An intranasal challenge model for testing Japanese encephalitis vaccines in rhesus monkeys.

Placebo-controlled field efficacy trials of new Japanese encephalitis (JE) vaccines may be impractical. Therefore, an animal model to evaluate efficacy of candidate JE vaccines is sought. Previous work has shown that exposure of monkeys to JE virus (JEV) via the intranasal route results in encephalitis. Here we report the further development of this model and the availability of titered virus stocks to assess the protective efficacy of JE vaccines. To determine the effective dose of our JE challenge virus, dilutions of a stock JEV (KE-93 isolate) were inoculated into four groups of three rhesus monkeys. A dose-dependent response was observed and the 50% effective dose (ED50) was determined to be 6.0 x 10(7) plaque forming units (pfu). Among animals that developed encephalitis, clinical signs occurred 9-14 days postinoculation. Infection with JEV was confirmed by detection of JEV in nervous tissues and IgM to JEV in the cerebrospinal fluid. Viremia with JEV was also detected intermittently throughout infection. Validation of the model was performed using a known effective JE vaccine and saline control. One ED90 of virus (2.0 x 10(9) pfu) was used as a challenge dose. Four of four animals that received saline control developed encephalitis while one of four monkeys administered the JE vaccine did so. This study demonstrates that the virus strain, route of inoculation, dose, and the outcome measure (encephalitis) are suitable for assessment of protective efficacy of candidate JE vaccines.     

The effect of interferon on Japanese encephalitis virus in vitro

The studies on the effect of the Recombinant Leukocyte A Interferon (r IFN-alpha A) on 4 local strains of JE virus in Thailand were performed in vitro in our laboratories in Bangkok during August - October 1984. The procedures consisted of the plaque reduction assay and the Rhesus monkey kidney cell line, LLC-MK2 cells. These 4 strains namely Vip, KE-093, KE-094 and KE-095 were isolated from the JE patients in Thailand during 1983-1984. The results revealed that all of the JE virus strains tested were sensitive to the r IFN-alpha A with its minimal effective doses ranging from 30 I.U./ml to 1,500 I.U./ml. The studies on the effects of r IFN-alpha A on JE virus replicating in the cell culture for 0 hour, 1 hour and 6 hours indicated that if the virus had more hours to replicate in the cell culture, higher concentration of the IFN was needed in order to combat the replication of the virus in the cell culture. r IFN-alpha A at higher concentrations showed more efficacy in combating the replication of the JE virus in vitro.     

Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone,which Demonstrates Attenuation and Immunogenicity in Mice

The live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine, produced in primary hamster kidney cells, is safe and effective. Past attempts to adapt this virus to replicate in cells that are more favorable for vaccine production resulted in mutations that significantly reduced immunogenicity. In this study, 10 genetically distinct Vero cell-adapted JEV SA14-14-2 variants were isolated and a recombinant wild-type JEV clone, modified to contain the JEV SA14-14-2 polyprotein amino acid sequence, was recovered in Vero cells. A single capsid protein mutation (S66L) was important for Vero cell-adaptation. Mutations were also identified that modulated virus sensitivity to type I interferon-stimulation in Vero cells. A subset of JEV SA14-14-2 variants and the recombinant clone were evaluated in vivo and exhibited levels of attenuation that varied significantly in suckling mice, but were avirulent and highly immunogenic in weanling mice and are promising candidates for the development of a second-generation, recombinant vaccine.     

日本脑炎病毒受体功能缺陷细胞的筛选和鉴定

目的筛选并鉴定日本脑炎病毒（JEV）受体功能缺陷的BHK-21细胞突变株。方法用化学诱变剂ICR-191人工诱变JEV易感细胞系BHK-21,经JEV攻击,对存活细胞克隆化,RT-PCR筛选JEV RNA阴性细胞,用流式细胞术,免疫荧光和空斑实验鉴定其对JEV的敏感性。结果筛选到1株JEV RNA阴性细胞3A10-3F。流式细胞术检测3A10-3F细胞与JEV的结合率为2.10%,免疫荧光和空斑实验证实其对JEV感染有相当程度的抵抗,在JEV感染复数MOI 1时3A10-3F细胞培养上清中JEV最高滴度比BHK-21细胞中JEV最高滴度下降2个数量级。结论用化学诱变法筛选到1株JEV受体功能缺陷细胞,为进一步鉴定JEV受体,深入研究JEV致病机理提供了有益线索。     

Nonconjugative R plasmid-mediated antibiotic resistance in Escherichia coli isolated from animals in Peninsular Malaysia

Four of the five veterinary E. coli strains, which were unable to transfer their antibiotic resistance by conjugation, were found to harbour plasmids. Evidence from transformation, agarose gel electrophoresis and curing experiments showed that in strains KE-3, KE-4 and KE-14 a nonconjugative R plasmid carried the gene for resistance to tetracycline. The plasmids in KE-9 were cryptic.     

