Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence.These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.     

Mumps vaccine L-Zagreb, prepared in chick fibroblasts. I. Production and field trials

Leningrad-L3 Mumps Vaccine virus has been further attenuated by adaptation and passage on SPF chick embryo fibroblast cell cultures. This new mumps strain has been designated L-Zagreb and has been used to prepare mumps vaccines which meet the WHO requirements. Observations during both the field trial period prior to registration and during the later use of the vaccine showed that the few reactions observed were mild and that seroconversion was obtained in 88-98% of vaccines. The morbidity of mumps in Croatia declined more than tenfold after the introduction of the new vaccine. During a mumps epidemic, vaccine efficiency was calculated to be 97-100%.     

The F gene of rodent brain-adapted mumps virus is a major determinant of neurovirulence

Prior to the introduction of live-attenuated vaccines, mumps virus (MuV) was the leading cause of virus-induced meningitis. Although vaccination has been effective at controlling the disease, the use of insufficiently attenuated strains has been associated with high rates of aseptic meningitis in vaccinees. The molecular basis of MuV attenuation is poorly understood, and no reliable molecular markers of virulence have been identified. In this study, reverse genetics has been used to identify molecular determinants of MuV neuropathogenesis. Recombinant viruses, containing the envelope-associated genes from the Kilham (MuV(KH)) rodent brain-adapted strain of MuV, were generated in the Jeryl Lynn 5 (MuV(JL5)) vaccine strain background. The syncytium phenotypes of the recombinant viruses on Vero cells differed depending on the source of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, with heterologous combinations showing either an increase or a decrease in the level of cell fusion compared to that of the homologous parental combinations. This was confirmed by transiently cotransfecting eukaryotic F and HN glycoprotein expression constructs. A Lewis rat model that discriminates between neurovirulent and nonneurovirulent MuV strains based on the extent of hydrocephalus induced in the rat brain after intracerebral inoculation was used to assess the phenotype of the recombinant viruses. Expression of the matrix (M), small hydrophobic (SH), or HN gene in isolation did not confer a neurovirulent phenotype. Expression of the F gene of the neurovirulent strain alone was sufficient to induce significant levels of hydrocephalus. Coexpression of the homologous HN gene led to a marginal increase in the level of hydrocephalus.     

Prevention of mumps in preschool institutions using a live mumps vaccine made from strain L-3

The effectiveness of the emergency vaccinal prophylaxis of epidemic parotitis was studied in 19 children's day-care centers. As revealed in this study, the immunological effectiveness of vaccination did not depend on the age of vaccinees, but sharply decreased if live parotitis vaccine contained less than 10,000 HADU50 per immunization dose. After a single administration of the vaccine 91.1 +/- 0.98% of children were found to produce mumps antibodies. The immunization of children with live parotitis vaccine prepared from strain l-3 immediately after the first case of parotitis had been registered proved to be a highly effective measure. The coefficient of epidemiological effectiveness was 96.4%.     

Neurovirulence of mumps virus: intraspinal inoculation test in marmosets.

An intraspinal inoculation test of mumps virus using marmosets was performed in order to develop a neurovirulence test of mumps vaccines. In the group inoculated with non-neurovirulent Jeryl Lynn vaccine strain at 10(2.0) pfu/dose, there were only minimal histopathological changes in 3 of the 5 marmosets. In contrast, all marmosets inoculated with neurovirulent Urabe and NK-M46 vaccine strains developed extensive encephalitis and meningitis. Thus, this marmoset model, which can distinguish between non-neurovirulent and neurovirulent vaccine strains, is useful for evaluating neurovirulence of vaccine strains and elucidating the molecular pathogenesis of mumps.     

The reactogenicity and immunogenicity of the Urabe Am 9 live mumps vaccine and persistence of vaccine induced antibodies in healthy young children

The immunogenicity and reactogenicity of the Urabe Am 9 mumps virus vaccine strain were studied after the administration of different doses of the vaccine to 197 children ranging in age from seven and a half months to nine years and without a history of mumps. There was no effect of dose on the response in serum neutralizing antibodies in the range of 10(2.9) to 10(4.7) TCID50/dose. In the 90 subjects without detectable serum neutralization antibodies before vaccination seroconversion was obtained in 94.4% after 42 days. Half of a group of 34 seropositive children who were tested also showed a fourfold or greater rise in antibodies. Persistence of vaccine-enhanced haemagluttinin-inhibition (EHI) antibodies was satisfactory as only two of 46 vaccinees followed-up for between 27 and 32 months had undetectable levels of EHI antibodies and the geometric mean titre of vaccine-induced EHI antibodies had only fallen to about one-third by 32 months after vaccination. Although there was serological evidence of a subclinical re-infection in three subjects, to date none of the vaccinees has had clinical mumps indicating that the vaccine confers protection against disease. The vaccine was well tolerated. Furthermore, the majority of the few 'reactions' reported were probably not vaccine-related. It is concluded that the Urabe Am 9 is an acceptable strain for use in live mumps vaccines.     

