Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Genetic Characteristics of Wuxiang Virus in Shanxi Province, China.

Wuxiang virus (WUXV) is the first sandfly-borne Phlebovirus isolated from Phlebotomus chinensis collected in China and has been established as a consistent viral presence in the local sandfly populations of both Wuxiang County and Yangquan City. However, its distribution in the Shanxi Province remains unclear. In this study, three novel WUXV strains were isolated from sandflies collected from Jiexiu City, Shanxi Province, China, in 2022. Subsequently, whole-genome sequences of these novel strains were generated using next-generation sequencing. The open reading frame (ORF) sequences of the WUXV strains from the three locations were subjected to gene analysis. Phylogenetic analysis revealed that WUXV belongs to two distinct clades with geographical differences. Strains from Wuxiang County and Yangquan City belonged to clade 1, whereas strains from Jiexiu City belonged to clade 2. Reassortment and recombination analyses indicated no gene reassortment or recombination between the two clades. However, four reassortments or recombination events could be detected in clade 1 strains. By aligning the amino acid sequences, eighty-seven mutation sites were identified between the two clades, with seventeen, sixty, nine, and one site(s) in the proteins RdRp, M, NSs, and N, respectively. Additionally, selection pressure analysis identified 17 positively selected sites across the entire genome of WUXV, with two, thirteen, one, and one site(s) in the proteins RdRp, M, NSs, and N, respectively. Notably, sites M-312 and M-340 in the M segment not only represented mutation sites but also showed positive selective pressure effects. These findings highlight the need for continuous nationwide surveillance of WUXV.

Metagenomic analysis of individual mosquitos reveals the ecology of insect viruses.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Using a meta-transcriptomic approach, we analysed the virome of 2,438 individual mosquitos (79 species), spanning ~4000 km along latitudes and longitudes in China. From these data we identified 393 core viral species associated with mosquitos, including seven (putative) arbovirus species. We identified potential species and geographic hotspots of viral richness and arbovirus occurrence, and demonstrated that host phylogeny had a strong impact on the composition of individual mosquito viromes. Our data revealed a large number of viruses shared among mosquito species or genera, expanding our knowledge of host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, possibly facilitated by long-distance mosquito migrations. Together, our results greatly expand the known mosquito virome, linked the viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the ecology of viruses of insect vectors.

Human and animal exposure to newly discovered sand fly viruses, China.

Introduction: The Hedi virus (HEDV) and Wuxiang virus (WUXV) are newly discovered Bunyaviruses transmitted by sandflies. The geographical distribution of isolation of these two viruses continues to expand and it has been reported that WUXV causes neurological symptoms and even death in suckling mice. However, little is known about the prevalence of the two viruses in mammalian infections. Methods: In order to understand the infection status of HEDV and WUXV in humans and animals from regions where the viruses have been isolated, this study used Western blotting to detect the positive rates of HEDV and WUXV IgG antibodies in serum samples from febrile patients, dogs, and chickens in the forementioned regions. Results: The results showed that of the 29 human serum samples, 17.24% (5/29) tested positive for HEDV, while 68.96% (20/29) were positive for WUXV. In the 31 dog serum samples, 87.10% (27/31) were positive for HEDV and 70.97% (22/31) were positive for WUXV, while in the 36 chicken serum samples, 47.22% (17/36) were positive for HEDV, and 52.78% (19/36) were positive for WUXV. Discussion: These findings suggest there are widespread infections of HEDV and WUXV in mammals (dogs, chickens) and humans from the regions where these viruses have been isolated. Moreover, the positive rate of HEDV infections was higher in local animals compared to that measured in human specimens. This is the first seroepidemiological study of these two sandfly-transmitted viruses. The findings of the study have practical implications for vector-borne viral infections and related zoonotic infections in China, as well as providing an important reference for studies on the relationship between sandfly-transmitted viruses and zoonotic infections outside of China.

Isolation and Identification of Sandfly-Borne Viruses from Sandflies Collected from June to August, 2019, in Yangquan County, China.

In Yangquan County, the sandfly-transmitted virus (Wuxiang virus) was first isolated from sandflies in 2018. However, relationships between the abundance and seasonal fluctuations of local sandflies and sandfly-transmitted viruses are unknown. Herein, we report that sandfly specimens were collected in three villages in Yangquan County, from June to August, 2019. A total of 8363 sandflies were collected (June, 7927; July, 428; August, 8). Eighteen virus strains (June, 18; July, 0; August, 0) were isolated in pools of Phlebotomus chinensis. The genome sequence of the newly isolated virus strain was highly similar to that of the Wuxiang virus (WUXV), isolated from sandflies in Yangquan County in 2018. Our results suggested that the sandfly-transmitted viruses, and the local sandfly population, are stable in Yangquan County, and that June is the peak period for the virus carried by sandflies in this area.

