Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.     

Pyramidal and extrapyramidal involvement in experimental infection of cynomolgus monkeys with enterovirus 71

Among the enteroviruses, polioviruses and enterovirus 71 (EV71) are two major neurotropic viruses causing serious neurological manifestations. While polioviruses are being eradicated globally by vaccination, EV71 still has the potential to cause a large outbreak such as that in Taiwan in 1998, in which there were many fatalities. In this study, we determined the neurovirulence of EV71 by neuropathological analysis of cynomolgus monkeys after experimental infection with five EV71 strains, which were isolated from individual patients with fatal encephalitis; meningitis; and hand, foot, and mouth disease. After intraspinal inoculation, the monkeys developed neurological manifestations within 1-6 days post-inoculation, irrespective of the inoculated strains. These manifestations included not only pyramidal tract signs such as flaccid paralysis, but also extrapyramidal tract signs such as tremor and ataxia. Histological and viral examinations confirmed virus replication in the spinal cord, brainstem, cerebellar cortex, and dentate nuclei, and cerebrum. The strains isolated during the 1970s and 1990s showed no particular differences with respect to neurotropism. Thus, it is clear that EV71 has a wider neurotropism than that of polioviruses.     

Neurovirulence of herpes simplex virus types 1 and 2 isolates in diseases of the central nervous system

Herpes simplex virus (HSV) isolates derived from the central nervous system of ten patients with HSV-1-induced encephalitis, one patient with multiple sclerosis, and 14 patients with HSV-2-induced meningitis were investigated for neurovirulence by assaying the LD₅₀ after nose and intracerebral (i.c.) inoculation of mice. HSV-1 encephalitis strains were significantly more virulent after nose inoculation (i.e. neuroinvasive) when compared with HSV-1 isolates from patients with oral lesions only, whereas HSV-2 meningitis strains were significantly more virulent after i.c. inoculation when compared with HSV-2 isolates from patients with genital lesions only. No correlation between high neurovirulence (defined as low LD₅₀ for both routes of infection) and replication in cell cultures of neuronal and non-neuronal cell lines was found, but the weakly neurovirulent HSV-1 strain isolated from a patient with multiple sclerosis gave low replication yields. After nose inoculation, a highly neuroinvasive HSV-1 laboratory reference strain replicated to high titers in nose tissue, the trigeminal ganglia and brainstem, while a strain with low neuroinvasiveness but high i.c. virulence replicated less well in the brainstem. Neuroinvasiveness of the virus strain might be one factor of relevance in the pathogenesis of HSV-1 encephalitis in man.     

烟台市手足口病合并脑炎病毒分离及基因序列分析

目的 了解烟台地区手足口病合并脑炎病原谱及其基因学特征.方法 采集烟台地区手足口病合并脑炎患者粪便及脑脊液标本,通过细胞培养分离病毒,实时荧光PCR鉴定病毒型别,RT-PCR扩增VP1区基因部分序列并测序,进行序列分析.结果 10份粪便标本分离病毒3株,均为EV71.与C4a型代表株核苷酸同源性为98％～99％,氨基酸同源性98.90％～99.45％.系统发生树中,烟台分离株与C4亚型C4a簇代表株位于同一分支.结论 烟台地区手足口病合并脑炎病原主要为EV71,属于C4亚型C4a簇.     

2008至2009年北京地区分离的肠道病毒71型全基因组序列分析

目的了解2008至2009年从北京地区手足口病（HFMD）患儿分离到的肠道病毒71型（EV71）全基因组序列特点（未包括多聚腺苷尾）,以探讨基因序列的改变是否与病毒的致病性有关。方法选取首都儿科研究所病毒研究室2008年分离到的5株EV71毒株和2009年分离到的4株EV71毒株,其中4株来源于重症HFMD患儿（伴高热、持续抽搐及意识丧失等中枢神经系统症状）,5株来源于轻症HFMD患儿。设计覆盖病毒全基因组的10对特异性引物,对9株EV71毒株进行RT-PCR扩增、全基因组序列测定和分析。结果 9株EV71毒株的全基因组长度为7406bp或7405bp,部分毒株在5′UTR存在1处1个碱基的缺失。9株EV71毒株的全基因组核苷酸和氨基酸同源性分别为96.3%～99.4%和98.2%～99.6%,在VP1区核苷酸和氨基酸同源性分别为96.9%～99.9%和98.3%～100.0%。重症HFMD来源的4株毒株中有3株在VP2蛋白第144位及3D聚合酶（3Dpol）第140和263位同时出现相同的氨基酸变异（T144S、R140K和I263V）,并且在5′UTR区第208和254位同时出现相同的碱基变异（G208A和A254G）。9株EV71毒株的全基因组与C4亚型毒株具有最高的核苷酸同源性,在VP1区为94.3%～95.5%;在3D及3′UTR区与CV-A16/G10的同源性（84.3%～85.0%和89.0%～91.5%）高于与EV71-B型、A型及C型（C1～3、C5）的同源性。VP1和3D基因的遗传进化分析显示,9株EV71毒株与C4亚型毒株属同一分支。结论 VP2蛋白第144位氨基酸突变（T→S）、3Dpol第140和263位氨基酸突变（R→K和I→V）及5′UTR区第254位碱基突变（A→G）可能与EV71感染后引起的不同临床症状有关。根据VP1核苷酸序列,2008至2009年北京地区流行的EV71属于C4亚型;非结构蛋白基因在EV71进化中可能有一定的作用。     

