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.

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.

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.

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.

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.

Isolation and genetic characteristics of human genotype 1 Japanese encephalitis virus, China, 2009.

BACKGROUND: Several studies have shown that the predominant genotype of Chinese Japanese encephalitis virus (JEV) is evolving from genotype 3 to genotype 1. However, in recent years, almost all genotype 1 isolates were from mosquitoes, and genotype 1 has been less associated with human disease than genotype 3. This study reports the isolation of human genotype 1 JEV and its genetic characteristics to provide additional insights into human JE pathogens that are currently circulating in China. METHODS AND RESULTS: In 2009, 31 cerebrospinal fluid samples were collected from patients living in Yunnan and Shanxi provinces and were used to inoculate Aedes albopictus C6/36 cells for virus isolation. The JEV strains were identified using immunofluorescent assays and the reverse transcription-polymerase chain reaction. Phylogenetic analyses based on the partial capsid/pre-membrane and full envelope (E) sequences were performed using Clustalx 1.8 software. Three JEV isolates were obtained from a 4-year-old girl and a 2-year-old boy living in Yunnan and an 82-year-old woman in Shanxi. The boy had been immunized with one dose of JE live attenuated vaccine. New isolates were grouped into genotype 1. Amino acid sequence for the viral E protein indicated 95% to 100% identity with each other and with other JEV strains. When compared with a consensus sequence of E protein, two amino acid substitutions were found: Ser(E-123)-Asn in the two Yunnan isolates and Lys(E-166)-Arg in the Shanxi isolate. CONCLUSIONS: Our findings indicate that the genotype 1 of JEV is causing human infections in China. Our observation of a previously vaccinated boy developing JE from genotype 1 virus infection also calls for more detailed studies, both in vitro and in vivo neutralization tests as well as active surveillance, to examine the possibility of a lack of complete protection conferred by the live attenuated JE vaccine against genotype 1 virus.

Geo-spatial hotspots of hemorrhagic fever with renal syndrome and genetic characterization of Seoul variants in Beijing, China.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) is highly endemic in mainland China, and has extended from rural areas to cities recently. Beijing metropolis is a novel affected region, where the HFRS incidence seems to be diverse from place to place. METHODOLOGY/PRINCIPAL FINDINGS: The spatial scan analysis based on geographical information system (GIS) identified three geo-spatial "hotspots" of HFRS in Beijing when the passive surveillance data from 2004 to 2006 were used. The Relative Risk (RR) of the three "hotspots" was 5.45, 3.57 and 3.30, respectively. The Phylogenetic analysis based on entire coding region sequence of S segment and partial L segment sequence of Seoul virus (SEOV) revealed that the SEOV strains circulating in Beijing could be classified into at least three lineages regardless of their host origins. Two potential recombination events that happened in lineage #1 were detected and supported by comparative phylogenetic analysis. The SEOV strains in different lineages and strains with distinct special amino acid substitutions for N protein were partially associated with different spatial clustered areas of HFRS. CONCLUSION/SIGNIFICANCE: Hotspots of HFRS were found in Beijing, a novel endemic region, where intervention should be enhanced. Our data suggested that the genetic variation and recombination of SEOV strains was related to the high risk areas of HFRS, which merited further investigation.

Genetic analysis of a hantavirus strain carried by Niviventer confucianus in Yunnan province, China.

Hantavirus genome sequences were recovered from lung tissues of Chinese white-bellied rats (Niviventer confucianus) captured in Yunnan province, China. Pairwise comparison of the nucleotide and deduced amino acid sequences of the entire S and partial M and L segments indicated that the newly discovered virus strain, which was designated as strain YN509, was very different from other rodent-borne hantaviruses. Phylogenetic analysis showed that the new strain fit into a clade containing Da Bie Shan virus (DBSV) (also carried by N. confucianus), which is mainly found in Anhui Province in mainland China. Strain YN509 appears to be in a sister taxa of the DBSV group described previously. These data suggest that strain YN509 is a new subtype of DBSV, which appears to be widely distributed in China with a higher genetic diversity than expected.

Anaplasma phagocytophilum in livestock and small rodents.

To identify the prevalence of Anaplasma phagocytophilum in both wild rodents and domestic animals and to make clear the genetic characteristics of the agents from different animals in China, a total of 105 livestock and 159 small rodents were analyzed by real-time-PCR and sequence analysis. The prevalence rate was 6.7% (7/105) and 14.5% (23/159), respectively. The nucleotide sequences of 16S rRNA (rrs) from the positive livestock and rodents were identical to each other. The phylogenetic analysis based on partial A. phagocytophilum p44ESup1 gene revealed that A. phagocytophilum identified in this study was placed on a separate clade distinct from those in other continents. These findings indicated A. phagocytophilum in rodents might be able to infect livestock and intensified the threats of anaplasmosis to livestock in the area. Further studies on public health significance of the agent are worth investigation in future.

Anaplasma phagocytophilum from Rodents and Sheep, China.

To characterize the strains of Anaplasma phagocytophilum in wild and domestic animals in China, we isolated the organism from rodents and sheep in northeastern China. We isolated 3 strains (2 from rodents and 1 from sick sheep) through propagation in BALB/c mice and then cell culture in HL60 cells. The 3 isolates were identified by Wright-Giemsa staining, immunofluorescence, and electronic microscopy and were characterized by sequence analyses of the 16S rRNA gene, partial citrate synthase gene, major surface protein 4 gene, and heat shock protein gene. The multiple sequences of the 3 isolates were identical to each other but different from all known strains from other countries. The public health and veterinary relevance of the isolates deserves further investigation.

Tick-borne agents in rodents, China, 2004-2006.

A total of 705 rodents from 6 provinces and autonomous regions of mainland People's Republic of China were tested by PCRs for tick-borne agents (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, spotted fever group rickettsiae, and Francisella tularensis). Infection rates were 5.5%, 6.7%, 9.1% and 5.0%, respectively. Eighteen (2.6%) rodents of 10 species were positive for 2 or 3 agents. Sequence analysis of PCR products confirmed the presence and genotypes of detected agents. These findings demonstrate that these tick-borne agents cocirculate and that a variety of rodent species may be involved in their enzootic maintenance.

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.

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.

Seoul virus in patients and rodents from Beijing, China.

Hemorrhagic fever with renal syndrome (HFRS) is a significant public health problem with an increasing incidence in Beijing, China (report of disease surveillance from the Center for Disease Control and Prevention of Beijing, China). Hantaviruses were detected using RT-PCR method in blood samples of HFRS patients and lung tissues of rodents captured in Beijing. Phylogenetic analyses of 724bp partial S segment of the hantavirus gene showed that the detected Seoul virus (SEOV) fell into three different lineages, two of which circulated in Beijing. A nucleotide sequence identity of 99.7% for one of the cases of HFRS--the human- and Rattus norvegicus-originated SEOV sequences--had only two silent substitutions, suggesting genetic analysis is an essential tool for "case-investigation."

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.

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.