A longitudinal study of Japanese encephalitis in suburban Bangkok, Thailand

A one-year study of Japanese encephalitis (JE) in a small focus of transmission was conducted in suburban Bangkok in 1985. Monthly data were collected on weather, vector density, sentinel pig and chick JE antibody seroconversions, and epidemiology as related to human JE cases. The primary vector species were found to be Culex gelidus and Culex tritaeniorhynchus; from which one isolate each was obtained in March and June, respectively. Pig JE antibody seroconversion peaked in April (the hottest month), with secondary peaks following in July and December. Chick seroconversions were found only in June and July. Human cases (7) in the primary focus occurred from May-July, and started 2 months following the finding of the first JEV isolate in mosquitoes and 1 month following mass JEV seroconversion in pigs. Overall, the attack rate in the focus (0.83/10(5] was greater than 4 times that of the rest of Bangkok (0.19/10(5]. Attack rates were highest in 0-9 and 10-19 year-old groups, respectively. Indications are that JEV is transmitted to humans in Bangkok at least 10 out of 12 months per year, but that cases are concentrated in the May to July period.     

Short report: genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate.

We determined the full-length genome sequence of Japanese encephalitis virus (JEV) K94P05 isolated in Korea. Sequence analysis showed that the 10,963-nucleotide-long RNA genome of K94P05 was 13 or 14 nucleotides shorter than the genome of other JEV isolates because of a deletion in the 3' noncoding region of K94P05. Compared with sequences of other JEV isolates, the full-length nucleotide sequence showed 89.0-89.6% homology, and the deduced amino acid sequence showed between 96.4-97.3% homology. A region of approximately 60 nucleotides immediately downstream of the open reading frame stop codon of K94P05 showed high sequence variability as compared with other JEV isolates. K94P05 formed a distinct group within a phylogenetic tree established with the full-length genome sequences. Cross-neutralization studies showed that polyclonal antibodies to Korean isolates were 3 times better at neutralizing the Korean isolates than antibodies to Nakayama-NIH. These findings suggest that Korean JEV K94P05 is genetically and antigenically distinct from other Asian JEV isolates.     

Presence of hemagglutination inhibition and neutralization antibodies to Japanese encephalitis virus in wild pigs on an offshore island in Singapore

A study was conducted to determine the presence of hemagglutination inhibition (HI) and neutralization antibodies against Japanese encephalitis virus (JEV) in wild pigs on an offshore island in Singapore. Blood samples were collected from 28 wild pigs on the island. All the sera tested with HI assay and plaque reduction neutralization tests were found to be positive for antibodies against JEV. Our results indicate that the wild pigs have been infected with JEV on the offshore island and there is JEV transmission.     

一株特殊抗原性乙脑毒株的分子生物学特征研究

目的对一株抗原性特殊的乙脑毒株进行生物学和分子生物学特征研究,以找出特殊抗原性的分子生物学基础。方法通过空斑减少交叉中和试验对乙脑病毒KT株的抗原性进行比较。提取KT株RNA,逆转录后扩增其E基因片段并测序,通过Blast与GenBank中所有乙脑病毒基因进行核苷酸和氨基酸序列比对,利用PdbViewer对KT株关键位点突变后的空间构象进行预测比对。结果交叉中和试验显示,KT株免疫血清对其他株乙脑病毒的中和作用较低,而其他乙脑病毒免疫血清对KT株的中和作用也较低。对E基因序列进行比对,KT株属于基因Ⅲ型,氨基酸序列上只在E62位存在一个独特的位点突变:组氨酸(H)→精氨酸(R)。空间构象显示,该位点位于结构域Ⅰ与结构域Ⅱ的连接交界处,当发生由组氨酸(H)到精氨酸(R)突变时,其与周围氨基酸形成的氢键数量和长度发生改变,空间构象也随之发生改变。结论乙脑病毒KT株的抗原性与其他乙脑病毒株存在较大差异,E62位组氨酸(H)→精氨酸(R)的突变是导致其抗原性差异的主要原因。     

