Mumps Virus Gene Research Articles

A recombinant varicella vaccine harboring a respiratory syncytial virus gene induces humoral immunity

The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is highly efficient and causes few adverse events; therefore, it is used worldwide. We previously constructed recombinant vOka (rvOka) harboring the mumps virus gene. Immunizing guinea pigs with rvOka induced the production of neutralizing antibodies against the mumps virus and VZV.Here, we constructed recombinant vOka viruses containing either the respiratory syncytial virus (RSV) subgroup A fusion glycoprotein (RSV A–F) gene or RSV subgroup B fusion glycoprotein (RSV B–F) gene (rvOka-RSV A–F or rvOka-RSV B–F). Indirect immunofluorescence and Western blot analyses confirmed the expression of each recombinant RSV protein in virus-infected cells. Immunizing guinea pigs with rvOka-RSV A–F or rvOka-RSV B–F led to the induction of antibodies against RSV proteins. These results suggest that the current varicella vaccine genome can be used to generate custom-made vaccine vectors to develop the next generation of live vaccines.

Real‐time PCR and its application to mumps rapid diagnosis

A real-time polymerase chain reaction assay was initially developed in China to detect mumps genome. The primers and TaqMan-MGB probe were selected from regions of the hemagglutinin gene of mumps virus. The primers and probe for the real-time PCR were evaluated by both laboratories in China and in the UK using three different pieces of equipment, LightCycler (Roche), MJ DNA Engine Option 2 (BIO-RAD) and TaqMan (ABI Prism) on different samples. The reaction was performed with either a one-step (China) or two-step (UK) process. The sensitivity (10 copies) was estimated using a serial dilution of constructed mumps-plasmid DNA and a linear standard curve was obtained between 10 and 10(7) DNA copies/reaction, which can be used to quantify viral loads. The detection limit on cell culture-grown virus was approximately 2 pfu/ml with a two-step assay on TaqMan, which was equivalent to the sensitivity of the nested PCR routinely used in the UK. The specificity was proved by testing a range of respiratory viruses and several genotypes of mumps strains. The concentration of primers and probe is 22 pmol and 6.25 or 7 pmol respectively for a 25 microl reaction. The assay took 3 hr from viral RNA extraction to complete the detection using any of the three pieces of equipment. Three hundred forty-one (35 in China and 306 in the UK) clinical specimens were tested, the results showing that this real-time PCR assay is suitable for rapid and accurate detection of mumps virus RNA in various types of clinical specimens.

Changes in Mumps Virus Gene Sequence Associated with Variability in Neurovirulent Phenotype

Changes in Mumps Virus Gene Sequence Associated with Variability in Neurovirulent Phenotype

Rapid and quantitative detection of mumps virus RNA by one-step real-time RT-PCR

We developed a new TaqMan-based one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection and quantification of mumps virus RNA. Oligos targeting the matrix protein gene of mumps virus were designed by using our oligo designing and analyzing software, Oligoware 1.0. Oligos's specificity was tested with 5 strains (4 laboratory isolated and 1 Jeryl Lynn strain) of mumps virus. The suggested TaqMan-based one-step real-time RT-PCR assay correctly detected the 4 laboratory-isolated strains and 1 Jeryl Lynn strain. To confirm the specificity of the TaqMan PCR assay, parainfluenza type 1, 2, 3 strains, sendai virus, and measles virus (vaccine strain) were tested, and no cross-reactivity was observed between mumps and tested strains. In addition, a BLAST (NCBI) search showed no genomic cross-reactivity with other viruses or cells. Testing of the assay's reproducibility was repeated several times, and the same results were achieved. The new assay was able to quantify the concentrations of mumps virus gene ranging from 10 1 to 10 8 copies per reaction sensitively with generated plasmid standards. In addition, it was shown that a significant correlation ( R 2 = 0.9564) between genome number as determined by one-step real-time RT-PCR and the corresponding number of plaque in paired samples was found with regression analysis. The results of one-step real-time RT-PCR assay also corresponded well to those of nested PCR. We conclude that our one-step real-time RT-PCR assay is a reliable, specific, and sensitive tool for the diagnosis of mumps virus. We consider that these results come from highly conserved primers and probe set that were designed with Oligoware 1.0.

Changes in Mumps Virus Gene Sequence Associatedwith Variability in NeurovirulentPhenotype

Changes in Mumps Virus Gene Sequence Associatedwith Variability in NeurovirulentPhenotype

Volume 77, no. 21, 2003, Table of Contents andAuthor Index

Table of Contents and Author Index: “Zhongqi Li” should be added to the list of authors for the article “Changes in Mumps Virus Gene Sequence Associated with Variability in Neurovirulent Phenotype,” p. 11616-11624.

Changes in mumps virus gene sequence associated with variability in neurovirulent phenotype.

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.

