Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing.

Objective: Viral encephalitis is an infectious disease severely affecting human health. It is caused by a wide variety of viral pathogens, including herpes viruses, flaviviruses, enteroviruses, and other viruses. The laboratory diagnosis of viral encephalitis is a worldwide challenge. Recently, high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections. Thus, In this study, we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods: We designed nine pairs of specific polymerase chain reaction (PCR) primers for the 12 viruses by reviewing the relevant literature. The detection ability of the primers was verified by software simulation and the detection of known positive samples. Amplicon sequencing was used to validate the samples, and consistency was compared with Sanger sequencing. Results: The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×, and the sequence lengths were consistent with the sizes of the predicted amplicons. The sequences were verified using the National Center for Biotechnology Information BLAST, and all results were consistent with the results of Sanger sequencing. Conclusion: Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis. It is also a useful tool for the high-volume screening of clinical samples.

Full length genome sequencing and phylogenetic analysis of two strains of Getah virus / 中国热带医学

@#Abstract: Objective　The main aim of the study is to sequence the complete genome of two Getah virus strains (GS11-155 and HNDZ1712-1) isolated in Gansu Province and Hainan Province in 2011 and 2017 respectively and analyze the molecular and genetic evolution of the two strains compared with M1, which was first isolated in 1964 in Hainan Province, China. Methods　Genome of two newly isolated Getah viruses were sequenced by virus gene amplification technique, and the genomic database of Getah viruses was established. The molecular characteristics and genetic evolution of the viruses were analyzed by bioinformatics software. Results　The genome length of two new isolated Getah virus strains (GS11-155 and HNDZ1712-1) was 11 690 nt and 11 621 nt, respectively. Both strains had the structural characteristics of Alphavirus genome. Although the nucleotide sequence lengths of structural genes, non-structural genes and non-coding junction regions of the two strains were identical, the nucleotide sequence lengths of the 5' and 3' non-coding regions of the viral genomes were a few different. The 3'UTR repeats elements in the genomes of the two virus strains did not change. It was 97.7% and 98.1% different of nucleotide and amino acid homology between both strains of Getah virus, HNDZ1712-1 isolated in 2017 and M1 isolated in 1964 in Hainan Province. Interesting, Gansu 2011 cluster and Hainan 2017 cluster were emerged leading by both strains GS11-155 and HNDZ1712-1 respectively, those two clusters totally independent with M1 virus isolated from Hainan in 1964 in whole genome phylogenetic analysis first. Conclusions　Although the HNDZ1712-1 was also isolated from mosquito samples in Hainan Province, it was in a completely different evolutionary branch from the M1 isolated from Hainan Island in 1964, and was closely related to the strain isolated from Gansu Province (GS11-155) thousands of kilometers away. It is suggested that the two new strains of Getah virus are different from the Getah virus isolated in 1964.

Detection of Pseudorabies Virus Antibodies in Human Encephalitis Cases.

Pseudorabies virus (PRV), a veterinary pathogen that infects domestic animals as well as wild animals such as wild boar and feral swine, was recently reported to infect human and led to endophthalmitis and encephalitis. A retrospective seroepidemiologic survey was conducted using 1,335 serum samples collected from patients with encephalitis and ELISA positive rates were 12.16%, 14.25%, and 6.52% in 2012, 2013, and 2017, respectively. The virus neutralizing antibody titers of positive samples correlated well with ELISA results. The pseudorabies virus antibody positive rate of patients with encephalitis were higher than that of healthy people in 2017. The above results suggest that some undefined human encephalitis cases may be caused by PRV infection.

A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus.

OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION: A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.

Serological Survey of Zika Virus in Humans and Animals in Dejiang Prefecture, Guizhou Province, China.

OBJECTIVE: The current outbreak of Zika virus (ZIKV) poses a severe threat to human health. Two ZIKV strains were isolated from mosquitoes collected from the Dejiang prefecture in China in 2016, which was the first isolation of ZIKV in nature in China. METHODS: In this study, serum samples were collected from 366 healthy individuals and 104 animals from Dejiang prefecture in 2017, and the plaque reduction neutralization test (PRNT) was used to evaluate the seroprevalence of ZIKV. RESULTS: None of the 366 residents from whom the samples were collected were seropositive for ZIKV. None of the 11 pigs from whom the samples were collected were seropositive for ZIKV, while 1 of 63 (1.59%) chickens and 2 of 30 (6.67%) sheep were seropositive for ZIKV. CONCLUSION: The extremely low seropositivity rate of ZIKV antibodies in animals in the Dejiang prefecture, Guizhou province in this study indicates that ZIKV can infect animals; however, there is a low risk of ZIKV circulating in the local population.

TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus.

OBJECTIVE: To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed. METHODS: By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay. RESULTS: With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%. CONCLUSION: A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

The Relationship between Japanese Encephalitis and Environmental Factors in China Explored Using National Surveillance Data.

