Genotyping of varicella zoster virus strains isolated in Mongolia

This study analyzed 50 varicella zoster virus (VZV) samples collected during 2004 to 2007 from patients with VZV infection, who were treated at the National Center of Communicable Diseases, Ulan-Bator, Mongolia. The method based on amplification of specific DNA fragments of the ORF21, ORF22, and ORF50 genes was used, followed by the sequencing and detection of the status of characteristic point mutations in these fragments. The results indicated that the collected samples belonged to genotypes J (62%), M1 (18%), E1 (12%), E2 (4%), and M2 (4%). Moreover, restriction endonuclease polymorphism in ORF 62 for the cleavage site Smal and Mspl, in ORF 38 and ORF 54 for the cleavage site Pstl and Bgll were analyzed. All the samples were Sma- Msp-. All samples with genotype E were Pst+ Bgl-; all samples with genotype M1 and M2 were Pst+ Bgl+. Out of 31 samples with genotype J, 29 and 2 were Pst+ Bgl+ and Pst+ Bgl+, respectively. The study could identify the genotypes of VZV circulating in Mongolia and confirmed the absence of mutations characteristic for the vaccine strain.     

水痘-带状疱疹病毒临床分离株基因型别分析以及与Oka疫苗株的区别

目的 运用分子生物学方法 对从水痘或带状疱疹患者皮肤疱疹液中分离得到的水痘-带状疱疹(VZV)株进行基闪型研究,并区分感染是南野牛株还是由Oka疫苗株引起的.方法 从水痘或带状疱疹患者的皮肤水疱液中分离VZV,然后利用聚合酶链反应和限制性片段长度多态性分析对病毒株的ORF38、54、62和R5可变区基因进行分析.结果 在所分离的19株VZV中,存在PstⅠ+ Bgl Ⅰ+ R5A和Pst Ⅰ+ Bgl Ⅰ+RSB两种基因型,其中Pst Ⅰ+ Bgl Ⅰ+ R5A占52.7%,Pst Ⅰ+ Bgl Ⅰ+R5B占47.3%,没有发现与Oka疫苗株相同的基因型.结论 本研究中所分离的VZV毒株均系野生株,它们的基因型与欧洲、美国、日本的VZV分离株均不相同.利用病毒基因组中ORF38和ORF62区域的单一核苷酸多态性,能够区分疫苗株和野毒株.     

Growth of vaccine strains of avian pneumovirus in different cell lines.

The isolation of avian pneumovirus (APV) (avian metapneumovirus) is usually performed in embryonated chicken eggs or chicken embryo fibroblast cell cultures followed by adaptation in continuous cell lines such as Vero cells. This study was conducted to find a suitable cell line that could be used to propagate vaccine strains of APV to high titre. For this purpose, we compared the growth of two Vero cell-adapted vaccine strains of APV (P63 and ca-APV) in seven different cell types with their growth in Vero cells. The cell types used were BGM-70, MA-104, QT-35, BHK-21, McCoy and DF-1 cells and primary turkey embryo fibroblast cells. When compared with growth in Vero cells, both viruses yielded higher titres in BGM-70 cells, while P63 also produced higher titres in MA-104 cells. In another experiment, the two viruses were grown and titrated in Vero cells under various cell culture conditions, such as age of cells, seeding concentration, and time of harvest. None of these cell culture variables were found to affect virus titres.     

Genotyping of varicella-zoster virus and the discrimination of Oka vaccine strains by TaqMan real-time PCR