Spatial statistical and environmental correlation analyses on vector density, vector infection index and Japanese encephalitis cases at the village and pigsty levels in Liyi County, Shanxi Province, China.

BACKGROUND: In the eco-epidemiological context of Japanese encephalitis (JE), geo-environmental features influence the spatial spread of the vector (Culex tritaeniorhynchus, Giles 1901) density, vector infection, and JE cases. METHODS: In Liyi County, Shanxi Province, China, the spatial autocorrelation of mosquito vector density, vector infection indices, and JE cases were investigated at the pigsty and village scales. The map and Enhanced Thematic Mapper (ETM) remote sensing databases on township JE cases and geo-environmental features were combined in a Geographic Information System (GIS), and the connections among these variables were analyzed with regression and spatial analyses. RESULTS: At the pigsty level, the vector density but not the infection index of the vector was spatially autocorrelated. For the pigsty vector density, the cotton field area was positively related, whereas the road length and the distance between pigsties and gullies were negatively related. In addition, the vector infection index was correlated with the pigsty vector density (PVD) and the number of pigs. At the village level, the vector density, vector infection index, and number of JE cases were not spatially autocorrelated. In the study area, the geo-environmental features, vector density, vector infection index, and JE case number comprised the Geo-Environment-Vector-JE (GEVJ) intercorrelation net system. In this system, pig abundance and cotton area were positive factors influencing the vector density first. Second, the infection index was primarily influenced by the vector density. Lastly, the JE case number was determined by the vector infection index and the wheat area. CONCLUSIONS: This study provided quantitative associations among geo-environmental features, vectors, and the incidence of JE in study sties, one typical northern Chinese JE epidemiological area without rice cultivation. The results highlighted the importance of using a diverse range of environmental management methods to control mosquito disease vectors and provided useful information for improving the control of vector mosquitoes and reducing the incidence of JE in the northern Chinese agricultural context.

Correlations between uncertainty in illness and anxiety, depression and quality of life in patients receiving maintenance haemodialysis: A cross-sectional study.

AIM: To investigate the uncertainty in illness, anxiety, depression and quality of life in patients receiving maintenance haemodialysis and confirm the correlations between these variables. DESIGN: A cross-sectional study was conducted among 396 patients receiving maintenance haemodialysis in four tertiary hospitals in China. METHODS: Uncertainty in illness was measured by Mishel's uncertainty in illness scale. The scores of self-rating anxiety scale, self-rating depression scale and medical outcomes study short form 36 were collected and compared with available norms. The Pearson correlation coefficient was calculated to investigate the correlation between uncertainties in illness with these vital variables. RESULTS: The mean score of uncertainty in illness was 78.16 out of 160. Compared with the norm, patients receiving maintenance haemodialysis had a statistically significantly lower score of depression and higher scores of most domains of quality of life. Uncertainty in illness is corrected with anxiety, depression positively and with quality of life negatively.

Factors influencing nurses' behavioral intention toward caring for COVID-19 patients on mechanical ventilation: A cross-sectional study.

OBJECTIVES: To investigate nurses' behavioral intention toward caring for COVID-19 patients on mechanical ventilation, as well as the factors affecting their intention. BACKGROUND: COVID-19 patients undergoing mechanical ventilation have many care needs and pose more challenges for nurses, which might adversely affect nurses' intention toward caring behavior. METHODS: A cross-sectional study was conducted by using simple random sampling to recruit 598 nurses from five tertiary hospitals in Sichuan Province, China. The participants responded to an online questionnaire that included questions on demographic characteristics; the Attitude, Subjective Norms, and Behavioral Intention of Nurses toward Mechanically Ventilated Patients (ASIMP) questionnaire; the Nursing Professional Identity Scale (NPIS); and the Compassion Fatigue-Short Scale (CF-Short Scale). ANOVA, Spearman correlation analysis, and multiple linear regression were performed to analyze the data. RESULTS: The mean total behavioral intention score was 179.46 (± 14.83) out of a total score of 189.00, which represented a high level of intention toward caring for patients on mechanical ventilation. Multiple linear regression revealed that subjective norms (ß = 0.390, P<0.001), perceived behavioral control (ß = 0.149, P<0.001), professional identity (ß = 0.101, P = 0.009), and compassion fatigue (ß = 0.088 P = 0.024) were significant predictors of nurses' behavioral intention. CONCLUSIONS: Most nurses have a positive behavioral intention to care for COVID-19 patients undergoing mechanical ventilation. The findings in this study provide some insight for developing effective and tailored strategies to promote nurses' behavioral intention toward caring for ventilated patients under the pandemic situation.

