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.     

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.     

Comparison of the Neurovirulence of a Vaccine and a Wild-Type Mumps Virus Strain in the Developing Rat Brain

Prior to the adoption of widespread vaccination programs, mumps virus was the leading cause of virus-induced central nervous system (CNS) disease. Mumps virus-associated CNS complications in vaccinees continue to be reported; outside the United States, some of these complications have been attributed to vaccination with insufficiently attenuated neurovirulent vaccine strains. The development of potentially neurovirulent, live, attenuated mumps virus vaccines stems largely from the lack of an animal model that can reliably predict the neurovirulence of mumps virus vaccine candidates in humans. The lack of an effective safety test with which to measure mumps virus neurovirulence has also hindered analysis of the neuropathogenesis of mumps virus infection and the identification of molecular determinants of neurovirulence. In this report we show, for the first time, that mumps virus infection of the neonatal rat leads to developmental abnormalities in the cerebellum due to cerebellar granule cell migration defects. The incidence of the cerebellar abnormalities and other neuropathological and clinical outcomes of mumps virus infection of the neonatal rat brain demonstrated the ability of this model to distinguish neurovirulent (Kilham) from nonneurovirulent (Jeryl Lynn) mumps virus strains. Thus, this neonatal rat model may prove useful in evaluating the neurovirulence potential of new live, attenuated vaccine strains and may also be of value in elucidating the molecular basis of mumps virus neurovirulence.     

The effect of serial passage on Sabin oral poliomyelitis vaccine virus in secondary monkey kidney versus Vero cells as measured by the monkey neurovirulence test

The three Sabin strains of poliomyelitis seed virus were serially passaged in either secondary monkey kidney or Vero cell cultures and the tenth passage of each virus harvest compared to non-passaged Sabin reference virus of the same type using the monkey neurovirulence test. All three types were further attenuated by passage in Vero cells, whereas only type 2 became further attenuated after passage in secondary monkey kidney cells. After passage in Vero cells, type 3 poliomyelitis virus became more heat stable, as measured by its replicative capacity at 40°C.     

Dengue type 4 live-attenuated vaccine viruses passaged in vero cells affect genetic stability and dengue-induced hemorrhaging in mice

Most live-attenuated tetravalent dengue virus vaccines in current clinical trials are produced from Vero cells. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DEN-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this study we investigated two DEN-4 viruses: the infectious cDNA clone-derived DEN-4 2A and its derived 3' NCR 30-nucleotide deletion mutant DEN-4 2AΔ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared following cell passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Although no significant increases in virus titers were observed following cell passages, DEN-4 2A and DEN-4 2AΔ30 virus titers following Vero cell passages were 17-fold to 25-fold higher than titers following MRC-5 cell passages. Neurovirulence for DEN-4 2A and DEN-4 2AΔ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 2A and DEN-4 2AΔ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 2A infectious clone indicated that single point mutation of E-Q(438)H, E-V(463)L, NS2B-Q(78)H, and NS2B-A(113)T imperatively increased mouse hemorrhaging severity. The relationship between amino acid mutations acquired during Vero cell passage and enhanced DEN-induced hemorrhages in mice may be important for understanding DHF pathogenesis, as well as for the development of live-attenuated dengue vaccines. Taken together, the genetic stability, virus yield, and DEN-induced hemorrhaging all require further investigation in the context of live-attenuated DEN vaccine development.     

Analysis of mumps virus defectiveness in vitro

The efficiency of mumps virus replication (EOR) was determined by comparing the multiplicity of infectious input (MOI) with the multiplicity of viral production (MOP). The replication kinetics of low passage wild, high passage laboratory and attenuated vaccine virus were evaluated using regular stock and cloned pools of virus (Table). The EOR of stock wild virus was 2.8, 4-fold greater than stock vaccine virus (0.7), and 300-fold greater than stock laboratory virus (0.009). The EOR of vaccine and laboratory virus was increased strikingly by double cloning, 0.7 to 2.0 and 0.009 to 3.7, respectively, while such limiting dilution hemadsorption plaquing techniques, employed to eliminate defective virions, reduced the EOR of wild virus (2.8 to 0.6).     

Detection of measles,mumps and rubella viruses by immuno‐colorimetric assay and its application in focus reduction neutralization tests

Measles, mumps and rubella are vaccine‐preventable diseases; however limited epidemiological data are available from low‐income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture‐based rapid and reliable immuno‐colorimetric assay (ICA) was established and its utility studied. Twenty‐three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT‐PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post‐infection in Vero or Vero‐human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post‐infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero‐epidemiological, cross‐neutralization and pre/post‐vaccine studies.     