Single nucleotide polymorphisms (SNPs) in five genes have been used to identify four major subtypes of wild-type varicella-zoster virus (VZV) A, B, C, and J. Additional SNPs, located in the IE62 major transactivating gene can be used to differentiate the Oka vaccine strain (vOka) from wild-type VZV. Primer-probe sets for the detection of the five polymorphic loci were designed by Applied Biosystems for the ABI 7900HT platform. Probes for each allele were labeled with VIC or 6-carboxyfluorescein fluorogenic markers. Each primer-probe set was validated to establish assay sensitivity and specificity using VZV DNA of predetermined copy number and genotype. Further evaluation was carried out using DNA samples from the vesicle fluid or skin swab of the rash of adult patients with herpes zoster or rashes due to vOka. Assay sensitivity ranged from 10 and 10(8) copies/ml of VZV DNA (equivalent to 2 to 20 copies per reaction). Statistical analyses showed that for each genotype, a set of two probes clearly differentiated the nucleotide present (allele) at that locus (P < 0.0001). It was possible to determine the genotype of wild-type VZV using one of four SNP assays and also to differentiate wild type from vOka using a single SNP assay. The assay can be used for diagnostic and epidemiological studies of VZV, including the differentiation of vOka from wild-type strains, investigation of breakthrough infections, and varicella outbreaks following immunization.     

VZV meningitis following varicella vaccine

Live attenuated varicella zoster virus is administered to prevent varicella in children and herpes zoster in the elderly. We report a case of disseminated herpes zoster in a previously healthy elderly woman hours after Oka strain vaccination. PCR and restriction enzyme analysis revealed that symptoms were caused by wild type virus.     

Review of the Varilrix varicella vaccine

Varicella zoster virus causes an acute infection that affects most children globally, but the age of infection can be greater in residents of tropical areas. It has generally been considered a mild disease, although there are accumulating data to show that it can cause significant morbidity and mortality in immunocompetent as well as immunocompromised children and adults. Oka-strain live attenuated varicella vaccines were developed in the 1970s. Varilrix developed by GlaxoSmithKline Biologicals (Rixensart, Belgium), is one of the vaccines produced and marketed in over 80 countries. Similar to the other Oka-strain vaccines, Varilrix is safe, immunogenic and efficacious in both immunocompromised and immunocompetent children and adults.     

Mumps virus strains isolated in Croatia in 1998 and 2005: Genotyping and putative antigenic relatedness to vaccine strains

Two mumps virus strains 9218/Zg98 and Du/CRO05 were isolated in two locations in Croatia in 1998 and 2005, respectively. Genetic characterization of these temporally distinct mumps virus isolates was carried out in order to determine their genotype and putative antigenic relatedness to mumps virus vaccine strains. Sequence analysis of the small hydrophobic (SH) gene revealed that isolate 9218/Zg98 shows less than 95% of similarity to any reference strain, thus representing a potential reference strain for a new genotype. Isolate Du/CRO05 clearly belongs to genotype G with the 97% of homology to the reference strain Glouc1/UK96. When compared to each other, the two Croatian strains have extremely low level of homology of only 89% indicating no relatedness between them. Putative antigenic properties of the HN protein of these two isolates were compared to different vaccine strains. The results reveal a higher level of homology of antigenic determinants to non-A genotype vaccine strains than to A genotype vaccine strain.     

Rabies virus strains: a comparison study by polypeptide analysis of vaccine strains with different pathogenic patterns.

The five constitutent polypeptides of the rabies vaccine strains ERA, HEP, CVS, and PM (serotype 1) and Mokola (serotype 3) have been examined by tryptic peptide analysis. A comparison of the tryptic peptides of the N proteins of Mokola and the four serotype 1 strains revealed a general similarity. In contrast only few tryptic peptides of the G or M proteins were shared between different serotypes. While the tryptic peptide patterns of the corresponding M₁, M₂, N, and L proteins of the four vaccine strains show a great similarity the analysis of the tryptic peptides revealed characteristic differences in the G proteins. An overall comparison of the tryptic peptide patterns of the structural proteins of the four vaccine strains indicates that CVS and PM are more closely related to each other than to ERA or HEP. Characteristic qualitative and quantitative differences between the four vaccine strains were also demonstrated by their polypeptide patterns in SDS-PAGE. HEP, CVS, and PM each have two M₁ proteins which differ in their phosphate content. ERA, on the other hand, contains only a single M₁ protein which is characterized by a very low phosphate content. In contrast to ERA and HEP, CVS and PM each contain two glycoproteins, which have identical leucine-containing tryptic peptides but differ in the carbohydrate content. Whereas the pathogenic strains CVS and PM possess 150 and 104 L protein molecules per virion, 17 and 37 L protein molecules per virion were found to be present in the less pathogenic strains ERA and HEP.     