血清cathepsin S在冠心病不稳定斑块中的价值及意义

目的探讨血清中cathepsin S、基质金属蛋白酶（MMP）-2、MMP-9及高敏C反应蛋白（hs-CRP）水平在冠状动脉不同类型斑块中的价值。方法84例患者入选,其中急性心肌梗死19例,不稳定型心绞痛27例,稳定型心绞痛38例。介入手术之前对＂罪犯＂血管进行血管内超声（IVUS）检查,然后根据斑块的类型将患者分为稳定斑块、不稳定斑块及破裂斑块组,同时测定血清中上述指标的变化。结果84例患者中,血管内超声发现有36处稳定斑块,18处不稳定斑块,30处破裂斑块。三组患者在参考面积,参考血管管腔面积、病变面积以及重构指数之间差异没有统计学意义。血清测定结果显示,不稳定斑块组血清中cathepsin S浓度较稳定斑块组明显升高（不稳定斑块组0.408±0.136 IU/L,稳定斑块组0.355±0.056 IU/L,P〈0.05）,余各组之间相比差异无统计学意义;测定MMP-2及MMP-9在三组之间没有明显变化;不稳定斑块组hs-CRP较稳定斑块组明显升高（5.23±4.28 pg/mL比2.30±2.71 pg/mL,P〈0.01）,不稳定斑块组较破裂斑块组hs-CRP也有明显升高（5.23±4.28 pg/mL比3.19±2.33 pg/mL,P〈0.05）,但破裂斑块组与稳定斑块组相比差异无统计学意义。结论cathepsin S和hs-CRP升高对于斑块的不稳定具有预测价值,对于患者的预后没有预测价值。     

广西新分离流行性乙型脑炎病毒GP0722株全基因组分子特性研究

目的 对广西新分离乙脑病毒GP0722株进行全基因序列测定和分析,了解其基因组结构及毒力特征。方法 应用乙脑病毒全基因组扩增引物进行RT-PCR扩增,PCR产物直接测序,拼接后得到全基因序列。应用Clustal X(1.8)、DNASTAR、Mega 4. 1等生物软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析。结果 广西新分离乙脑病毒GP0722全基因长10 965个核苷酸,从97到10 395位编码一个开放阅读框,编码3 432个氨基酸,与目前使用的减毒活疫苗株SA-14-14-2株比较,只有88.9%的核苷酸同源性,97.6%的氨基酸同源性,全基因组共存在1 222个核苷酸差异,83个氨基酸差异。与GenBank中选择的21株乙脑病毒全基因序列比较发现,其核苷酸总体差异率为0.9%~18.8%,氨基酸总体差异率为0.1%~5.2%。通过PrM/C区段、E区段、3′NTR区段和全基因序列进行系统进化分析显示该毒株属于基因1型乙脑病毒。结论 新分离的乙脑病毒GP0722株属于基因1型,与JEV/sw/Mie/40/2004进化关系最近,与疫苗株SA-14-14-2相比关键位点氨基酸未见变异,现行使用的疫苗仍能保护GP0722引起的感染。     

The new antirheumatic drug KE-298 suppresses monocyte chemoattractant protein (MCP)-1 and RANTES production in rats with adjuvant-induced arthritis and in IL-1β-stimulated synoviocytes of patients with rheumatoid arthritis

We analyzed the effects of the new antirheumatic drug KE-298 on monocyte chemoattractant protein (MCP)-1 and regulated on activation normal T-cell expressed and secreted (RANTES) production in rats with adjuvant-induced arthritis and in interleukin (IL)-1beta-stimulated rheumatoid arthritis (RA) synoviocytes. In rats with adjuvant-induced arthritis, the enhanced production of MCP-1 and RANTES and the development of arthritis were suppressed by oral treatment with 100 mg/kg per day of KE-298 for 18 days. Furthermore, KE-298 (10-100 microg/ml) suppressed MCP-1 and RANTES production by IL-1beta-stimulated RA synoviocytes through inhibition of NF-kappaB and AP-1 activation. These results suggest that the inhibitory effect of KE-298 on MCP-1 and RANTES production might partly explain its efficacy in rats with adjuvant-induced arthritis and in patients with RA.     