Japanese encephalitis (JE) is a serious public health issue. This study was undertaken to better understand the relationship between JE distribution and environmental factors in China. JE data from 2005 to 2010 were retrieved from National Notifiable Disease Report System. ArcGIS, remote sensing techniques, and R software was used to exhibit and explore the relationship between JE distribution and environmental factors. Our results indicated that JE cases were mostly concentrated in warm-temperate, semitropical and tropical zones with annual precipitation > 400 mm; Broad-leaved evergreen forest, shrubs, paddy field, irrigated land, dryland, evergreen coniferous forest, and shrubland were risk factors for JE occurrence, and the former five were risk factors for counties with high JE incidence. These findings will inform the effective allocation of limited health resources such as intensive vaccination, surveillance and training in areas with high environmental risk factors.

From discovery to spread: The evolution and phylogeny of Getah virus.

Getah virus (GETV) was first isolated in Malaysia in 1955. Since then, epidemics in horses and pigs caused by GETV have resulted in huge economic losses. At present, GETV has spread across Eurasia and Southeast Asia, including mainland China, Korea, Japan, Mongolia, and Russia. Data show that the Most Recent Common Ancestor (MRCA) of GETV existed about 145years ago (95% HPD: 75-244) and gradually evolved into four distinct evolutionary populations: Groups I-IV. The MRCA of GETVs in Group III, which includes all GETVs isolated from mosquitoes, pigs, horses, and other animals since the 1960s (from latitude 19°N to 60°N), existed about 51years ago (95% HPD: 51-72). Group III is responsible for most viral epidemics among domestic animals. An analysis of the GETV E2 protein sequence and structure revealed seven common amino acid mutation sites. These sites are responsible for the structural and electrostatic differences detected between widespread Group III isolates and the prototype strain MM2021. These differences may account for the recent geographical radiation of the virus. Considering the economic significance of GETV infection in pigs and horses, we recommend the implementation of strict viral screening and monitoring programs.

Identification of a Newly Isolated Getah Virus in the China-Laos Border, China.

In this study, we isolated a virus strain (YN12031) from specimens of Armigeres subalbatus collected in the China-Laos border. BHK-21 cells infected with YN12031 exhibited an evident cytopathic effect (CPE) 32 h post-infection. The virus particles were spherical, 70 nm in diameter, and enveloped; they also featured surface fibers. Molecular genetic analysis revealed that YN12031 was closely related to alpha viruses such as Chikungunya virus and Sindbis virus, and located in the same clade as MM2021, the prototype of Getahvirus (GETV) isolated in Malaysia in 1955. Phylogenetic analysis of the E2 and capsid genes further revealed that YN12031 was located in the same clade as the Russian isolate LEIV/16275/Mag. Analysis of the homology of nucleotides and amino acids in the coding area and E2 gene demonstrated that the YN12031 isolated from the China-Laos border (tropical region) was related closest to the LEIV/16275/Mag isolate obtained in Russia (North frigid zone area) among other isolates studied. These results suggest that GETV can adapt to different geographical environments to propagate and evolve. Thus, strengthening the detection and monitoring of GETV and its related diseases is very crucial.

Molecular evolution of emerging Banna virus.

Banna virus (BAV) is an emerging pathogen that causes human viral encephalitis and has been isolated from types of blood-sucking insects and mammals in Asia. However, there are no reported systematic studies that describe the origin and evolution of BAV. Here, a phylogenetic analysis of BAVs isolated from a variety of potential vectors and vertebrate hosts worldwide revealed that BAVs emerged in the beginning of the 20th century and do not exhibit a species barrier. The mean substitution rate of BAVs was 2.467×10-2substitution/site/year (95% HPD, 1.093×10-3 to 5.628×10-2). The lineage is mainly composed of BAVs from high-latitude regions, which are the most recently emerged viruses with significantly higher substitution rates compared with the lineage comprised of the isolates from middle or low-latitude regions. The genetic differences between BAV strains are positively correlated with the geographic distribution. Strains from the same latitude regions are almost 100% identical, whereas the differences between strains from long distance regions with different latitudes could be >60%. Our results demonstrate that BAV is an emerging virus at a stage that involves rapid evolution and has great potential for introduction into non-endemic areas. Thus, enhanced surveillance of BAV is highly recommended worldwide.

A Centralized Report on Pediatric Japanese Encephalitis Cases from Beijing Children's Hospital, 2013.

