAL FINDINGS: The incidence and mortality rates were calculated, and a phylogenetic analysis was performed after laboratory testing of the virus in rodents. Spatial epidemiology analysis was applied to investigate the epidemic foci, including their sources. A total of 6,206 HFRS cases and 59 related deaths were reported in Shandong Province. The virus carriage rates of the rodents Rattus norvegicus, Apodemus agrarius and Mus musculus were 10.24%, 6.31% and 0.27%, respectively. The phylogenetic analysis indicated that two novel viruses obtained from R. norvegicus in Anqiu City and Qingzhou City were dissimilar to the other strains, but closely related to strains previously isolated in northeastern China. Three epidemic foci were defined, two of which were derived from the Jining and Linyi epidemic foci, respectively, while the other was the residue of the Jining epidemic focus. CONCLUSIONS/SIGNIFICANCE: The southeastern and central Shandong Province are current key HFRS epidemic foci dominated by A. agrarius and R. norvegicus, respectively. Our study could help local departments to strengthen prevention and control measures in key areas to reduce the hazards of HFRS.

Prevalence and genetic diversities of hantaviruses in rodents in Beijing, China.

A total of 835 rodents captured in Beijing, China, were tested for hantavirus infection. Fifty-five (6.6%) were positive for viral RNA when lung tissue samples were examined by reverse transcriptase-polymerase chain reaction. Of 666 sera collected from the above rodents, 50 (7.5%) were positive for IgG antibody by ELISA. Among the 50 seropositive rodents, 37 were positive for viral RNA. In addition, five rodents were positive for viral RNA but negative for IgG antibody. The infection rates among study sites (chi(2) = 28.93, df = 8, P = 0.001) and habitats (chi(2) = 22.88,df = 7, P = 0.02) were significantly different. The sequences of partial M-segment of hantaviruses detected in 11 representative rodents had 0.1-8.2% divergence. Phylogenetic analysis showed that our hantavirus sequences fell into three different lineages regardless of geographical origin or rodent species. A strain detected from a trading center of agricultural products, which might be imported from other provinces, was genetically different from other strains of Beijing.

Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes.

The hemagglutination inhibition (HI) assay is a widely used serological method to measure the levels of protective antibody responses against influenza viruses. However, the traditional HI assay which uses chicken erythrocytes is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes was able to increase the sensitivity of HI assay. The current report describes further optimization of this modified HI assay. It was shown that this method was able to increase detection of HI activities in rabbit sera immunized with H5 HA antigens, and proved that this increased sensitivity is useful in dissecting the strain specificity of HI antibody responses. In addition, the modified HI assay using horse erythrocytes increased the sensitivity of detecting HI antibodies specific for three major serotypes of avian influenza viruses, H5, H7 and H9, in people who may have asymptomatic infection with avian influenza viruses. Based on these results, the optimized use of horse erythrocytes should be standard practice for detecting HI activities against avian influenza viruses.

Genetic Diversity and the Spatio-Temporal Analyses of Hantaviruses in Shandong Province, China.

Hemorrhagic fever with renal syndrome (HFRS) is a serious public health problem in Shandong Province, China. We conducted an epizootiologic investigation and phylogeographic and phylodynamic analyses to infer the phylogenetic relationships of hantaviruses in space and time, and gain further insights into their evolutionary dynamics in Shandong Province. Our data indicated that the Seoul virus (SEOV) is distributed throughout Shandong, whereas Hantaan virus (HTNV) co-circulates with SEOV in the eastern and southern areas of Shandong. Their distribution showed strong geographic clustering. In addition, our analyses indicated multiple evolutionary paths, long-distance transmission, and demographic expansion events for SEOV in some areas. Selection pressure analyses revealed that negative selection on hantaviruses acted as the principal evolutionary force, whereas a little evidence of positive selection exists. We found that several positively selected sites were located within major functional regions and indicated the importance of these residues for adaptive evolution of hantaviruses.

A new Hantaan-like virus in rodents (Apodemus peninsulae) from Northeastern China.