Re-isolation of Wuxiang Virus from Wild Sandflies Collected from Yangquan County, China.

We previously isolated a new species of the genus Phlebovirus from wild sandflies collected from Wuxiang County in central China, which named the Wuxiang virus (WUXV). In this study, we re-isolated the WUXV from wild sandflies collected from two villages in Yangquan County, China in 2019. Four virus isolates that caused cytopathic effects in BHK-21 cells were successfully isolated from sandfly specimens collected from chicken pens and sheep pens. Phylogenetic analyses of the L, M and S gene segments of the viruses revealed that the four virus strains represented the previously isolated WUXV. The minimum infection rate (MIR) of the virus isolated from the sheep pen was 3.21, and the MIR of the virus isolated from the chicken pen was 3.45. The positive rates of Wuxiang virus neutralizing antibodies in serum samples of local healthy people and domestic chickens were 8.7% (4/46) and 100% (4/4), respectively, suggesting that Wuxiang virus can infect human and animal. In view of the fact that Wuxiang virus is infectious to humans and animals and has a relatively wide geographical distribution in China, it is of great public health significance to strengthen the investigation and study on the infection status of Wuxiang virus in humans and animals.

Distribution of Dermacentor silvarum and Associated Pathogens: Meta-Analysis of Global Published Data and a Field Survey in China.

Dermacentor silvarum is an obligate blood sucking arthropod and transmits various pathogens to humans and domestic animals. Recently several new viruses were detected in D. silvarum as an emerging disease threat. In this study, we aimed to analyze its geographical distribution and associated pathogens. Data were collected from multiple sources, including a field survey, reference book, and literature review. We searched various electronic databases with the terms "Dermacentor silvarum" OR "D. silvarum" for studies published since 1963 and the positive rates for Dermacentor silvarum-associated pathogens were estimated by meta-analysis. D. silvarum was found only in four countries in Eurasia, ranging from 22° N to 57° N latitude. At least 20 human pathogens were associated with D. silvarum, including five species of spotted fever group rickettsiae, three species in the family of Anaplasmataceae, three genospecies in the complex Borrelia burgdorferi sensu lato, Francisella tularensis, Babesia venatorum, Coxiella buenetii, Borrelia miyamotoi, and five species of virus. Among them, Rickettsia raoultii was widely detected in D. silvarum, showing the highest pooled positive rate (25.15%; 95% CI 13.31-39.27). Our work presents the most comprehensive data and analysis (to our knowledge) for the geographical distribution of D. silvarum and associated pathogens, revealing an emerging threat to public health and stocking farming. Continued surveillance and further investigations should be enhanced.

Isolation and Characterization of Wuxiang Virus from Sandflies Collected in Yangquan County, Shanxi Province, China.

In this study, we isolated a group of viruses that can cause a cytopathic effect in BHK-21 cells from sandfly specimens collected in Yangquan County, Shanxi Province, central China. The representative virus strain is SXYQ1860 and, with increased viral injection, the suckling mice became ill and died, with viral titers continually increasing. The results of molecular genetic evolution analysis of the three gene segments of the virus, L, M, and S, indicate that the newly isolated viruses from Yangquan County are the same as the Wuxiang virus (WUXV) previously isolated from sandflies collected in Wuxiang county, China. This is the first time that the WUXV was also isolated outside Wuxiang County. Therefore, strengthening the surveillance of neglected sandflies and the viruses they transmit to help prevent and control arboviruses and the associated diseases is essential for public health.

Professional Identity During the COVID-19 Pandemic: A Cross-Sectional Survey of Nurses in China.