An outbreak of enterovirus 71 infection in Taiwan 1998: a comprehensive pathological, virological, and molecular study on a case of fulminant encephalitis.

BACKGROUND: In a recent enterovirus outbreak in Taiwan, serotype 71 was the culprit of encephalitis causing rapid clinical deterioration and death among young children. OBJECTIVES: Since knowledge of enterovirus 71 (EV71) infection in the central nervous system is still limited, the purpose of the present case study was attempted to uncover the pathogenesis of the virus. STUDY DESIGN: We performed a detailed pathological examination, virological and molecular studies on a case of EV71 infection with a rapidly fatal outcome. In addition, the whole genome of the virus was sequenced to determine the genetic relationships to other enteroviruses and two other EV71 strains (a prototype BrCr and a neurovirulent MS strain), and to provide the genetic basis of its neurovirulence of the new isolate, NCKU9822 strain. RESULTS: Characteristic features of acute encephalomyelitis were observed, with most prominent lesions in the spinal cord and brain stem. Mild myocarditis and pancreatitis were also noticed. EV71 antigen was localized to neurons on immunohistochemical staining. EV71 was recovered from all organs with inflammatory reaction. Sequence analysis showed that overall NCKU9822 and the two EV71 strains shared 80% nucleotide identity and 95% amino acid identity. It had only 45% amino acid and 52% nucleotide identities with polioviral P1 capsid region. CONCLUSION: The spinal cord and brain stem were the main targets of EV71 in the fatal cases in this outbreak, however, heart and pancreas might also be involved. Since the amino acid sequences in the P1 region are conserved (97% identity) among the three EV71 strains as compared to other enteroviruses and polioviruses, these EV71 neurovirulent strains might share the same mechanisms of neurovirulence, and the mechanisms might be different from those in polioviruses.     

Relationship between neurovirulence and temperature sensitivity of an attenuated western equine encephalitis virus

Summary An attenuated strain of western equine encephalitis virus which arose spontaneously during a persistent infection was compared with the virulent parent strain for its growth potential over the temperature range 32° to 42° C. At 42° C, the attenuated strain proved unable to grow in a one-step growth experiment and failed to produce plaques. The temperature sensitivity was found to be due not to inactivation at 42° C of the attenuated virus particles, but to some temperature sensitive step occurring late in the replicative cycle. When the attenuated strain was passaged at 41 °C and clones selected for growth at this temperature, the resulting population did not regain the neurovirulence of the wild-type virus. In contrast, when virulent-revertants were selected from the attenuated, temperature-sensitive virus, temperature sensitivity remained unchanged. These findings indicated a lack of covariation of the temperature sensitivity and neurovirulence of western equine encephalitis virus, and suggest that mutation to avirulence and temperature sensitivity probably occurred independently.     

Genetic diversity of enterovirus 71 isolated from cases of hand,foot and mouth disease in Yokohama City between 1982 and 2000