Lung tissue samples of 76 Korean field mice (Apodemus peninsulae) collected from northeastern China bordering with Far East Russia and Korea were detected for hantavirus partial M-segment or entire S-segment sequences by RT-PCR and 481-nt mitochondrial DNA fragment of the rodents. Four A. peninsulae mice were found positive for partial M-segment of hantavirus. Sequence analyses of partial M-segment or/and entire S-segment of the hantaviruses revealed that three were closely related to Hantaan virus (HTNV) strain 76-118. One new variant of HTNV-like virus designated as "Jilin-AP06" was much different from other rodent-borne hantavirus from China, and clustered with Amur (AMR) virus strains, which represent a distinct genetic lineage. These findings imply that hantavirus Jililn-AP06 strain from A. peninsulae is a new record of rodent-borne AMR virus in China. A. peninsulae might be a natural carrier of two distinct hantaviruses, AMR virus and HTNV in China.

The spatiotemporal expansion of human rabies and its probable explanation in mainland China, 2004-2013.

BACKGROUND: Human rabies is a significant public health concern in mainland China. However, the neglect of rabies expansion and scarce analyses of the dynamics have made the spatiotemporal spread pattern of human rabies and its determinants being poorly understood. METHODS: We collected geographic locations and timeline of reported human rabies cases, rabies sequences and socioeconomic variables for the years 2004-2013, and integrated multidisciplinary approaches, including epidemiological characterization, hotspots identification, risk factors analysis and phylogeographic inference, to explore the spread pattern of human rabies in mainland China during the last decade. RESULTS: The results show that human rabies distribution and hotspots were expanding from southeastern regions to north or west regions, which could be associated with the evolution of the virus, especially the clade I-G. A Panel Poisson Regression analysis reveals that human rabies incidences had significant correlation with the education level, GDP per capita, temperature at one-month lag and canine rabies outbreak at two-month lag. CONCLUSIONS: The reduction in the overall human rabies incidence was accompanied by a westward and northward expansion of the circulating region in mainland China. Higher risk of human rabies was associated with lower level of education and economic status. New clades of rabies, especial Clade I-G, played an important role in recent spread. Our findings provide valuable information for rabies control and prevention in the future.

The association between hantavirus infection and selenium deficiency in mainland China.

Hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses and transmitted by rodents is a significant public health problem in China, and occurs more frequently in selenium-deficient regions. To study the role of selenium concentration in HFRS incidence we used a multidisciplinary approach combining ecological analysis with preliminary experimental data. The incidence of HFRS in humans was about six times higher in severe selenium-deficient and double in moderate deficient areas compared to non-deficient areas. This association became statistically stronger after correction for other significant environment-related factors (low elevation, few grasslands, or an abundance of forests) and was independent of geographical scale by separate analyses for different climate regions. A case-control study of HFRS patients admitted to the hospital revealed increased activity and plasma levels of selenium binding proteins while selenium supplementation in vitro decreased viral replication in an endothelial cell model after infection with a low multiplicity of infection (MOI). Viral replication with a higher MOI was not affected by selenium supplementation. Our findings indicate that selenium deficiency may contribute to an increased prevalence of hantavirus infections in both humans and rodents. Future studies are needed to further examine the exact mechanism behind this observation before selenium supplementation in deficient areas could be implemented for HFRS prevention.

Emerging tick-borne infections in mainland China: an increasing public health threat.

Since the beginning of the 1980s, 33 emerging tick-borne agents have been identified in mainland China, including eight species of spotted fever group rickettsiae, seven species in the family Anaplasmataceae, six genospecies in the complex Borrelia burgdorferi sensu lato, 11 species of Babesia, and the virus causing severe fever with thrombocytopenia syndrome. In this Review we have mapped the geographical distributions of human cases of infection. 15 of the 33 emerging tick-borne agents have been reported to cause human disease, and their clinical characteristics have been described. The non-specific clinical manifestations caused by tick-borne pathogens present a major diagnostic challenge and most physicians are unfamiliar with the many tick-borne diseases that present with non-specific symptoms in the early stages of the illness. Advances in and application of modern molecular techniques should help with identification of emerging tick-borne pathogens and improve laboratory diagnosis of human infections. We expect that more novel tick-borne infections in ticks and animals will be identified and additional emerging tick-borne diseases in human beings will be discovered.

Detection of Quang Binh virus from mosquitoes in China.

