Discrimination of West Nile virus and Japanese encephalitis virus strains using RT-PCR RFLP analysis

West Nile (WN) virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. Since an outbreak of WN encephalitis in humans and horses occurred in New York City in late August 1999, the possibility exists that WN virus will invade regions that have close links with the United States, such as Japan. We developed a genetic diagnostic method that discriminates between strains of WN virus and Japanese encephalitis (JE) virus. The method involves RT-PCR restriction fragment length polymorphism (RFLP) analysis with a RT-PCR primer set, a nested PCR primer set, and a restriction enzyme. We detected WN and JE viruses in experimentally infected animal brain, spleen, and serum samples. Our method is useful in distinguishing WN viruses from the endemic background of JE viruses, and in discriminating the highly virulent WN strain, which was isolated in New York in 1999, from other WN virus strains.     

Molecular Epidemiology of Japanese Encephalitis Virus in Okinawa

In order to elucidate the molecular characteristics of Japanese encephalitis (JE) virus in Okinawa, 23 strains of JE virus isolated in a 25-year span were sequenced for the 240 nucleotides of the C-preM junction region and 111 nucleotides of the E gene region and compared with those of reference strains isolated in mainland Japan. The results of phylogenic analysis showed that although all the Okinawan isolates showed more than 96% homology in the nucleotide sequence in each region, they were chronologically divided into two groups: the old group (nine strains) and a new group (14 strains). On the other hand, in a comparison with reference strains in mainland Japan, the Okinawan isolates showed more than 94% nucleotide sequence homology in both regions, indicating that the Okinawan strains belong to the same genotype as that of JE strains in mainland Japan. The nucleotide homology of the old group was relatively higher than that of the new group. Among the 14 strains in the new group, 13 strains were isolated from mosquitoes collected from a pig farm from 1986 through 1992. These strains showed higher nucleotide divergence than the old group strains, isolated from mosquitoes and swine sera collected at several sites, in both regions. A nucleotide substitution at the position 1920 in the E gene was identified in three isolates. This substitution generated an asparagine-proline-threonine sequence capable of serving as an attachment site of carbohydrate.     

Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

Recently it has been reported that Japanese encephalitis virus (JEV)-specific RNAs can be synthesized in vitro in the subcellular fraction including outer-nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane-associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.     

Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa,Subtropical Area in Japan. II. Prevalence of Japanese Encephalitis Antibody in Residents in Okinawa,Miyako and Ishigaki Islands

During 1989 to 1990, human sera were collected by age groups in Okinawa (the northern, central and southern areas), Miyako and Ishigaki islands and examined for the neutralization (N) antibodies to two strains, Nakayama (vaccine strain) and C307 (Okinawan strain), of Japanese encephalitis (JE) virus. In Okinawa island, the N antibody positive rate to C307 was higher than that to Nakayama, while in Miyako and Ishigaki islands, the positive rate to Nakayama was higher than that to C307, suggesting that JE virus transmission rate was higher in Okinawa than in Miyako and Ishigaki islands. In Okinawa Prefecture, JE vaccine had not been administered to most of residents over 31 years of age at the time of serum collection. In residents over 31 years old, the positive rate to C307 was highest in the north of Okinawa (83.3%) and was lowest in Miyako (26.3%), with the second lowest in Ishigaki (33.3%). The distribution of N antibody titers to C307 gave hyperbolic patterns in the 0–5 age groups in Miyako and Ishigaki, and also in the 31–40, 41–50 age groups in Miyako and the 41–50 age group in Ishigaki, suggesting low rates of natural infection in these 4–5 decades in both islands. In residents of ages subjected to JE vaccine, a characteristic pattern was obtained, in which the curves to Nakayama shifted to higher titers than those to C307, suggesting that the first antigenic stimulation was caused by vaccine, not by natural infection of JE virus.     

Epidemiological and Ecological Studies of Japanese Encephalitis in Okinawa,Subtropical Area in Japan. I. Investigations on Antibody Levels to Japanese Encephalitis Virus in Swine Sera and Vector Mosquito in Okinawa,Miyako and Ishigaki Islands