Summary.  Enterovirus 71 (EV71) is known as one of the major causative agents of hand, foot and mouse disease (HFMD) and is also associated with neurological manifestations such as aseptic meningitis, polio-like paralysis and encephalitis. Recently, large HFMD outbreaks, involving severe neurological complications, have been experienced in Malaysia, Taiwan and some other countries in the Western-Pacific region. To investigate the genetic diversity of EV71 isolates in a single community in Japan, nucleotide sequences of the VP4 region of 52 EV71 isolates in Yokohama City from 1982 to 2000 were determined and the phylogenetic relationship was compared with other referential EV71 strains in Japan and in the world. There were two major genotypes of EV71 in Yokohama City through the 1980’s and 1990’s. Six EV71 isolates in the early 1980’s in Yokohama City were closely related to those from HFMD outbreaks in Japan and from outbreaks of polio-like paralysis in Europe in the 1970’s. During recent HFMD outbreaks in 1997 and 2000, two distinct genotypes of EV71 were co-circulating in Yokohama City as in HFMD outbreaks in Malaysia and Taiwan. However, the genetic diversity of EV71 in Yokohama City was not directly correlated with the severity of HFMD. The results confirmed the circulation of two distinct genotypes of EV71 over the past 20 years in Japan. Received June 25, 2002; accepted September 16, 2002     

青海省分离到肠道病毒71型及其VP1区基因特征分析

目的 研究2008年青海省手足口病（Hand,Foot and Mouth Disease,HFMD）的致病病原体中肠道病毒71型（Human Enterovirus 71,HEV71）的VP1区基因特征.方法 采集青海省HFMD患者的粪便、疱疹液和咽拭子标本共335份,并进行病毒分离,然后对阳性分离株病毒进行肠道病毒血清型别的分子定型.对分离到的HEV71阳性分离株进行VP1编码区基因扩增,核苷酸序列测定和同源进化分析.结果 在335份临床标本中,共分离到45株阳性病毒分离物,其中30株鉴定为HEV71（占66.7%）,是主要病原体.对30株HEV71进行VP1区核苷酸序列测定后,分析发现它们在VP1区核苷酸水平和氨基酸水平上有一定差异,同源性分别在95.2%～100.0%和96.6%～100%之间.它们与1998年以来在我国分离的HEV71在核苷酸和氨基酸水平上的同源性都较高.与其他35株各基因型和基因亚型的HEV71代表株构建的亲缘进化树中显示,青海省HEY71分离株与C4亚型代表株聚为一簇,属于C4基因亚型C4a进化分支.结论 青海省从HFMD病例中分离到HEV71,也确认了青海省HFMD的病原体HEV71流行株的基因型为C4基因型中的C4a进化分支,并且存在多个传播链.     

Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase

In 1998, an enterovirus 71 (EV71) epidemic in Taiwan resulted in 78 deaths; however, the molecular basis of EV71 pathogenicity remains poorly understood. Comparison of the deduced amino acid sequences in 3D polymerases of EV71clinical isolates showed the T251V or T251I substitution from 1986 and 1998 outbreaks. An EV71 replicon system showed that introducing an I251T mutation did not affect luciferase activities at 35 °C when compared with wild type; however, lower luciferase activities were observed when they were incubated at 39.5 °C. In addition, the I251T mutation in the EV71 infectious clone not only reduced viral replication at 39.5 °C in vitro but also decreased the virulence of the mouse adaptive strain MP4 in neonatal mice in an i.p. infection model. Therefore, these results suggested that the threonine at position 251 results in a temperature sensitivity phenotype of EV71 which may contribute to the attenuation of circulating strains.     

Genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients

Summary We examined four type 1 polioviruses isolated from the stools of patients with vaccine-associated paralytic poliomyelitis in China. All of these isolates were shown to be Sabin derived viruses by restriction fragment length polymorphism assay after polymerase chain reaction and by sequencing of the viral genome encoding the viral coat protein, VP1. However, the same analysis of the 3D coding region suggested that two of the four isolates had the sequence of wild type poliovirus in the tested region. Furthermore there were also point mutations in the 5 non-coding region. One was a single base change from U to C at nucleotide position 525, and the other three were from G to A at position 480. All the four strains were more neurovirulent than Sabin type 1 virus in transgenic mice with human poliovirus receptor gene. The data showed that the nucleotide positions of type 1 poliovirus which were identified to be in favor of the high neurovirulence were indeed changed during natural transmission, and suggested that the point mutation alone or a recombination of the vaccine type with wild type genome results in an acquisition of neurovirulence.     