Cell-mediated immune response to mumps virus infection in man.

The antibody and cell-mediated immune response to mumps virus infection was studied in groups of subjects after natrually acquired mumps virus infection, after parenteral immunization with live attenuated mumps vaccine, and in a population of mumps seronegative subjects. The technique of neutralization of tissue culture infectivity was utilized to study mumps specific antibody. The cell-mediated immunity (CMI) was detected by specific immune release (SIR) of radioactivity by purified lymphocytes after they were reacted with radioactive chromium (51Cr) labeled human conjunctival cell cultures chronically infected with mumps virus. No SIR activity was observed in lymphocytes obtained from cord blood and young individuals seronegative for antibody to mumps virus. Detectable SIR activity was observed in a few older seronegative subjects; however, immunization with mumps vaccine in such antibody negative subjects failed to result in the development of any antibody response in the serum. High SIR activity was observed in the lymphocytes of naturally infected and vaccinated subjects. Although all naturally infected or immunized subjects had varying levels of mumps specific antibody activity in the serum, no correlation existed between the levels of antibody and SIR activity. These observations suggest the development of mumps specific in vitro correlates of CMI after naturally acquired or vaccine-induced mumps virus infection.     

Characterization of mumps virus isolated in saitama prefecture, Japan, by sequence analysis of the SH gene

Mumps virus (MuV) strains isolated from cerebrospinal fluid and throat swabs from patients in Saitama Prefecture and Tokyo, Japan, from 1997 to 2000 were examined by analyzing the SH gene nucleotide sequence (316-nt). Eighteen of the 20 strains studied were divided into three genotypes, recognized as B, G, and H in previous reports. Two genotypes (G and H) are believed to be new in Japan. Two of the 20 strains belonged to none of the previously reported genotypes (A-I), but were closely related to two known strains, MP94-H and Loug1/UK97. We propose that the two strains identified in this study together with the previously reported strains, MP94-H and Loug1/UK97, form a new genotype, designated J, based on the divergence of the SH gene nucleotide sequences between these four strains and other strains reported (genotypes A-I). Our results also suggest that more than two genotypes circulated in Saitama Prefecture from 1997 to 1999, but only one, genotype G, was in evidence in 2000. Genotype B was earlier reported as the predominant strain in Japan, but it became undetectable by the year 2000. These results provide important epidemiological data on mumps in Japan.     

Discrimination of mumps virus small hydrophobic gene deletion effects from gene translation effects on virus virulence

Deletion of the small hydrophobic (SH) protein of certain paramyxoviruses has been found to result in attenuation, suggesting that the SH protein is a virulence factor. To investigate the role of the mumps virus (MuV) SH protein in virulence, multiple stop codons were introduced into the open reading frame (ORF) of a MuV molecular clone (r88-1961(SHstop)), preserving genome structure but precluding production of the SH protein. No differences in neurovirulence were seen between the wild-type and the SH(stop) viruses. In contrast, upon deletion of the SH gene, significant neuroattenuation was observed. These data indicate that the MuV SH protein is not a neurovirulence factor and highlight the importance of distinguishing gene deletion effects from protein-specific effects.     