2013—2017年北京市通州区不同剂次水痘疫苗免疫史突破病例特征分析

目的了解2013—2017年北京市通州区不同剂次水痘疫苗免疫史突破病例流行病学及临床特征。方法通过现场调查和查阅中国疾病控制信息系统和北京市免疫规划信息系统获得水痘发病数据和免疫史资料,采用描述流行病学方法进行分析。结果2013—2017年通州区共报告中小学、托幼机构水痘病例数2102例,原发水痘病例989例,有明确1剂次免疫史的水痘突破病例966例,2剂次免疫史的突破病例147例。不同免疫史患者的性别、年龄、职业构成比差异有统计学意义。原发病例中,发热病例占46.71%,中重度出疹病例占34.68%;1剂次突破病例中发热病例占41.20%,中重度出疹病例占17.39%;2剂次水痘突破病例中发热病例占26.53%,中重度出疹比例占7.48%。3组的发热、出疹构成比差异有统计学意义。1剂次突破病例发病间隔中位数为5.11年,2剂次突破病例发病间隔中位数为2.44年,1剂次和2剂次突破病例发病间隔差异有统计学意义。结论随着免疫接种剂次的增加,水痘突破病例发热和出疹症状越来越不典型,由于临床症状相对较轻,突破病例在集体单位中作为传染源更容易被忽视。     

Molecular studies of the Oka varicella vaccine

Varicella zoster virus (VZV) is one of eight members of the Herpesviridae family for which humans are the primary host; it causes two distinct diseases, varicella (chickenpox) and zoster (shingles). Varicella results from primary infection, during which the virus establishes latency in sensory neurons, a characteristic of all members of the Alphaherpesvirinae subfamily. Zoster is caused by reactivation of latent virus, which typically occurs when cellular immunity is impaired. VZV is the first human herpesvirus for which a vaccine has been licensed. The vaccine preparation, v-Oka, is a live-attenuated virus stock produced by the classic method of tissue culture passage in animal and human cell lines. Over 90 million doses of the vaccine have been administered in countries worldwide, including the USA, where varicella morbidity and mortality has declined dramatically. Over the last decade, several laboratories have been committed to investigating the mechanism by which the Oka vaccine is attenuated. Mutations have accumulated across the genome of the vaccine during the attenuation process; however, studies of the contribution of these changes to vaccine attenuation have been hampered by the lack of a suitable animal model of VZV disease and by the heterogeneity that exists among the viral population within the vaccine preparation. Notwithstanding, a wealth of data has been generated using various laboratory methodologies. Studies of the vaccine virus in human xenografts implanted in severe combined immunodeficiency-hu mice, have enabled analyses of the replication dynamics of the vaccine in dorsal root ganglia, T lymphocytes and skin. In vitro assays have been used to investigate the effect of vaccine mutations on viral gene expression and sequence analysis of vaccine rash viruses has permitted investigations into spread of the vaccine virus in a human host. We present here a review of what has been learned thus far about the molecular and phenotypic characteristics of the Oka vaccine.     

A simplified plaque assay for varicella vaccine

A simple and accurate plaque assay is described for potency testing of attenuated varicella vaccine. Assays were performed on the African green monkey kidney continuous cell line CV-1, in multidish-plates, under a semi-solid carboxymethylcellulose overlay. The test is economical and yields accurate individual titre estimates, the reliability of which may be assessed by parallel titration of reference preparations.     

Comparison of immune responses of mice immunized with five different Mycobacterium bovis BCG vaccine strains.

