Infection with severe fever with thrombocytopenia virus in healthy population: a cohort study in a high endemic region, China.

BACKGROUND: Severe fever with thrombocytopenia (SFTS) caused by SFTS virus (SFTSV) was a tick-borne hemorrhagic fever that posed significant threat to human health in Eastern Asia. The study was designed to measure the seroprevalence of SFTSV antibody in healthy population residing in a high endemic region. METHODS: A cohort study was performed on healthy residents in Shangcheng County in Xinyang City from April to December in 2018, where the highest SFTS incidence in China was reported. Anti-SFTSV IgG was measured by indirect enzyme-linked immunosorbent assay and neutralizing antibody (NAb) was detected by using PRNT50. The logistic regression models were performed to analyze the variables that were associated with seropositive rates. RESULTS: Totally 886 individuals were recruited. The baseline seroprevalence that was tested before the epidemic season was 11.9% (70/587) for IgG and 6.8% (40/587) for NAb, which was increased to 13.4% (47/350) and 7.7% (27/350) during the epidemic season, and further to 15.8% (80/508) and 9.8% (50/508) post epidemic. The IgG antibody-based seropositivity was significantly related to the patients aged ≥ 70 years old [adjusted odds ratio (OR) = 2.440, 95% confidence interval (CI): 1.334-4.461 compared to the group of < 50 years old, P = 0.004], recent contact with cats (adjusted OR = 2.195, 95% CI: 1.261-3.818, P = 0.005), and working in tea garden (adjusted OR = 1.698, 95% CI: 1.002-2.880, P = 0.049) by applying multivariate logistic regression model. The NAb based seropositivity was similarly related to the patients aged ≥ 70 years old (adjusted OR = 2.691, 95% CI: 1.271-5.695 compared to the group of < 50 years old, P = 0.010), and recent contact with cats (OR = 2.648, 95% CI: 1.419-4.941, P = 0.002). For a cohort of individuals continually sampled with 1-year apart, the anti-SFTSV IgG were maintained at a stable level, while the NAb level reduced. CONCLUSIONS: Subclinical infection might not provide adequate immunity to protect reinfection of SFTSV, thus highlighting the ongoing threats of SFTS in endemic regions, which called for an imperative need for vaccine development. Identification of risk factors might help to target high-risk population for public health education and vaccination in the future.

Correlation between thrombocytopenia and host response in severe fever with thrombocytopenia syndrome.

Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging infectious disease caused by a novel bunyavirus, SFTS virus (SFTSV), with fatal outcome developed in approximately 17% of the cases. Thrombocytopenia is a hallmark feature of SFTS, and associated with a higher risk of fatal outcome, however, the pathophysiological involvement of platelet in the clinical outcome of SFTS remained under-investigated. In the current study, by retrospectively analyzing 1538 confirmed SFTS patients, we observed that thrombocytopenia was associated with enhanced activation of the cytokine network and the vascular endothelium, also with a disturbed coagulation response. The platelet phenotypes were also extensively altered in the process of thrombocytopenia development of SFTS patients. More importantly, all these disturbed host responses were related to the severity of thrombocytopenia, thus were considered to play in a synergistic way to influence the disease outcome. Moreover, the clinical effect of platelet transfusion was assessed by comparing two groups of patients with or without receiving this therapy. As a result, we observed no therapy effect in altering frequencies of fatal outcome, clinical bleeding development, or dynamic change of platelet count during the hospitalization. It's suggested that platelet supplementation alone acted a minor role in improving disease outcome, therefore new therapeutic intervention to regulate host response should be proposed. The current results revealed some evidence of interrelationship between platelet count and clinical outcome of SFTS disease from the perspective of activation of the cytokine network, the vascular endothelium, and the coagulation/fibrinolysis system. These evaluations might help to attain a better understanding of the pathogenesis and therapy choice in SFTS.

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.

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.

Discovery of two novel totiviruses from Culex tritaeniorhynchus classifiable in a distinct clade with arthropod-infecting viruses within the family Totiviridae.

Two double-stranded RNA viruses, named Culex tritaeniorhynchus totivirus NJ2 (CTV_NJ2) and NJ3 (CTV_NJ3), were discovered from wild-captured Culex tritaeniorhynchus mosquitoes. The complete genomes (7,624 and 7,612 bp in length) were obtained using RNA sequencing. Both CTV_NJ2 and CTV_NJ3 encode a putative capsid protein and an RNA-dependent RNA polymerase. The most similar strain to CTV_NJ2/3 is Omono River virus strain AK4 (ORV-AK4). The CP and RdRp identities of AK4 are different to CTV_NJ2 (84% and 87%) and CTV_NJ3 (47% and 62%). Phylogenetic analysis showed that taxonomically speaking CTV_NJ2/3 grouped within the unclassified Totiviridae and formed a distinct clade with other arthropod-infecting viruses.