辽宁省2008～2011年流行性腮腺炎野病毒SH蛋白基因特征分析

本研究用Vero/Slam细胞从辽宁省2008～2011年流行性腮腺炎暴发和散发患者的临床标本中分离到13株流行性腮腺炎野病毒(Mumps virus,MuV),应用逆转录-聚合酶链反应(RT-PCR)针对MuV分离株的SH基因的316个核苷酸片段进行扩增,并对该产物进行序列测定。将这13株MuV与从GenBank下载的世界卫生组织(WHO)MuV基因型参考株一起进行分子流行病学研究。结果提示:除2011-015株外,辽宁省2008～2011年12株MuV分离株均属于F基因型,核苷酸和氨基酸同源性为94.9%～100%和83.3%～100%。与F基因型参考株序列相比,核苷酸和氨基酸同源性分别为92.4%～97.2%和96.5%～84.2%。表明2008～2011年辽宁省流行的F基因型MuV发生较大的型内变异。另外还发现F基因型MuV在SH基因上存在着特异性突变(CNt65,CNt105,G Nt137,C Nt192,C Nt239,GNT262),而其它基因型MuV在这些位点上均未发生改变。F基因型MuV在SH基因编码的氨基酸保守位点也发生变化。如:第2位上由S→P,第6位上由P→L,第23位上由T→N,第48位上由L→P/R。与基因分型有关的氨基酸三联体,2008-01-007毒株也发生了改变,由IML变为TMP。2011-015株病毒与F基因型参考株平均核苷酸和氨基酸同源性分别为87.5%和79.8%,与G型参考株平均核苷酸和氨基酸同源性分别为96.8%和97.4%,属于G基因型。该基因型为中国内地首次发现。     

Gene-specific contributions to mumps virus neurovirulence and neuroattenuation

Mumps virus (MuV) is highly neurotropic and was the leading cause of aseptic meningitis in the Western Hemisphere prior to widespread use of live attenuated MuV vaccines. Due to the absence of markers of virus neuroattenuation and neurovirulence, ensuring mumps vaccine safety has proven problematic, as demonstrated by the occurrence of aseptic meningitis in recipients of certain vaccine strains. Here we examined the genetic basis of MuV neuroattenuation and neurovirulence by generating a series of recombinant viruses consisting of combinations of genes derived from a cDNA clone of the neurovirulent wild-type 88-1961 strain (r88) and from a cDNA clone of the highly attenuated Jeryl Lynn vaccine strain (rJL). Testing of these viruses in rats demonstrated the ability of several individual rJL genes and gene combinations to significantly neuroattenuate r88, with the greatest effect imparted by the rJL nucleoprotein/matrix protein combination. Interestingly, no tested combination of r88 genes, including the nucleoprotein/matrix protein combination, was able to convert rJL into a highly neurovirulent virus, highlighting mechanistic differences between processes involved in neuroattenuation and neurovirulence.     

Development of a new live attenuated mumps virus vaccine in human diploid cells

A new live attenuated mumps vaccine was developed in human diploid cells. The S-12 virus was isolated from a 10-year-old girl showing typical symptoms of mumps infection, the diagnosis was confirmed by a pediatrician. The virus was isolated in green monkey kidney cells, without passage in chick embryo cavity or chick embryo fibroblasts. Attenuation of the wild virus was performed by serial passages in human diploid cells (MRC-5). The attenuated virus was characterized by identity tests, as well as by a reduction in plaque size, as marker tests. The virus was free from adventitious agents and safe for laboratory animals as well as for monkeys. The reactogenicity and immunogenicity of the S-12 virus for man was investigated by administration of a monovalent vaccine to 20 seronegative adult male volunteers and 30 children aged 1 to 5 years without history of mumps infection or vaccination. Seroconversion was obtained in 95% of the vaccinees. The new vaccine has the advantage of not requiring specific pathogen-free eggs, and being free from avian proteins and therefore can be used in sensitized patients.     

Identification and development of a promising novel mumps vaccine candidate strain

Mumps epidemics are usually caused by airborne transmission of mumps virus (MuV) and have high morbidity in non-immunized children. Epidemiological studies in many regions of China show that the genotype F viral strain is the most prevalent. However, the genotype A strain is currently used to prepare vaccines. Regional epidemiological MuV data suggest a significant application for the development of live attenuated mumps vaccines targeting specific genotypes. This article reports the isolation and culture of a genotype F MuV candidate strain that could be used to prepare a live attenuated mumps vaccine. This strain is shown to have good immunological efficacy and stability in neurovirulence evaluations. This work should facilitate the implementation of mumps vaccination in mainland China by targeting the most prevalent MuV genotype, genotype F.     

mRNA sequence and deduced amino acid sequence of the mumps virus small hydrophobic protein gene.

The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.     

Cloning and Characterization of the Genomic RNA Sequence of the Mumps Virus Strain Associated with a High Incidence of Aseptic Meningitis

cDNA clones of the mumps virus wild-type strain, associated with a high incidence of aseptic meningitis (ODATE-1 strain), were isolated and analyzed from genomic nucleotide position 22 to 8520 containing the NP, P, M., F, SH and HN protein coding region. The ODATE-1 strain exhibited a RFLP profile identical to that of the Urabe vaccine strain in spite of the fact that the virus was isolated from non-vaccinated cases. However, a comparison of nucleotide and amino acid sequences among the ODATE-1 strain, Urabe strain and Miyahara strain revealed that the ODATE-1 strain was not related to the Urabe strain.     