Amino acid change 335 E to K affects the sialic-acid-binding and neuraminidase activities of Urabe AM9 mumps virus hemagglutinin-neuraminidase glycoprotein

A mutation coding for the amino acid change E335 to K is frequently found in the hemagglutinin-neuraminidase (HN) gene of Urabe AM9 mumps viruses isolated during post-vaccination meningitis cases. To identify if this mutation modifies the biological activities of the HN glycoprotein, two variants of Urabe AM9 vaccine differing at amino acid 335 (HN-E335 and HN-K335) were isolated and their receptor-binding specificity was determined by means of competence assays. Pre-incubation of the viruses with sialic acids inhibited both syncytia formation in Vero cells and replication in SH-SY5Y cells. Thus, HN-K335 showed higher affinity towards sialylalpha2,6lactose, whereas HN-G335 preferred sialylalpha2,3lactose. These results are relevant because a high expression of sialylalpha2,6lactose in nerve cells was confirmed by means of Sambucus nigra lectin-cytochemistry. In addition, kinetics assays showed that HN-K335 and HN-E335 also differ in their hydrolysis rate (Vmax values of 37.5 vs. 3.5 nmol min-1mg-1, respectively). Therefore, HN-K335 variant presented a neuraminidase activity level 11-fold higher than that of HN-E335 variant. In conclusion, the mutation affects the receptor-binding and neuraminidase activities of Urabe AM9 mumps virus variants.     

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.     

RNA sequence variants in live poliovirus vaccine and their relation to neurovirulence.

Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) was used to study sequence heterogeneity and stability in attenuated poliovirus type 3 at positions in which the vaccine virus differs from its wild-type progenitor. Of seven genomic positions tested, only two (positions 472 and 2493) show nucleotide heterogeneity. Propagation of the vaccine virus in cell cultures leads to rapid selection of virus with reversions at these two positions of the genome. The relative abundance of reversions at position 472 correlates with the results of monkey neurovirulence tests, while the mutation at position 2493 is not directly associated with neurovirulence of the virus in monkeys. Instead, the abundance of mutations at the latter position correlates with the source of the seed virus and its passage level. These results further indicate that MAPREC at position 472 can be used to assess the quality of poliovirus type 3 vaccine.     

登革热Ⅳ型病毒在地鼠肾细胞培养内减毒的研究

本文报道登革热Ⅳ型病毒Ban18株通过原代地鼠肾细胞连续传代后的适应和减毒过程。 Ban18原株对原代地鼠肾细胞无病变或仅有极轻微病变,对乳小白鼠及断乳鼠的脑内毒力高达LD_(50)为Log6—7。通过连续传20—70代后,病变逐渐加重,出现时间缩短,二天即可达到细胞完全破坏。培养液内的病毒含量也随之提高达10~6—10~7/ml TCID_(50)。但随着传代代数增加,病毒对乳小白鼠的嗜神经毒力逐渐降低,脑内接种后仅个别发病或完全不致死。脑组织病理变化也较原株明显减轻,接种树鼩不产生病毒血症。以上结果符合弱毒株的减毒指标,是一株有希望的活疫苗毒株。     

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.     

Genetic Variation in the HN and SH Genes of Mumps Viruses: A Comparison of Strains from Mumps Cases with and without Neurological Symptoms

Background

It is known that mumps virus (MuV) strains may vary in their neurovirulent capacity, and certain MuV strains may be highly neurotropic. In animal models and epidemiological studies, mutations at specific amino acids (aa) have been proposed to be associated with neurovirulence. To assess whether these genetic variations can be observed in clinical samples from patients and if they correlate with neurovirulence as determined by clinical symptoms, 39 mumps patients with or without neurological symptoms were investigated.

Principal Findings

Respiratory samples, oral fluids, throat swabs, and neurological and cerebrospinal fluid samples were tested by RT-PCR and products sequenced. Sequences of the entire small hydrophobic (SH) gene and the partial hemagglutinin-neuraminidase (HN) gene were compared.

Conclusions

The results showed there was no significant difference between the samples of the two groups of patients at the aa sites in either the HN protein or the SH protein, which have previously been hypothesized to be associated with neurovirulence or antigenicity. The occurrence of neurological symptoms of mumps does not appear to be due to a single point mutation in either the HN or SH gene.     

狂犬病毒CTN—1株在Vero细胞上的适应传代研究

本文报导了用我国狂犬病毒固定毒人二倍体细胞适应株（ＣＴＮ－１）进行Ｖｅｒｏ细胞适应传代研究。通过连续传代培养,滴度可达８．０１ｏｇＬＤ５０／ｍｌ,达到了ＷＨＯ规定的不需浓缩的标准。病毒用０．０１ＭＯＩ感染细胞其产量与１Ｍｏｌ感染量相仿。病毒增殖高峰在４－５天,维持达１５天无明显下降,且可连续收获４－５次。因此,该毒种符合ＷＨＯ提出的疫苗生产毒种要求,可用于狂犬病疫苗生产。     

Genetic characterization of Sabin types 1 and 3 poliovaccine virus following serial passage in the human intestinal tract.