BACKGROUND: Emergency and intensive care unit nurses are the main workforce fighting against COVID-19. Their professional identity may affect whether they can actively participate and be competent in care tasks during the pandemic. OBJECTIVE: To examine the level of and changes in professional identity of Chinese emergency and intensive care unit nurses as the COVID-19 pandemic builds. METHODS: A cross-sectional survey composed of the Professional Identity Scale for Nurses plus 2 open-ended questions was administered to Chinese emergency and intensive care unit nurses through an online questionnaire. RESULTS: Emergency and intensive care unit nurses had a medium level of professional identity. Participants' total and item mean scores in 5 professional identity dimensions were higher than the professional identity norm established by Liu (P < .001). The greatest mean item score difference was in the dimension of professional identity evaluation (3.57 vs 2.88, P < .001). When asked about their feelings witnessing the COVID-19 situation and their feelings about participating in frontline work, 68.9% and 83.9%, respectively, reported positive changes in their professional identity. CONCLUSIONS: The professional identity of emergency and intensive care unit nurses greatly improved during the early stages of the COVID-19 pandemic. This finding may be attributed to more public attention and recognition of nurses' value, nurses' professional fulfillment, and nurses' feelings of being supported, motivated, respected, and valued.

Wuxiang Virus Is a Virus Circulated Naturally in Wuxiang County, China.

Background: Wuxiang virus was isolated from sandfly specimens collected in Wuxiang County, Shanxi Province, China in 2018, representing the first reported isolation of sandfly-borne virus from sandflies collected in a natural environment in China. The local sandfly density, seasonal fluctuations, and temporal and spatial distributions of the virus in Wuxiang County remain unclear. Materials and Methods: Four fixed sandfly collection sites were set up in Wuxiang County and sandfly specimens were collected continuously from June to August 2019. All sandfly specimens were subjected to viral isolation and molecular biological analysis. Results: The data on sandfly specimens collected in Wuxiang County from June to August 2019 showed a significant difference in the density of sandflies between June 26 and August 16 (p < 0.05). No statistical difference was found in sandfly density among collection sites (p > 0.05). A total of 33 virus isolates causing cytopathic effects in mammalian (BHK-21) cells were obtained from 7466 sandflies (91 pools) collected from June to August 2019. The results of molecular genetic evolution analysis of the nucleotide sequence of these isolates showed that the L and S genes (encoding NS and N proteins) of the 33 viruses isolated in 2019 are in the same evolutionary branch as the previously isolated Wuxiang virus. No significant difference was found in the virus isolation rate (the pool isolation rate of virus) among sandflies collected at different times from June to August (p > 0.05). The virus isolation rate of sandflies collected at different collection sites showed a statistically significant difference (p < 0.05). Conclusions: The results of this study suggest that the Wuxiang virus is a stable viral population in local sandflies. Strengthened research into Wuxiang virus infection of humans and animals and clarification of the public health hazards posed by Wuxiang virus to both humans and animals are urgently needed.

Developing a new individual earthquake resilience questionnaire: A reliability and validity test.

Earthquakes pose serious threats to the world. Good individual resilience can cope with disaster well, but there were few appropriate assessment tools. The purpose of this study was to develop a new individual earthquake resilience questionnaire and test its reliability and validity. First, we built the framework of the individual earthquake resilience questionnaire based on expert interviews. Then, we established the initial version of questionnaire and used the Delphi method and item selection to modify it by qualitative and quantitative methods. Finally, we built the final version of questionnaire (contained 4 dimensions and 17 items) and tested the reliability and validity. The Cronbach's α values of the four dimensions were between 0.79 and 0.91, the split-half reliabilities were between 0.85 and 0.93, and the test-retest reliabilities were between 0.72 and 0.80. The item content validity indexes were between 0.87-1, and the average questionnaire content validity index was 0.94. The correlation coefficients between each item and dimension with the total questionnaire ranged from 0.79-0.90 and 0.66-0.79, respectively. We used exploratory factor analysis to identify four common factors with a cumulative variance contribution rate of 74.97%. The questionnaire is a valid and reliable tool to measure individual resilience in the context of earthquake disasters.

Coronavirus disease 2019 pandemic promotes the sense of professional identity among nurses.

BACKGROUND: Under the COVID-19 pandemic, nurses are the mainstay in the fight against the pandemic. PURPOSE: To evaluate potential impact of the pandemic on nurses' professional identity. METHOD: Self-report questionnaires were distributed online. Data collected were compared with available norms. Multivariate logistic regression analyses were employed to calculate the OR of frontline vs. nonfrontline nurses. FINDINGS: The mean of the total score of the scale was 121.12 out of 150. Both the total score and scores on the five dimensions were significantly higher than norms. Frontline nurses had a significantly higher professional identity than non-frontline nurses (total score: odds ratio [OR], 1.19; professional identity evaluation: OR, 1.27; professional social support: OR, 1.18; professional social proficiency: OR, 1.33; and dealing with professional frustration: OR, 1.19). The most frequently mentioned tags were Hope, Frontline, Protection, Outbreak, Work, Situation. DISCUSSION: COVID-19 outbreak was associated with an enhancement in the professional identity of nurses.