From 1985 to 1989, serum specimens of swine raised in the northern, central and southern areas in Okinawa island were examined for Japanese encephalitis (JE) virus antibody by ELISA and hemagglutination-inhibition test. The antibody positive rate was found to be higher in the north and central than in the south. The 2-mercaptoethanol sensitive antibody to JE was detected mostly in June and July, and occasionally in other months except February and March. There was no month when all specimens from three areas turned antibody-negative simultaneously, indicating that JE virus transmission to swine lasted longer in Okinawa island than in other temperate areas in Japan. From 1986 to 1991, the vector mosquitoes (Culex tritaeniorhynchus) were collected in a pig farm in the south of Okinawa island. A total of 153 strains of JE virus was isolated from the vector mosquitoes mainly in June. In Miyako and Ishigaki islands, the antibody positive rate in swine sera was found to be extremely low, compared with that in Okinawa island. In Miyako island, where no paddy rice field is cultivated, a few adults as well as larvae of the vector mosquito were collected, while in Ishigaki island, where there are many watered rice fields, a lot of adults as well as larvae were collected. Although the environmental situation is quite different between the two islands, JE virus transmission appeared to be very low in both islands.     

Japanese Encephalitis Virus Infection in Vero Cells: The Involvement of Intracellular Acidic Vesicles in the Early Phase of Viral Infection Was Observed with the Treatment of a Specific Vacuolar Type H+-ATPase Inhibitor,Bafilomycin A1

The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H⁺ -ATPase (V-ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and ³H-uridine-labeled viral RNA were observed in fractions of a Percoll density gradient. At 10 min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04 g/ml corresponding to the endosome both in the control and bafilomycin A1-treated cells. At 60 min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08 g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05 g/ml and higher than 1.084 g/ml, respectively. At 60 min of the chasing period in bafilomycin A1-treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04 g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.     

西尼罗病毒的RT-PCR检测与鉴定

建立西尼罗病毒敏感、特异、快速的RT-PCR检测方法用于实验室诊断和流行病学监测。采用一步RT-PCR和套式PCR法对西尼罗病毒感染的乳鼠脑和细胞培养上清进行扩增,并对扩增产物进行序列测定。两种方法均可分别从两种组织中扩增出与预期大小相一致的片段,套式PCR法比一步RT-PCR法更为敏感,该扩增片段与西尼罗病毒埃及Eg101株相应序列的同源性为99％。     

流行性乙型脑炎病毒GSS株prM、E和NS1蛋白基因的克隆及在非复制型痘苗病毒中的表达

克隆流行性乙型脑炎(乙脑)病毒野毒(JEV)GSS株前膜蛋白信号序列、前膜蛋白(prM)、包膜蛋白(E)、非结构蛋白-1(NSl)和非结构蛋白NS2a的编码基因,并与非复制型痘苗病毒载体NTV进行同源重组,构建了乙脑病毒非复制型重组痘苗病毒疫苗株NTVA(E／L)JEV。通过：PCR和Southern blot检测证明,在非复制型痘苗病毒中有乙暗病毒prM信号序列、prM、E、NS1和NS2a基因的插入：Western blot检测证明,重组病毒可以在细胞内成功地表达prM、E和NSl蛋白,并可将prM、E和NSl蛋白分泌到细胞培养上清中;免疫荧光检测证明,E和NSl蛋白主要分布在细胞膜上。电镜下可见分泌到细胞外的病毒样颗粒。     

从云南省蝙蝠脑组织中分离出乙型脑炎病毒

为进一步阐明蝙蝠在保存乙脑病毒中的作用,于1997年7月,在云南省耿马县捕捉蝙蝠64只,取脑组织作病毒分离,从一只金管鼻蝠脑组织中分离出1株病毒,该毒株能引起BHK21细胞病变和乳鼠发病死亡,在pH5.75-7.4时能凝集鸽红血球,经用单克隆抗体血凝抑制和免疫荧光试验鉴定,证明为乙型脑炎病毒。进一步证明蝙蝠在乙型脑炎病毒保存和扩散中具有重要作用。从金管鼻蝠体内分离出乙型脑炎病毒属国内外首次报道。     

云南省4种伊蚊的乙型脑炎病毒分离物的研究

在云南省西南边境9县市捕获伊蚊属雌性成蚊16种19367只,用细胞法和乳鼠法分离病毒.从185批6491只白纹伊蚊中分离到病毒2株,从50批1605只剌扰伊蚊中分离到病毒2株,从23批772只窄翅伊蚊中分离到病毒2株,从4批103只阿萨姆伊蚊中分离到病毒1株.其它12种共10396只伊蚊的病毒分离物为阴性.分离到的7株病毒经免疫荧光、酶免疫、血凝抑制和中和试验鉴定,均为乙型脑炎病毒(JE virus).白纹伊蚊是野外竹林的优势蚊种.分析认为白纹伊蚊在当地乙型脑炎病毒保存和传播中起重要作用,刺扰伊蚊、窄翅伊蚊和阿萨姆伊蚊亦可参与该病毒的传播.     