Antigenic relationships between six genotypes of the small hydrophobic protein gene of mumps virus.

Six different genotypes of mumps virus, A, C, D, G, H and I, genotyped on the basis of the small hydrophobic protein gene sequence, were subjected to antigenic comparison. Monoclonal antibodies directed against the haemagglutinin-neuraminidase protein of the SBL-1 strain of genotype A were used in immunofluorescence tests with different mumps virus strains. In addition, the six virus genotypes were compared by cross-neutralization tests with human post-vaccination sera after vaccination with the Jeryl Lynn (JL) strain of mumps virus and with rabbit hyperimmune sera directed against the A or D genotypes of mumps virus. Genotypes C, D, G, H and I could not be antigenically separated. In contrast, three different virus strains of genotype A, SBL-1, JL and Kilham, were distinct and were found to represent three different serotypes within the A genotype of mumps virus. Vaccination of Swedish children with the JL strain of mumps virus resulted in clearly lower neutralization titres against the SBL-1 strain, which is endemic in Sweden, compared to the homologous vaccine titres.

Phylogenetic analysis of the small hydrophobic (SH) gene of mumps virus in Korea: identification of a new genotype.

Viral RNAs extracted from fifteen mumps virus isolated from throat swab, saliva, blood, urine or CSF during mumps epidemics between 1997-1998 in Korea were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared by nucleotide sequencing of the small hydrophobic (SH) gene. The deduced amino acid sequences of the SH gene were aligned with the published sequences of mumps virus isolated in different geographic areas. A comparison of the SH gene of mumps viruses in Korea indicated 96.2-100% and 91.2-100% similarity at the nucleotide and amino acid levels, respectively. Phylogenetic analysis, using the neighbor-joining method, showed that Korean mumps virus strains formed a genetically distinct monophyletic group from previously reported genotypes based on the 315-bp length nucleotide and 57 deduced amino acid sequences of the SH gene, and possibly be designated as a new genotype (I).

Genetic heterogeneity of mumps virus in the United Kingdom: identification of two new genotypes.

A reverse transcriptase nested polymerase chain reaction (PCR) was developed to detect the small hydrophobic (SH) gene of mumps virus (MuV). Phylogenetic analysis was performed on the entire SH gene sequence (318 nucleotides) and the putative SH protein (57 amino acids). At least 4 MuV genotypes were identified in the United Kingdom between 1995 and 1998 by direct sequencing of 26 PCR amplicons from a variety of specimens. Comparison of these and GenBank sequences identified 2 new genotypes in the United Kingdom. The results suggest that, after the introduction of universal mumps vaccination in the United Kingdom in 1988, there appears to have been a switch from a predominant genotype to a heterogeneous group of strains.

Characterization of three co-circulating genotypes of the small hydrophobic protein gene of mumps virus.

Eighteen virus isolates and 22 serum samples collected between 1971 and 1997 from patients with mumps were genotyped by PCR with specific primer pairs for the A, C and D genotypes of the small hydrophobic (SH) protein gene. All serum samples were subjected to nucleotide sequence analysis of the gene, and the deduced 57-amino-acid sequences were aligned with previously published sequences from the USA, Canada, Portugal, the UK, France, Germany, Switzerland, Denmark, Sweden, Russia, China and Japan. The existence of six genotypes of the SH protein gene, named A to F, was confirmed. In the Stockholm area, co-circulation of genotypes A, C and D at different times was found. There was a striking difference in genotype between the virus isolates and the serum samples. The 18 virus isolates represented genotypes C and D, whereas the 22 serum samples contained genotype A. In most cases, the amino acid sequences of the 22 genotype A specimens were identical to the previously described SBL-1 strain of genotype A. Genotypes C and D were always associated with meningitis, and in some cases parotitis, whereas infection with genotype A most often resulted in parotitis and seldom in meningitis.

In vitro transcription and replication of the mumps virus genome.

By the use of lysolecithin-permealized extracts from mumps virus-infected HeLa cells, we have developed an in vitro system, which not only directed the synthesis of mumps virus mRNAs but also supported replication of the genomic RNA. Furthermore, upon transcription of the P gene, both faithful and edited copies of the P gene were detected by RNase mapping with a riboprobe. Thus this system seems to promote biochemical analyses of underlying mechanisms operative in mumps virus gene expression and replication, including RNA editing.

Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence

We have cloned and determined the nucleotide sequences of the seventh gene of the Miyahara strain of mumps virus (MuV) encoding the L protein. The L gene is 6925 nucleotides in length and contains a single long open reading frame which is capable of coding for a protein of 2261 amino acids with a calculated molecular weight of 256,571 Da. The deduced amino acid sequence of the L protein of MuV showed significant homology with those of six other paramyxoviruses, human parainfluenza type 2 virus, Newcastle disease virus, Sendai virus, measles virus, human parainfluenza type 3 virus, and human respiratory syncytial virus. The predicted MuV L protein contained distinct elements thought to be essential for RNA polymerase activity. A noncoding sequence of 24 nucleotides downstream of the presumed polyadenylation site of the L gene showed significant complementarity with the leader sequence composed of 55 nucleotide at the 3′ end of the genomic RNA.