Pharmacodynamics of aminoglycosides and tetracycline derivatives against Japanese encephalitis virus

Objective:To explore the antiviral activity of antibiotic compounds,mainly aminoglycosides and tetracyclines against Japanese encephalitis virus(JEV) induced infection in vitro.Methods:Antiviral activity were evaluated against JEV using cytopathic effect inhibition assay,virus yield reduction assay,caspase 3 level,extracellular viral detection by antigen capture ELISA and viral RNA levels.Roults:JEV induced cytopathic effect along with reduction of viral progeny plaque formation indicated antiviral potential of the compounds suggesting that antibiotics had broad spectrum activity.Doxycycline and kanamycin administration in dose dependent manner declined viral RNA replication.Conclusions:The present study shows kanamycin and doxycyclinc can affect virion structure and alter replication causing inhibition of JEV induced pathogenesis in vitro.     

West Nile virus infection and serologic response among persons previously vaccinated against yellow fever and Japanese encephalitis viruses

It is hypothesized that previous heterologous flaviviral exposure may modulate clinical illness among persons infected with West Nile virus (WNV). Little is known about the serological response in such persons. In summer 2003, a WNV outbreak occurred in Colorado, the location of the Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases (DVBID). DVBID employees, most previously vaccinated with yellow fever virus (YFV) or Japanese encephalitis virus (JEV) vaccines, were studied to determine whether previous vaccination affected symptom development among those subsequently infected with WNV during the outbreak, as well as their serological response. Serum samples collected in December 2003 and previously banked samples were tested using the plaque reduction neutralization test (PRNT) against WNV, Saint Louis encephalitis virus, dengue- 4 virus, JEV, and YFV. Specimens shown to have WNV antibody by PRNT were tested by IgM and IgG enzymelinked immunosorbent assays (ELISAs). Ten (9%) of 113 serosurvey participants had WNV neutralizing antibody titers in December 2003. PRNT titers from previous specimens showed that one of the ten had seroconverted to WNV before 2003. Of the remaining nine participants, seven reported illness in the summer of 2003, two of which were unvaccinated and five previously vaccinated. In the December 2003 specimens, five persons previously unvaccinated or vaccinated only against YFV had a fourfold or greater neutralizing titer with WNV than with other flaviviruses, whereas no persons previously vaccinated against JEV or JEV and YFV showed a similar difference in neutralizing titers. Eight of nine persons infected in 2003 had negative or indeterminate WNV MAC-ELISA results in the December 2003 sample; the ninth person was vaccinated against YFV one month previously, and was also YFV positive by MAC-ELISA. We conclude that previous flaviviral vaccination does not markedly affect the development of WNV fever and that the IgM antibody response in patients without neuroinvasive WNV disease is transient.     

中国首次分离的二株基因Ⅰ型流行性乙型脑炎病毒全基因组序列分析

目的 分析中国首次分离的基因Ⅰ型流行性乙型脑炎病毒(JEV)全基因组的基因组特征。方法 设计JEV全基因组扩增引物,RT-PCR扩增片断,PCR产物直接测序,拼接后获得全基因组序列。采用生物学软件进行同源性和系统进化分析。结果 1977和1982年分离自云南蚊虫的M28和BN82215病毒株基因组全长分别为10 969 bp和10 970 bp,5′非编码区均含有96个核苷酸,3′非编码区含有573和574个核苷酸。它们的开放阅读框(ORF)都从97到10 396位,共10 299个核苷酸,编码3 433个氨基酸。M28和BN82215株与来自GenBank的5个基因型JEV株的全基因组核苷酸和氨基酸同源性分别为78.4％～96.3％和91.4％～99.6％,与近几年国内外基因Ⅰ型流行株的同源性最高,进化关系最接近,都属于基因Ⅰ型。这两株病毒与JEV疫苗株SA14-14-2相比,有56个氨基酸差异,其中E基因有11个氨基酸差异,但都不属抗原关键位点。结论 本研究阐明了中国首次分离的基因Ⅰ型JEV的全基因组分子特征,决定病毒抗原和毒力的E蛋白关键位点无明显变化。证实20世纪70和80年代云南省就有基因Ⅰ型JEV流行。     