腮腺炎病毒抗血清的制备

以制备适用于疫苗生产检定中病毒鉴别试验和外源因子检查的高效价腮腺炎病毒抗血清为目的。用腮腺炎病毒接种SPF鸡胚尿囊腔,培养收取病毒尿液免疫SPF鸡,采集抗血清。腮腺炎病毒接种Vero细胞,培养病毒抗原经PEG沉淀,超速离心法纯化后免疫家兔采集抗血清。比较两种免疫方法所得病毒抗血清效价。结果显示SPF鸡抗腮腺炎病毒血清中和抗体GMT为1:1716,兔抗腮腺炎病毒血清中和抗体GMT为1:732。两种动物抗血清均适用于疫苗生产相关检定。免疫SPF鸡制备的病毒抗血清无特定病原及抗体污染,是毒种外源因子检测和疫苗鉴别试验的理想试剂。免疫SPF鸡制备病毒抗血清的程序简单,结果易于验证,有利于生物试剂标准化。     

Protection of mumps in children with various underlying diseases: application of a live attenuated mumps and trivalent measles-rubella-mumps (MRM) vaccines in these children

A live attenuated mumps and trivalent measles-rubella-mumps (MRM) vaccines have been applied to 887 and 148 children with various underlying diseases at the vaccine clinic of Osaka University Hospital between 1975 and 1985, respectively. Clinical reactions after mumps vaccination occurred in only 7 children (0.8%) and those after MRM vaccination in 28 children (19%), but their underlying diseases were not deteriorated by either vaccination. Clinical follow up study revealed that 2 of the 430 children immunized with mumps vaccine had contracted the disease during 7 year period and 2 of the 123 children immunized with MRM vaccine had contracted clinical mumps or rubella during 3 year period. The seroconversion rates after mumps vaccination were 70% and 61% by the hemagglutination inhibition (HI) test and neutralization (NT) test, respectively, while 94% by the fluorescent antibody to membrane antigen (FAMA) test. Those after MRM vaccination were 87% for measles, 96% for rubella by the HI test and 89% for mumps by the FAMA test. Serological follow up study revealed that antibodies elicited by mumps vaccination were sustained without substantial decline for at least 7 years. These results suggest that a live attenuated mumps and MRM vaccines are safe and effective in children with various underlying diseases.     

Two clones obtained from Urabe AM9 mumps virus vaccine differ in their replicative efficiency in neuroblastoma cells

A high rate of post-vaccinal aseptic meningitis for Urabe AM9 mumps virus strain is well documented. This strain is composed of two virus variants differing at the nt 1081 (A/G) region in the hemagglutinin-neuraminidase (HN) gene. An association of HN-A(1081) variant with neurovirulence has been proposed. In order to test for neurotropism we isolated the HN-A(1081) and HN-G(1081) virus variants from Urabe AM9 mumps virus vaccine. Sequential passages were performed in monkey kidney Vero cells and human neuroblastoma SH-SY5Y cells. Viral replication was determined by conventional and real-time RT-PCR. The results show that clone HN-A(1081) can replicate efficiently in both cell types. However, a defective replication of clone HN-G(1081), lacking its genetic marker, was observed after the third passage in neuroblastoma cells. Kinetics assays showed that clone HN-A(1081) replicates faster than clone HN-G(1081). Viral clones were also inoculated into the brains of newborn rats. Clone HN-A(1081) replicated 14 times, while clone HN-G(1081) merely duplicated its level over the initial inoculum. These results suggest that there is a selective replication of HN-A(1081) mumps virus variants in cells of nervous origin.     

Recent mumps outbreaks in vaccinated populations: no evidence of immune escape

Recently, numerous large-scale mumps outbreaks have occurred in vaccinated populations. Clinical isolates sequenced from these outbreaks have invariably been of genotypes distinct from those of vaccine viruses, raising concern that certain mumps virus strains may escape vaccine-induced immunity. To investigate this concern, sera obtained from children 6 weeks after receipt of measles, mumps, and rubella (MMR) vaccine were tested for the ability to neutralize a carefully selected group of genetically diverse mumps virus strains. Although the geometric mean neutralizing antibody titer of the sera was lower against some virus strains than others, all viruses were readily neutralized, arguing against immune escape.