Association of Interleukin-17F Gene Polymorphism with Enterovirus 71 Encephalitis in Patients with Hand, Foot, and Mouth Disease

Enterovirus 71 (EV71) is one of the common pathogenic agents of hand, foot, and mouth disease (HFMD) and is associated with severe complications including encephalitis. Interleukin (IL)-17F plays an important role in tissue inflammation by inducing release of proinflammatory cytokines and chemokines. We investigated the association between EV71 encephalitis and of IL-17F 7488T/C (rs763780) gene polymorphism, which is known to cause a His-to-Arg substitution at amino acid 161. The study was performed in 58 Chinese patients with EV71 encephalitis and 127 Chinese patients with EV71-related HFMD without complications. Genotyping was determined by the polymerase chain reaction–restriction fragment length polymorphism technique. The patients with EV71 encephalitis had a significantly lower frequency of the IL-17F 7488TC+CC genotypes (10.3 %) as compared to the patients with EV71-related HFMD without complications (27.6 %, p?=?0.008). The frequency of IL-17F 7488C alleles was also significantly lower among the patients with EV71 encephalitis (5.2 %) as compared to that of the patients with EV71-related HFMD without complications (15 %, OR?=?0.310, 95 % CI?=?0.127–0.756, p?=?0.006). Furthermore, homozygotes with the T allele had significantly higher levels of C-reactive protein, white blood cell count, and neutrophil count as compared to the patients with CC+CT genotypes (p?=?0.004, 0.001, and 0.000, respectively). These findings suggested that the IL-17F 7488C allele could be significantly associated with protection against encephalitis in Chinese patients with EV71-related HFMD.     

Characterization of encephalitis in adult mice induced by intracerebral inoculation of herpes simplex virus type 1 (KOS) and comparison with mutants showing decreased virulence

The spread of herpes simplex virus type 1 (HSV-1) strain KOS, and two less neurovirulent mutants of the strain was studied in female DBA/2 mice during the 1- to 5-day postinoculation period after intracerebral inoculation. Immunohistopathology showed that wild-type KOS virus first infected the meninges and ependymal cells but did not infect cells at the inoculation sites. The virus continued to spread to some cells directly adjacent to ventricles; however, the most extensive and severe lesions were found in the pyriform lobes and other structures associated with the limbic system. The pattern of spread suggested that direct cell to cell viral spread is important but that retrograde axonal transport to distant sites probably accounts for the more severe lesions associated with the limbic system. Both less neurovirulent mutant viruses multiplied to a much lesser degree in the brain and spread less extensively than the wild type virus when equivalent doses were given; however, when a large dose of the least neurovirulent mar C10.1 mutant virus was inoculated, infection spread rapidly to the same regions of the brain affected by KOS. Studies of mar C10.1 showed that thymidine kinase deficiency, rather than a mutation in the gene coding for glycoprotein C, probably accounted for the decreased neurovirulence of this mutant. This mouse model of HSV-1 virus-induced encephalitis, in combination with appropriate studies of the molecular biology of the HSV-1 KOS strain, should be useful for the study of neurovirulence factors contributing to the pathogenesis of HSV-1.     

惠州市重症手足口病病原谱及EV71型VP1区基因特征分析

目的分析惠州市手足口病重症病例的病原谱构成,了解惠州市肠道病毒71型分离株的VPl区基因特征．为科学防治手足口病提供科学依据。方法采集300例重症手足口病（HFMD）患者标本,采用实时荧光RT-PCR检测肠道病毒核酸,并对肠道病毒7l型（EV71）和柯萨奇病毒A16型（CoxA16）进行分型检测;选择8株EV71分离株进行VPl区基因全长序列测定,测序结果利用DNASTAR软件进行核苷酸、氨基酸序列分析和同源性比较。并用Mega5．0软件构建亲缘性进化树。结果通过实时荧光RT-PCR特异性检测,EV71阳性结果154份,阳性率为51．33％;CoxAl6阳性结果38份,阳性率为12．67％。测序结果表明,8株EV71之间的VPl基因核苷酸同源性为96．9％～99．2％。氨基酸同源性为99．3％～100％。VPI区基因遗传进化分析表明。8株EV71分离株与c4基因亚型的代表株处于同一分支,均属于C4基因亚型的C4a进化分支。结论惠州市手足口病疫情主要病原EV71病毒均属于C4a基因亚型,与2004年以来的中国大陆EV71病毒流行的基因型一致,未产生明显的抗原漂移及变异。     

Changes in the neuraminidase of neurovirulent influenza virus strains

The influenza virus A/WS/33 has been adapted to mouse brain to produce two neurovirulent derivatives, A/NWS/33 (NWS) and A/WSN/33 (WSN), with the viral neuraminidase gene shown to be the major determinant of neurovirulence. The complete nucleotide sequence of the NA genes from each strain has been determined, which has allowed the identification of changes that have occurred during adaptation to mouse brain. Five changes are shared by the neurovirulent strains. Comparison to the known neuraminidase structure has identified four of these that may affect the active site of the enzyme. In addition, significant differences in the properties of the neuraminidase from the neurovirulent strains were observed relative to the parent strain. While no correlation was observed between neurovirulence and overall neuraminidase activity or preference for a particularN-substitution, the enzymes from both neurovirulent strains showed an increased preference for small substrates and those with 23 linkages, and their activity was potentiated by Ca²⁺ ions.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers L25815-7.     