Protection against Japanese encephalitis virus in mice and hamsters by treatment with carboxymethylacridanone, a potent interferon inducer

A low-molecular-weight chemical inducer of interferon, 10-carboxymethyl-9-acridanone (CMA), effectively prevented death caused by Japanese encephalitis virus (JEV) injected peripherally into weanling mice and baby hamsters. Marked reductions in mortality were seen in mice when a single dose of CMA was administered intraperitoneally, subcutaneously, or intramuscularly to animals challenged intraperitoneally with JEV. The degree of protection was dependent on dose and time of administration of CMA in relation to virus challenge: all hamsters given CMA on the same day as JEV survived, with lesser although still significant protection when CMA was given one or two days after JEV. Viremia, an important characteristic of the pathogenesis of natural JEV infection, was reduced nearly 10,000-fold in hamsters treated with CMA. Thus, in the experimental animal models developed for these studies, CMA provided marked therapeutic and prophylactic effect against JEV.     

Comparison of Plaque Size,Thermal Stability,and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha’s small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.     

脑梗死患者血浆溶血磷脂酸及基质金属蛋白酶-9与颈动脉斑块稳定性研究

目的 探讨脑梗死患者血浆溶血磷脂酸(lysophosphatidic acid,LPA)及基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)水平与颈动脉粥样斑块稳定性的关系.方法 选取87例脑梗死患者,根据颈动脉超声检查结果,分为无斑块组9例、内膜增厚组16例、不稳定性斑块组41例、稳定性斑块组21例.根据经颅多普勒(TCD)微栓子检测结果,分为微栓子检测阳性组27例和阴性组60例.分别用无机磷定量法和酶联免疫吸附法测定血浆LPA和MMP-9水平.结果不稳定性斑块组LPA、MMP-9显著高于其他各组(F=49.98、106.49,均P=0.00),稳定性斑块组、内膜增厚组MMP-9差异无统计学意义,但均高于无斑块组(q=7.04、7.51,均P=0.00),稳定性斑块组LPA高于内膜增厚组(q=7.37,P=0.00),并且两组均高于无斑块组(q=8.85,P=0.00;q=2.61,P=0.04).微栓子阳性组血浆LPA、MMP-9水平均显著高于微栓子阴性组(t=42.57、16.61,均P=0.00).LPA与MMP-9水平呈正相关(r=0.22,P=0.032). 结论 LPA与MMP-9参与了脑梗死的病理生理过程,其与颈动脉斑块不稳定性密切相关.     

早期神经功能恶化急性卒中患者的颈动脉粥样硬化超声特征

目的 探讨早期神经功能恶化(early neurological deterioration,END)急性卒中患者的颈动脉粥样硬化超声特征.方法 END定义为人院第7天时国立卫生研究院卒中量表评分较入院时至少增加2分.128例入院24 h内接受颈动脉超声检奁的急性缺血性卒中患者中,38例出现END的患者作为END组,40例危险因素相匹配的非END患者作为非END组.比较两组颈动脉粥样硬化超声特征.结果 END组斑块积分[(16.7±4.4)mm对(13.3±3.5)mm,t=2.673,P=0.009)、内膜-中膜横截面积[(26.4±8.5)mm2对(20.5±6.8)mm2,t=3.394,P=0.001]、动脉僵硬指数(28.94±4.29对21.22±5.85,t=6.618,P:0.000)以及小稳定斑块(66.7%对43.3%,χ2=9.164,P=0.003)、偏心性斑块(62.8%对45.6%,χ2=5.008,P=0.025)、狭窄≥50%(71.1%对37.5%,χ2=8.828,P=0.003)和负性重构(28.9%对7.5%,χ2=6.087,P=0.014)的构成比均显著高于非END组,而扩张系数[(14.74±8.66)×10-6/Pa对(19.16±9.35)×10-6/Pa,t=2.163,P=0.034]和顺应系数[(0.49±0.13)×10-4mm2/Pa对(0.58±0.11)×10-4 mm2/Pa,t=3.307,P:0.001]均显著低于END组.结论 斑块积分、内膜-中膜横截面积、动脉僵硬度、不稳定斑块、偏心性斑块、狭窄≥50%、负性重构、扩张性和顺应性等超声特征可能有助于预测急性卒中的END.