Fifteen pediatric cases of suspected Japanese encephalitis (JE) were reported in Beijing Children's Hospital during the late summer of 2013. The clinical manifestations in most cases included high fever, seizures, and abnormal magnetic resonance imaging findings. Twelve of 15 cases were laboratory-confirmed as JE cases by pathogen identification. Epidemiological investigations showed that five of the 12 laboratory-confirmed patients had an incomplete JE vaccination history. Follow-up investigations after discharge indicated that seven laboratory-confirmed JE patients without JE vaccinations had relatively poor prognoses, with an average Modified Rankin Scale (MRS) score of 2.6 when compared with the other five laboratory-confirmed, JE-vaccinated patients with an average MRS score of 0.5. The observation of pediatric JE cases among those with a history of JE vaccination warrants further attention.

Isolation and full-length genome analysis of mosquito-borne Manzanilla virus from Yunnan Province, China.

BACKGROUND: There have been four strains on Manzanilla virus (MANV) identified to date. Here, we identify a novel MANV strain (DHL10M107) isolated from Culex tritaeniorhynchus Giles mosquitoes from Ruili city, Dehong prefecture, Yunnan Province, in the People's Republic of China. RESULTS: The DHL10M107 L, M and S genes were sequenced at the nucleotide and deduced amino acid levels. The L, M and S gene sequences of DHL10M107 clustered with the MANV strains VN04-2108, TRVL3587, SA An 4165, and AV 782. DHL10M107 was most closely related to VN04-2108. Nucleotide homology ranged between 96 and 99% between DHL10M107 and VN04-2108. In terms of amino acid homology, all of the amino acid differences were in the L (96.3% homologous) and M (97.7% homologous) fragments. CONCLUSIONS: DHL10M107 is likely a MANV isolated from mosquitos in the Yunnan Province. This is the first reported isolation of MANV in mainland China.

Real-time RT-PCR Assay for the detection of Tahyna Virus.

A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

How environmental conditions impact mosquito ecology and Japanese encephalitis: an eco-epidemiological approach.

Japanese encephalitis (JE) is one of the major vector-borne diseases in Southeast Asia and the Western Pacific region, posing a threat to human health. In rural and suburban areas, traditional rice farming and intensive pig breeding provide an ideal environment for both mosquito development and the transmission of JEV among human beings. Combining surveillance data for mosquito vectors, human JE cases, and environmental conditions in Changsha, China, 2004-2009, generalized threshold models were constructed to project the mosquito and JE dynamics. Temperature and rainfall were found to be closely associated with mosquito density at 1, and 4month lag, respectively. The two thresholds, maximum temperature of 22-23°C for mosquito development and minimum temperature of 25-26°C for JEV transmission, play key roles in the ecology of JEV. The model predicts that, in the upper regime, a 1g/m(3) increase in absolute humidity would on average increase human cases by 68-84%. A shift in mosquito species composition in 2007 was observed, and possibly caused by a drought. Effective predictive models could be used in risk management to provide early warnings for potential JE transmission.

Molecular characterization of full-length genome of Japanese encephalitis virus genotype V isolated from Tibet, China.

OBJECTIVE: To determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V. METHODS: The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis. RESULTS: The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3'-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V. CONCLUSION: The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

Mosquitoes of Western Yunnan Province, China: seasonal abundance, diversity, and arbovirus associations.

OBJECTIVE: The western borderland between Yunnan Province, China, and Myanmar is characterized by a climate that facilitates year-round production of mosquitoes. Numerous mosquito-transmitted viruses, including Japanese encephalitis virus circulate in this area. This project was to describe seasonal patterns in mosquito species abundance and arbovirus activity in the mosquito populations. METHODS: Mosquitoes were collected in Mangshi and Ruili cities of Dehong Prefecture near the border of China and Burma in Yunnan Province, the Peoples Republic of China in 2010. We monitored mosquito species abundance for a 12-month period using ultraviolet light, carbon dioxide baited CDC light and gravid traps; and tested the captured mosquitoes for the presence of virus to evaluate mosquito-virus associations in rural/agricultural settings in the area. RESULTS: A total of 43 species of mosquitoes from seven genera were collected, including 15 Culex species, 15 Anopheles spp., four Aedes spp., three Armigeres spp., one Mimomyia spp., two Uranotaenia spp. and three Mansonia spp.. Species richness and diversity varied between Mangshi and Ruili. Culex tritaeniorhynchus, Culex quinquefasciatus, Anopheles sinensis and Anopheles peditaeniatus were the most abundant species in both sampling sites. Ultraviolet light traps collected more specimens than CDC light traps baited with dry ice, though both collected the same variety of mosquito species. The CDC gravid trap was the most effective trap for capture of Culex quinquefasciatus, a species underrepresented in light trap collections. A total of 26 virus strains were isolated, which included 13 strains of Japanese encephalitis virus, four strains of Getah virus, one strain of Oya virus, one strain from the orbivirus genus, and seven strains of Culex pipien pallens densovirus. CONCLUSIONS: The present study illustrates the value of monitoring mosquito populations and mosquito-transmitted viruses year-round in areas where the climate supports year-round adult mosquito activity.