Flaviviruses present a wide range of genetic diversity and exhibit diverse host relationships. Mosquito-borne flaviviruses have recently been isolated and characterized worldwide. Yunnan Province of China is one of the richest areas of species diversity and is the center of multi-species evolution in mainland Asia, which supports the circulation of numerous arthropod-borne viruses (arboviruses). In a screening program of arboviruses, mosquitoes were collected during the mosquito activity season in the Yunnan Province from 2007 to 2010. Eleven flavivirus strains, named Yunnan Culex flaviviruses (YNCxFVs), were obtained from Culex tritaeniorhynchus and Anopheles sinensis specimens. Sequence analyses based on partial nonstructural protein (NS) 5 gene indicated that the YNCxFVs shared 92.8-99.6% nucleotide identity with each other and were similar to the Culex-related flaviviruses. The complete genome of one representative isolate, LSFlaviV-A20-09, was sequenced. The genome was 10,865 nucleotides long and contained a single, long open reading frame (ORF) of 10,080 nucleotides that encoded a 3360-aa polyprotein. This genome was most closely related to the Quang Binh virus (QBV) VN180 strain, an insect-specific flavivirus isolated from Culex mosquitoes in Vietnam, but only had 83.0% nucleotide and 93.8% amino acid identities for the ORF sequence. The genome has approximately 66.3%-68.5% nucleotide sequence and 69.3-73.3% amino acid sequence identities to other Culex flaviviruses, and only has 47.9-57.9% nucleotide sequence and 38.7-55.1% amino acid sequence identities to Coquillettidia-related, Mansonia-related and Aedes-related flaviviruses. Phylogenetic analyses revealed that the LSFlaviV-A20-09 fell into the Culex-related flavivirus clade. Our discoveries provide more information regarding the heterogeneity of viruses that infect mosquitoes.

A new hantavirus from the stripe-backed shrew (Sorex cylindricauda) in the People's Republic of China.

Inspired by the recent discovery of genetically distinct hantaviruses from insectivore species worldwide, we performed a small-scale search for insectivore-borne hantaviruses. In this paper, we report the discovery of a new hantavirus, which was designated the Qian Hu Shan virus (QHSV). This virus was detected in the lung tissues of three stripe-backed shrews (Sorex cylindricauda), which were captured in the Yunnan Province, China. The full-length S genomic segment of the representative QHSV strain YN05-284 was 1661 nucleotides and is predicted to encode a nucleocapsid protein of 429 amino acids that starts at nucleotide position 48. It exhibited the highest similarity with other Sorex-related hantaviruses, with 68.1%-72.8% nucleotide and 71.9%-84.4% amino acid sequence identities. An analysis of a 1430-nucleotide region of the partial M segment exhibited approximately 54.4%-79.5% nucleotide and 43.2%-90.8% amino acid sequence identities to other hantaviruses. A comparison of a 432-nucleotide region of the L segment also showed similar degrees of identity, with 68.9%-78.4% nucleotide and 71.1%-93.8% amino acid sequence identities to other hantaviruses. Phylogenetic analyses using Bayesian methods indicated that QHSV shared the most recent common ancestor with other Sorex-related hantaviruses. The host was identified using a morphological assessment and verified using mitochondrial cytochrome b (mt-Cyt b) gene sequencing. A pair-wise comparison of the 1140-nucleotide mt-Cyt b gene sequence from the host demonstrated that the host was close to S. cylindricauda from Nepal with 94.3% identity. The virus-host association tanglegram, which was constructed using the Dendroscope software, indicated that the QHSV phylogeny and the host phylogeny were approximately matched, which suggests no evidence of host switching for QHSV. Our results contribute to a wider viewpoint regarding the heterogeneity of viruses that infect shrews.

Cytokine and chemokine responses to Japanese encephalitis live attenuated vaccine in a human population.

OBJECTIVES: The SA14-14-2 Japanese encephalitis (JE) live attenuated vaccine is licensed for use only in China, and has provided excellent efficacy in reducing the incidence of JE. The humoral immune response related to the JE vaccination has been well characterized, however cellular immune responses are less well known. METHODS: Thirty-four healthy males who had recently received inoculation with the SA14-14-2 live attenuated vaccine were recruited. Serum samples from these subjects were analyzed for cytokine and chemokine levels using the FlowCytomix method. RESULTS: Eighteen of 34 subjects were positive for JE virus-specific IgG antibodies. Levels of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and MIP-1ß were significantly higher in the vaccinees than in a control group (p<0.0001, p<0.0001, p=0.021, and p<0.0001, respectively). IL-6 was detectable in 64.7% of vaccinees, but was not detectable in any of the controls. IL-1ß, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-22, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were detected in very few subjects or were undetectable in both groups. CONCLUSIONS: IL-6, IL-8, MCP-1, MIP-1α, and MIP-1ß may play important roles in the immune response to JE live attenuated vaccine.