Investigation on Mosquito-Borne Viruses at Lancang River and Nu River Watersheds in Southwestern China.

During 2007 and 2010, an extensive entomological survey was performed to assess the distribution of mosquitoes and mosquito-borne arboviruses at Lancang River and Nu River watersheds in southwestern China. A total of 20,450 mosquitoes consisting 20 species was trapped and submitted 261 pools according to species and location. Culex tritaeniorhynchus and Anopheles sinensis were the most abundant species. Eighty-seven isolates representing 11 virus species in 8 genera were obtained from 6 mosquito species. The new isolates were identified as Getah virus (GETV), Japanese encephalitis virus (JEV), Yunnan Culex-related flavivirus (YNCxFV), Yunnan Aedes-related flavivirus (YNAeFV), Banna virus (BAV), Yunnan orbivirus (YUOV), Banna orbivirus (BAOV), Yunnan totivirus (YNToV), Nam Dinh virus (NDiV), Menghai rhabdovirus (MRV), and Anopheles minimus iridovirus (AMIV). These viruses included confirmed or potential pathogen of human disease, such as JEV, BAV, and NDiV, and several novel or reassortant arboviruses, such as YNAeFV, MRV, AMIV, and BAOV. GETV, JEV, YNCxFV, and NDiV were widely prevalent in the whole basin of the two rivers. The findings contribute to our understanding of the diversity and wide distribution of mosquito-borne arboviruses in the area, and are helpful to explore pathogenic evidence for fevers and viral encephalitis of unknown etiology.

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.

Molecular Characterization and Viral Origin of the First Dengue Outbreak in Xishuangbanna, Yunnan Province, China, 2013.

In August 2013, Xishuangbanna, Yunnan Province, China, had its first dengue outbreak. Dengue virus (DENV) RNA detection in sera or viral isolates revealed that all 222 autochthonous patients detected and three Chinese travelers from Laos (imported cases) were positive for DENV-3 serotype, while DENV-1 and DENV-4 were detected in travelers from Myanmar and Thailand during the outbreak. For 33 suspected dengue cases collected before the outbreak, two imported cases from Laos and nine residents living in Laos (Laotian cases) were positive for DENV-3. Further, a random subset of 33 positive cases for DENV-3 was sequenced for the full envelope gene of DENV. Phylogenetic analysis showed that all of the 25 autochthonous cases sequenced were grouped into the same clade, genotype II of DENV-3, with imported cases from Laos and Laotian cases. These results suggest that the genotype II of DENV-3 was associated with the outbreak and may have originated from the virus circulating in Laos.

Isolation and characterization of a novel invertebrate iridovirus from adult Anopheles minimus (AMIV) in China.

An invertebrate iridovirus (designated AMIV) was isolated from adult wild-captured Anopheles minimus mosquitoes in China. AMIV was pathologically and morphologically characterized and sequenced using the Ion Torrent™ sequencing platform. Phylogenetic analysis based on both the major capsid protein and core genes revealed that AMIV differs from all the members of the family Iridoviridae. The AMIV negatively strained virion has a diameter of about 130nm. AMIV contains a linear DNA molecule of 163,023bp, with 39% G+C content and 148 coding sequences. The genome analysis revealed that AMIV genome encodes a high content of replication associated genes including BRO-like genes. This is the ninth complete genome of IIV reported.

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.

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.

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.

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.

Perinatal outcomes and congenital abnormalities in the newborns of women affected by the 2009 pandemic influenza A (H1N1) in Beijing, China.

OBJECTIVE: To investigate the pregnancy complications, perinatal outcomes, and congenital abnormalities (CAs) that occurred in Beijing, China, when pregnant women became infected with the 2009 pandemic influenza A (H1N1) (H1N1 pdm). METHODS: Pregnancy complications, perinatal outcomes, and CAs were compared among 3 groups of pregnant women. The 23 women in group 1 were confirmed to harbor viral RNA; the 23 in group 2 had serum levels of virus-specific antibodies against H1N1 pdm, meaning that they were suspected of being infected with the virus; and the 93 in group 3 had no detectable virus-specific antibodies. RESULTS: Perinatal outcomes and pregnancy complications were not significantly different in groups 1 and 3. Higher percentages of stillbirths (12.0%) and placental disorders (13.0%) were observed in group 2 than in group 3. Many women in group 2 (62.5%) experienced symptoms of having a cold during pregnancy and most took no medication. Two cases of CA occurred in group 1, in the offspring of women infected in the second trimester. CONCLUSION: When left untreated, infection with the 2009 H1N1 pdm virus during pregnancy appears to have increased fetal mortality and morbidity. Because CAs are traumatic for all concerned, their possible association with the virus should be further evaluated.

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.