云南省4种伊蚊的乙型脑炎病毒分离物的研究

在云南省西南边境９县市捕获伊蚊属雌性成蚊１６种１９３６７只,用细胞法和乳鼠法分离病毒。从１８５批６４９１只白纹伊蚊中分离到病毒２株,从５０批１６０５只剌扰伊蚊中分离到病毒２株,从２３批７７２只窄翅伊蚊中分离到病毒２株,从４批１０３只阿萨姆伊蚊中分离到病毒１株。其它１２种共１０３９６只伊蚊的病毒分离物为阴性。分离到的７株病毒经免疫荧光、酶免疫、血凝抑制和中和试验鉴定,均为乙型脑炎病毒（ＪＥｖｉｒｕｓ）。白纹伊蚊是野外竹林的优势蚊种。分析认为白纹伊蚊在当地乙型脑炎病毒保存和传播中起重要作用,刺扰伊蚊、窄翅伊蚊和阿萨姆伊蚊亦可参与该病毒的传播。     

快速IgM抗体捕捉ELISA的建立及其在乙脑早期诊断中的初步应用

用辣根过氧化物酶标记的抗日本脑炎病毒(JEV)种特异性单克隆抗体为结合物,建立了一种快速IgM抗体捕捉酶联免疫测定法(RMAC-ELISA),并初步用于乙脑的快速诊断,取得了良好的效果。该法通过用鞣酸预处理微板,提高反应温度,在稀释液中加入PEG并提高NaCl浓度,用鸡蛋清代替牛血清白蛋白或鸡血清,用自来水代替PBS作为洗液,用快速漱洗3次代替3×5分钟洗涤,用TMBS代替OPD底物,用酶标单克隆抗体代替多克隆结合物等措施,加快了反应速度,缩短了反应时间,简化了操作步骤,提高了敏感性,保证了特异性。从微板预处理至出结果,仅需1小时。由于该法具有简便、快速、特异、敏感等特性,不仅可以用于乙脑的早期诊断及实验室研究,还有可能推广到其它的免疫学检测方法中去。     

Japanese Encephalitis Virus NS2B-3 Protein Complex Promotes Cell Apoptosis and Viral Particle Release by Down-Regulating the Expression of AXL

Virologica Sinica - Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a...     

乙脑病毒持续感染株preM区序列分析

为了研究乙型脑炎病毒持续感染株preM区域基因序列变异及其意义,我们将两种乙脑病毒野生株(JaGAr-01株和Nakayama株)分别感染人肝癌KN73细胞,经过多次细胞传代后建立乙脑病毒持续感染模型,收集感染细胞经反复冻融获取变异病毒.利用preM区特异引物进行RT-PCR法得到两种病毒的preM区基因片段,应用基因测序反应进行序列分析,并对两种病毒株preM区序列进行比较.preM区基因测序结果显示,与JaGAr-01野生株比较,JaGAr-01持续感染变异株(JaG-per)有1个核苷酸上碱基发生变异(第26位U→G)并导致相应氨基酸发生置换(第9位亮氨酸→精氨酸);Nakayama持续感染变异株(Nak-per)与其野生株相比则有11个核苷酸上碱基存在差异(第26位U→G,第37位G→A,第39位C→U,第45位U→C,第51位U→C,第99位U→C,第126位U→C,第165位C→U,第189位C→U,第195位C→U,第198位U→C),但仅有其中第26位、第37位、第39位的碱基变异引起相应编码的氨基酸发生置换(第9位亮氨酸→精氨酸及第13位缬氨酸→异亮氨酸).对比还发现变异后的JaGAr-01持续感染株与Nakayama持续感染株的基因序列相同.认为乙脑病毒持续感染变异株preM区存在基因变异,这种变异可能与该区参与病毒持续感染及维持病毒生物学特性有关.     