Among the various parameters which may contribute to Mycobacterium bovis BCG vaccination efficiency, the choice of the vaccine strain may play an important role. In the present study, we therefore compared the immunogenicity of five different BCG strains that are commonly used for BCG vaccine production (Glaxo 1077, Japanese 172, Pasteur 1173P2, Prague, and Russian strains). The comparison of the growth capacity of these BCG strains in BALB/c and C3H mice demonstrated that a great difference exists between the capacity of various BCG strains to multiply and persist in target organs. A much lower recovery of BCG could be shown in mice immunized with Prague and Japanese BCG strains. T-cell responses of BCG-immunized mice were also examined by analyzing T-cell proliferative responses, cytokine production, delayed-type hypersensitivity responses, and cytotoxic activity. All these assays demonstrated that BCG immunization induced strong CD4+ T-cell responses, mostly of the Th1 type, as demonstrated by interleukin-2 and gamma interferon production. These studies also demonstrated that there are differences between BCG strains in stimulating these T-cell responses. A lack of induction of cytotoxic activity was observed following immunization with the Japanese strain. Lower anti-purified protein derivative antibody responses were also observed after intravenous or oral immunization with this BCG strain. Finally, the protective activity of these BCG strains was tested by measuring the capacity of immunized mice to eliminate recombinant Pasteur and Japanese BCG strains which expressed beta-galactosidase. The results of these experiments clearly demonstrated that the Prague and Japanese strains were unable to protect mice against a second mycobacterial challenge whereas mice immunized with the Glaxo, Pasteur, or Russian strain eliminated the recombinant BCG very efficiently. Altogether, the results of the present study strongly support the view that there are considerable differences in the immunogenicity of various BCG vaccine strains and that these differences may play a major role in BCG vaccination efficiency.     

Genotyping of Toxoplasma gondii strains from immunocompromised patients

The genotypes of Toxoplasma gondii strains isolated from HIV and non-HIV immunocompromised patients with cerebral and extracerebral toxoplasmosis were determined and compared to those of strains isolated from non-immunocompromised patients in order to identify the possible relationships between parasite genotype and morbidity of toxoplasmosis. One hundred and ten strains of T. gondii were obtained, either by cell culture (n = 73), brain biopsy (n = 17) or mouse inoculation (n = 20). Ninety strains isolated from immunocompromised patients (74 HIV+ and 16 non-HIV patients) were compared to 20 strains isolated from immunocompetent patients (17 cases of congenital toxoplasmosis, and three cases of primary acquired infection). Genotyping was performed by PCR/RFLP on locus SAG2, and T. gondii strains were classified as Type I, II or III. Ninety out of 110 strains were successfully genotyped, including 20 strains that had been maintained in mice, 69/73 strains maintained in cell cultures, but only 1/17 strains from formalin-fixed paraffin-embedded brain biopsies. 76.7% of the strains in the study population were of type II, 15.6% were type I and 7.7% were type III. The distribution of strain genotypes in immunocompromised and non-immunocompromised patients was comparable: 14.1% and 21% for type I, 76.1% and 79% for type II and 9.8% and 0% for type III, respectively; no correlation could be established between genotype and clinical presentation, i.e., cerebral or extracerebral toxoplasmosis. These results suggest that the type of infecting parasitic strain does not predominantly influence the pathogenesis of toxoplasmosis in immunocompromised patients and fully supports the need for specific prophylaxis in patients infected by T. gondii, regardless of the strain genotype.     

Persistence of immunity to varicella in children with leukemia immunized with live attenuated varicella vaccine