Differentiation of the mumps vaccine strains from the wild viruses by the nucleotide sequences of the P gene

Nucleotide sequence analysis of a part of the P gene of mumps virus allowed the differentiation of live attenuated mumps vaccine strains from each other and from wild mumps viruses. Restriction enzyme analysis was found to serve as a convenient method of screening for one strain of mumps vaccine. Examination by the method of virus isolates obtained from the patients who developed mumps parotitis or meningitis following vaccination revealed that most of those viruses were related to the respective vaccine viruses used for immunization.

Strain-variable editing during transcription of the P gene of mumps virus may lead to the generation of non-structural proteins NS1 (V) and NS2

The sequence of the P (phosphoprotein) gene of mumps virus has been determined. It has two open reading frames, the first of which probably encodes the NS1 (or V) protein of mumps virus. Expression of the P protein requires the insertion of two non-templated residues to link the two ORFs in a process analogous to that observed in the P/V gene of simian virus type 5 to which mumps virus is closely related. Strain differences in the accuracy of insertion of non-templated G residues in the P/V gene transcripts have been described.

Nucleotide sequence of the leader and nucleocapsid protein gene of mumps virus and epitope mapping with the in vitro expressed nucleocapsid protein

The nucleotide sequence of the leader and the gene encoding the nucleocapsid protein (NP) of mumps virus Miyahara strain have been determined. The leader sequence is 55 nucleotides in length and the NP gene is 1845 nucleotides in length, exclusive of poly(A). The NP gene codes for a protein of 549 amino acids, with a calculated molecular weight of 61,365. For epitope mapping, a series of NPs from which C-termini were serially deleted were expressed in vitro from five mRNA constructs and were examined by radioimmunoprecipitation assay (RIPA) with eight nonoverlapping monoclonal antibodies (MoAbs) against the mumps virus NP. It was found that seven out of eight MoAbs reacted with the NP synthesized in vitro. Five recognized the epitopes located within the C-terminal 74 amino acids region and one within the adjacent 64 amino acids upstream. The epitope of the remaining one was in the N-terminal half of the NP.

Nucleotide sequence of the matrix, fusion and putative SH protein genes of mumps virus and their deduced amino acid sequences

cDNA clones representing the M, F and a putative SH gene of the SBL strain of mumps virus have been prepared and their nucleotide sequence determined. The M gene of mumps virus appears to contain 1253 nucleotides and codes for a protein of 375 amino acid residues ( M r 41589). The protein is hydrophobic and the deduced amino acid sequence shows homologies with those of other paramyxoviruses. The F gene of the SBL strain was compared to that of the RW strain [Waxham et al. (1987) Virology 159, 381–388]. The F gene is 1727 nucleotides long and codes for a protein of 538 amino acids ( M r 58789). There are substantial variations between the F gene sequences of various mumps virus strains. The F gene is followed by a small (315 nt) transcription unit which contains an open reading frame of 57 amino acids encoding a very hydrophobic protein ( M r 6712). This may be similar to the SH gene of SV5, although there appears to be no sequence homology between the SV5 SH protein and the putative SH protein of mumps virus. A physical and transcription map of mumps virus indicates the gene order to be 3'-N-P-M-F-SH-HN, similar to that of other paramyxoviruses and SV5 in particular.

Cloning and sequencing of the haemagglutinin-neuraminidase gene of mumps virus (Miyahara strain).

Cloning and sequencing of the haemagglutinin-neuraminidase gene of mumps virus (Miyahara strain).

Cloning and sequencing of the fusion protein gene of mumps virus (Miyahara strain).

Cloning and sequencing of the fusion protein gene of mumps virus (Miyahara strain).

Cloning and sequencing of the mumps virus fusion protein gene

The fusion protein (F) gene of mumps virus was cloned from a cDNA library constructed from infected cell mRNA. The F-specific plasmids were identified by hybridization to a degenerate oligonucleotide probe whose sequence was deduced from the N-terminal amino acid sequence of the F 2 protein. The complete nucleotide sequence of the F gene was determined. The gene is 1786 nucleotides long and encodes one long open reading frame of 538 amino acids. The F protein has a 19-amino acid signal peptide cleaved between Cys and Val residues. The cleavage site for activation of the F 0 protein into themature F 1,2 is ArgArgHisLysArg. A stretch of 30 hydrophobic amino acids near the C-terminus of the protein is followed by serveral charged amino acids and appears to serve as the anchoring domain for the protein in the lipid bilayer. The F gene of mumps virus is highly related to the F gene of the paramyxovirus SV-5.