乙脑病毒持续感染株preM区序列分析

为了研究乙型脑炎病毒持续感染株preM区域基因序列变异及其意义,我们将两种乙脑病毒野生株(JaGAr-01株和Nakayama株)分别感染人肝癌KN73细胞,经过多次细胞传代后建立乙脑病毒持续感染模型,收集感染细胞经反复冻融获取变异病毒。利用preM区特异引物进行RT-PCR法得到两种病毒的preM区基因片段,应用基因测序反应进行序列分析,并对两种病毒株preM区序列进行比较。preM区基因测序结果显示,与JaGAr-01野生株比较,JaGAr-01持续感染变异株(JaG-per)有1个核苷酸上碱基发生变异(第26位U→G)并导致相应氨基酸发生置换(第9位亮氨酸→精氨酸);Nakayama持续感染变异株(Nak-per)与其野生株相比则有11个核苷酸上碱基存在差异(第26位U→G,第37位G→A,第39位C→U,第45位U→C,第51位U→C,第99位U→C,第126位U→C,第165位C→U,第189位C→U,第195位C→U,第198位U→C),但仅有其中第26位、第37位、第39位的碱基变异引起相应编码的氨基酸发生置换(第9位亮氨酸→精氨酸及第13位缬氨酸→异亮氨酸)。对比还发现变异后的JaGAr-01持续感染株与Nakayama持续感染株的基因序列相同。认为乙脑病毒持续感染变异株preM区存在基因变异,这种变异可能与该区参与病毒持续感染及维持病毒生物学特性有关。     

乙脑/寨卡嵌合病毒包膜蛋白V343A及I341V/V343A联合突变对小鼠脑内神经毒力的影响

为探讨乙脑/寨卡嵌合病毒JE/ZIKV(MR766)的包膜蛋白V343A突变及I341V/V343A联合突变对小鼠神经毒力的影响，分别构建了V343A和I341V/V343A联合突变的嵌合病毒全长cDNA质粒，经酶切鉴定后，体外转录获得病毒RNA，并电转染入BHK21细胞拯救病毒。利用病毒测序和免疫荧光实验鉴定病毒；蚀斑实验和生长曲线对比突变病毒与亲本株生物学特性差异；动物实验比较神经毒力差异。基因测序与免疫荧光等证明突变病毒JE/ZIKV(V343A)和JE/ZIKV(I341V/V343A)拯救成功。突变病毒JE/ZIKV(V343A)和JE/ZIKV(I341V/V343A)的蚀斑直径分别为（1.09±0.15） mm和（1.15±0.29） mm，小于亲本株JE/ZIKV(MR766)蚀斑直径（2.09±0.36） mm（P<0.001）；生长曲线显示两突变病毒增殖速度均快于亲本株，且JE/ZIKV(I341V/V343A)快于JE/ZIKV(V343A)；动物实验结果显示，JE/ZIKV(V343A)的LD50为5.62 pfu/0.03 mL，JE/ZIKV(I341V/V343A)的LD50为34.84 pfu/0.03 mL，均高于亲本株（LD50=2.21 pfu/0.03 mL）。研究结果表明，寨卡病毒E蛋白V343A突变使嵌合病毒小鼠脑内神经毒力减弱，I341V/V343A联合突变使病毒毒力进一步减弱，表明E蛋白的341和343位氨基酸残基参与了嵌合病毒对小鼠的脑内神经毒力的调控。     

新疆伊犁地区马脑组织西尼罗病毒感染的调查

目的了解新疆伊犁地区草原放养马群中西尼罗病毒(W estN ile virus,WNV)中枢感染的流行状况。方法采用一步法实时荧光定量逆转录聚合酶链反应(Real Tim e RT-PCR)对采自新疆伊犁地区草原放养、未接种WNV疫苗的189例马脑组织进行WNV包膜蛋白(E)基因片段检测。结果被检马脑组织标本中未发现WNV E基因片段。结论目前尚没有证据表明我国新疆伊犁地区的放养马中存在WNV脑炎的感染,提示该地区出现WNV脑炎流行的可能性小。     

Serologic survey of West Nile virus in horses from Central-West,Northeast and Southeast Brazil

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.     

Decreases in Both the Seroprevalence of Serum Antibodies and Seroprotection against Japanese Encephalitis Virus among Vaccinated Children

The incidence of Japanese encephalitis(JE) has significantly decreased in China due to JE vaccines. In this study, we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus(JEV) to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children. A total of 300 serum samples were collected from vaccinated children aged 3–10 years in Zhaotong, Yunnan,China. The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test. The highest seropositivity of 82% was observed in vaccinated children during the first0.5–1.5 years after booster vaccination. Then, the seropositivity began to decline and remained lower than the original level observed in the 0.5–1.5-year group. An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination. Similarly, the neutralizing antibody(nAb) titres gradually decreased over time, and the levels showed a positive correlation with the protective efficacy in mice. This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine. Our results provide data that clarify the persistence and waning of antibodies to JEV, which may help elucidate the pathogenesis of JE.