To determine the safety and efficacy of varicella vaccine, we studied 437 children in remission from leukemia who were immunized with live attenuated varicella virus. Three hundred one of the patients received two doses of vaccine and 136 received a single dose of vaccine from 1 of 10 lots from two manufacturers. The patients have been followed for an average of three years (range, one to six). Seroconversion occurred in 88 percent of the 437 children after the first dose of vaccine and in 98 percent after one or two doses. The proportions of patients who were seronegative after one, three, and five years were 20, 25, and 30 percent, respectively, with little change over time in the geometric mean titers of specific antibody (6.3, 6.5, and 5.7, respectively). Chickenpox has been documented in 36 vaccinated patients (8 percent) who had 3 to 640 vesicles (mean, 100), mild illness, and no complications. Of the 83 vaccinated patients exposed to varicella within their families, 11 had chickenpox; the attack rate was 14 percent (8 percent among seropositive patients, 29 percent among seronegative patients). There was no relation between the time since vaccination and either the attack rate or the severity of the breakthrough illness. Two doses of vaccine appeared to be no more effective than a single dose. Of the 372 patients receiving maintenance chemotherapy when immunized, 149 (40 percent) had a rash, which was treated with acyclovir in 16 children (4 percent) and became a severe febrile illness in 4. These reactions were not fatal and were all associated with vaccine lots, the use of which has since been discontinued. We conclude that in children in remission from leukemia, varicella vaccine is safe and induces an immunity to chickenpox that persists for more than three years.     

The effectiveness of the varicella vaccine in clinical practice

BACKGROUND: A live attenuated varicella vaccine was approved for use in the United States in March 1995 and is recommended for all susceptible persons 12 months of age or older. METHODS: To assess the effectiveness of the varicella vaccine, we conducted a case-control study with two controls per child with chickenpox, matched according to both age and pediatric practice. Children with potential cases of chickenpox were identified by active surveillance of pediatric practices in the New Haven, Connecticut, area. Research assistants visited the children on day 3, 4, or 5 of the illness, assessed the severity of the illness, and collected samples from lesions to test for varicella-zoster virus by polymerase chain reaction (PCR). RESULTS: From March 1997 through November 2000, data collection was completed for 330 potential cases, of which 243 (74 percent) were in children who had positive PCR tests for varicella-zoster virus. Of the 56 vaccinated children with chickenpox, 86 percent had mild disease, whereas only 48 percent of the 187 unvaccinated children with chickenpox had mild disease (P<0.001). Among the 202 children with PCR-confirmed varicella-zoster virus and their 389 matched controls, 23 percent of the children with chickenpox and 61 percent of the matched controls had received the vaccine (vaccine effectiveness, 85 percent; 95 percent confidence interval, 78 to 90 percent; P<0.001). Against moderately severe and severe disease the vaccine was 97 percent effective (95 percent confidence interval, 93 to 99 percent). The effectiveness of the vaccine was virtually unchanged (87 percent) after adjustment for potential confounders by means of conditional logistic regression. CONCLUSIONS: Varicella vaccine is highly effective as used in clinical practice.     

人巨细胞病毒临床分离株糖蛋白B基因型分析

目的 分析巨细胞病毒临床分离株糖蛋白B基因型。方法 以巢式聚合酶链反应(nested-PCR)检测器官移植受者外周血细胞和输卵管妊娠患者生殖道组织中的巨细胞病毒gBn和gBcls基因并测序分型。结果 分离到44例HCMV gBn、gBcls阳性基因，32例有HCMV gBn基因分型结果，其中l型8例(25％)，2型2例(6％)，3型22例(69％)，未见4型，分布以gBn3型为主。HCMV gBcls基因分型结果为26例，其中l型14例(54％)，2型5例(20％)，3型7例(26％)，未见4型，gBcls基因型则散在分布，以1型为多见。21例同时得到gBn、gBcls基因分型结果，其中8例(36％)表现出基因型别不一致。表现为gBclsl-gBn3型。结论 HCMV临床分离株，其糖蛋白基因分别属于gB基因型l-3型，并存在较多的gBcls与gSn基因型别的不对应现象，提示HCMV临床株有较高频率株内基因重组发生，是否由此导致病毒抗原性发生改变，值得进一步研究.     

Evolution of vaccine poliovirus strains

The paper considers different aspects of the molecular evolution of poliovirus vaccine strains: mutational and recombinant variability; mechanisms of fixation of occurring genetic changes. Matters concerning the policy of vaccination of the population against poliomyelitis are covered.