Emerging Sand Fly-Borne Phlebovirus in China.

We isolated 17 viral strains capable of causing cytopathic effects in mammalian cells and death in neonatal mice from sand flies in China. Phylogenetic analysis showed that these strains belonged to the genus Phlebovirus. These findings highlight the need to control this potentially emerging virus to help safeguard public health.

Pigsties near dwellings as a potential risk factor for the prevalence of Japanese encephalitis virus in adult in Shanxi, China.

BACKGROUND: The increasing trend of adult cases of Japanese encephalitis (JE) in China, particularly in northern China, has become an important public health issue. We conducted an epidemiological investigation in the south of Shanxi Province to examine the relationships between mosquitoes, Japanese encephalitis virus (JEV), and adult JE cases. METHODS: Mosquito specimens were collected from the courtyards of farmers' households and pig farms in Shanxi Province. Mosquitoes were pooled, homogenized, and centrifuged. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mosquito-borne arbovirus genes in homogenates. Specimens positive for these genes were inoculated into the baby hamster kidney cell line (BHK-21) to isolate virus. Minimum infection rate was calculated and phylogenetic analyses were performed. RESULTS: A total of 7 943 mosquitoes belonging to six species in four genera were collected; Culex tritaeniorhynchus accounted for 73.08% (5 805/7 943), C. pipiens pallens for 24.75% (1 966/7 943), and the remaining 3% (104/ 7943) consisted of Anopheles sinensis, Aedes vexans, Ae. dorsalis, and Armigeres subalbatus. Sixteen pools were positive for JEV based on RT-PCR using JEV pre-membrane gene nested primers. Phylogenetic analyses showed that all JEVs belonged to genotype I; two pools were positive using Getah Virus (GETV) gene primers. In addition, one JEV strain (SXYC1523) was isolated from C. pipiens pallens specimens. These results indicate that the minimum infection rate of JEV in mosquito specimens collected from the courtyards of farmers' households with pigsties was 7.39/1 000; the rate for pig farms was 2.68/1 000; and the rate for farmers' courtyards without pigsties was zero. CONCLUSIONS: The high-prevalence regions of adult JE investigated in this study are still the natural epidemic focus of JEV. Having pigsties near dwellings is a potential risk factor contributing to the prevalence of adult JE. To prevent the occurrence of local adult JE cases, a recommendation was raised that, besides continuing to implement the Expanded Program on Immunization for children, the government should urge local farmers to cease raising pigs in their own courtyards to reduce the probability of infection with JEV.

[Isolation and identification of mosquito-borne arboviruses in Yuncheng city, Shanxi province, 2012].

OBJECTIVE: To investigate the species and distribution of mosquitoes and mosquito-borne arboviruses in Yuncheng city of Shanxi province, China. METHODS: Mosquito samples were collected in 19 collection sites from Linyi county and Yongji city in Yuncheng city, in August, 2012. After identification and classification, all the specimens were homogenized and centrifuged to acquire supernatant before being inoculated to both C6/36 and BHK21 cells for viral isolation. Positive isolates were identified with arbovirus species-specific primers under RT-PCR, for further sequencing and phylogenetic analysis. RESULTS: A total of 10 455 mosquitoes of 7 species in 4 genuese were collected. The predominant mosquito species in Linyi county was Culex pipens pallens (91.96%, 3 911/4 253), but the one in Yongji city was Culex tritaeniorhynchus (72.85%, 4 518/6 202). A total of 23 strains of viruses were isolated from the mosquito pools. 15 strains from Culex tritaeniorhynchus and Culex pipens pallens were identified as genotype I Japanese encephalitis virus (JEV). Four strains from Culex pipens pallens were identified as Culex flavivirus (CxFV). Three strains from Culex pipens pallens were identified as Culex pipiens pallens densovirus (CppDNV). One strain from Armigeres subalbatus and Aedes albopictus was identified as Getah virus (GETV). CONCLUSION: Four kinds of arboviruses were isolated from the mosquito pools, including GETV and CxFV, which were isolated and documented in Shanxi province for the first time. In the city of Yuncheng, Culex tritaeniorhynchus had been the predominant species and major vector for transmitting JEV. Genotype I JEV remained the major JEV circulating in the local natural environment.

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.