Neurovirulence of wild and laboratory Junin virus strains in animal hosts

The neurovirulence of Candid #1 and XJCL3 laboratory strains and CbalV4454 and CbaFHA5069 wild strains of Junin virus was studied in albino mice, guinea pigs, and a South American wild rodent, Calomys musculinus, of different ages inoculated by the intracerebral route. Infectivity in brain and organs, lethality, and neuropathological lesions were determined. The laboratory and wild strains showed similar neurovirulence only in 2-day-old mice. The neurovirulence of laboratory strains decreased with the age of the animal, and the Candid #1 strain affected only 2-day-old mice. In guinea pigs, the 2 wild strains and XJCL3 laboratory strain were neurovirulent for 11-day-old and adult animals giving moderate lymphocytic infiltration in the brain and mild lesions in the spinal cord. Virus titres from the brain and the spinal cord were lower with the XJCL3 and CbalV4454 strains than with the CbaFHA5069 strain; with the latter, virus was recovered only from the lymph nodes, the lung, kidney, liver, and spleen. The Candid #1 strain was not neurovirulent even for 11-day-old animals. In contrast, the laboratory strains were neurovirulent for Calomys musculinus, depending on the age of the animal. Virus was recovered from the brains showing lymphocyte infiltration but not from other organs. The CbaFHA5069 strain was not neurovirulent, although virus was recovered from the brain, spleen, liver, lymph nodes, and salivary glands. These results with the 3 hosts indicate that Junin virus neurovirulence is virus strain-dependent, and host species and age-dependent, with the Candid #1 strain proving the least neurovirulent of the strains studied.     

Molecular epidemiology of enterovirus 71 in peninsular Malaysia, 1997–2000

Summary. Human enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae) has been responsible for sporadic cases and outbreaks of hand-foot-and-mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like disease in Europe, the U.S.A., Australia and Asia. Recently, there has been an increase in EV71 activity in the Asia-Pacific region, with many outbreaks of HFMD associated with brainstem encephalitis manifesting as neurogenic pulmonary oedema with a high case fatality rate. In 1997, and again in 2000, EV71 outbreaks occurred in peninsular Malaysia. Variations in VP1 gene sequences have been shown to divide all known EV71 field isolates into three distinct genogroups (A, B and C). Consequently we examined the VP1 gene sequences of 43 EV71 strains isolated in peninsular Malaysia between 1997 and 2000 in order to determine the genogroup prevalence over the period. In this study we show that four subgenogroups (B3, B4, C1 and C2) of EV71 circulated in peninsular Malaysia between 1997 and 2000. Subgenogroups B3, B4 and C1 have been identified as the primary cause of the outbreaks of EV71 in peninsular Malaysia. Subgenogroup C1 also displayed endemic circulation from 1997 to 2000 and subgenogroup C2 was present at a low level during the 1997 outbreak.Received September 6, 2002; accepted January 20, 2003 Published online June 2, 2003     

北京市2008年肠道病毒71型的RT-PCR鉴定及VP1区基因特征分析

目的 研究2008年北京市手足口病（Hand,foot and mouth disease,HFMD）患者中肠道病毒71型（Human enterovirus 71,HEV71）的VP1编码区基因特征.方法 采集北京市朝阳区医院和幼儿园的HFMD患者标本共285份,进行HEV71特异性RT-PCR鉴定和病毒分离,随机选取10株HEV71阳性分离株进行VP1编码区基因扩增和核苷酸序列测定和分析.结果 129份标本RT-PCR鉴定结果 为阳性,阳性率为45.26%.10株HEV71在VP1区核苷酸水平和氨基酸水平上的差异分别在94.6%～99.6%和95.9%～100%之间.北京朝阳HEV71分离株属于C4基因亚型C4a进化分支.结论 RT-PCR法可以快速、准确地鉴定HEV71.引起本次HFMD流行的HEV71为C4基因亚型C4a进化分支,并且存在多个传播链,提示自1998年起,C4亚型的HEV71在中国大陆有较广泛的传播.加强对HEV71的分子流行病学监测,了解HEV71的基因特征,对预防和控制HEV71在中国的暴发具有重要意义.