Poliovirus isolates types 1 and 3 were obtained from five and seven successive passages respectively, in infants who had been fed monovalent OPV in two separate clinical trials conducted in 1960. The purpose of these trials was to answer the question how much the vaccine virus would revert to its original neurovirulent phenotype following multiplication in the intestinal tract. Human passages were performed either by contact exposure or by feeding the excreted virus while the infants were maintained in isolation. Several virus isolates were obtained at each passage level. Infants participating in both studies showed no symptoms of disease. Antigenic studies (McBride, van Wezel) and protein analysis (PAGE) of the isolates, reported earlier from this laboratory, had shown that the isolates remained vaccine-like, although isolates from the later passages revealed some differences. Monkey neurovirulence test results showed that for both types 1 and 3 viruses the loss of attenuation of the vaccine strain upon passage was gradual, although the loss was faster for type 3. Examination of the oligonucleotide maps demonstrated that the oligonucleotide configuration of the isolates remained the same as for the vaccine strain but there was an increase of individual spot differences with increasing passage. The nucleotide sequence analysis of selected regions of the virus genomes revealed that there was no change from a G to A in nucleotide 480 of type 1 isolates; however, nucleotide 476 changed from a U to an A in type 1 passages 3, 4 and 5. Conversely, for type 3 the change of nucleotide 472 from a U to a C changed at the early first passage (4 days following administration of OPV), and remained a C in the six following passages; type 3 nucleotide 2034 did not change in the first passage from a U to a C, but it became a C in all further passages tested. The nucleotide changes mentioned for both virus types remained stable in successive passages. However, there was another nucleotide change for type 3 from a U to a C at position 1973 only for passages 5 and 6 which reverted to a U for passages 7L and 7LL. Study of selected human passage virus strains could further contribute to the identification of the critical nucleotides that are responsible for the attenuation of these two polio types of vaccine viruses.     

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.     

Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines

A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met-->Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.     

Further-attenuated measles vaccine: Virus passages affect viral surface protein expression,immunogenicity and histopathology pattern in vivo

Measles vaccine viruses Leningrad-16 (L-16) and Moscow-5 (M-5, an L-16-derived clonal variant), at passage levels used for vaccination and after ten further low-multiplicity passages on quail embryo (QE) cells, were compared for (1) immunogenicity, (2) histopathological lesions induced in vivo and (3) surface protein expression within infected cells and on the virion surface. At the 10th passage, viruses evoked a poorer neutralizing antibody response in guinea pigs, induced an earlier appearance of more pronounced pathological lesions and replicated faster in Vero cells that the original viruses. H protein expression increased 1.8–2.3-fold after 10 passages of the L-16 variant, but remained virtually unaltered for the M-5 variant. F protein expression of both 10th-passage variants was 0.5–0.8 that of the original virus variants. A similar two-fold decrease in F protein expression was noted after a single virus passage in guinea pigs. The data implicate the loss of F protein as a cause of reduced immunogenicity of further attenuated measles vaccines.     

A host-specific, temperature-sensitive translation defect determines the attenuation phenotype of a human rhinovirus/poliovirus chimera, PV1(RIPO)

By using a rhinosvirus/poliovirus type 1 chimera, PV1(RIPO), with the cognate internal ribosome entry site (IRES) of human rhinovirus type 2 (HRV2), we set out to shed light on the mechanism by which this variant expresses its attenuated phenotype in poliovirus-sensitive, CD155 transgenic (tg) mice and cynomolgus monkeys. Here we report that replication of PV1(RIPO) is restricted not only in human cells of neuronal origin, as was reported previously, but also in cells of murine origin at physiological temperature. This block in replication was enhanced at 39.5°C but, remarkably, it was absent at 33°C. PV1(RIPO) variants that overcame the replication block were derived by serial passage under restrictive conditions in either mouse cells or human neuronal cells. All adapting mutations mapped to the 5'-nontranslated region of PV1(RIPO). Variants selected in mouse cells, but not in human neuronal cells, exhibited increased mouse neurovirulence in vivo. The observed strong mouse-specific defect of PV1(RIPO) at nonpermissive temperature correlated with the translational activity of the HRV2 IRES in this chimeric virus. These unexpected results must be kept in mind when poliovirus variants are tested in CD155 tg mice for their neurovirulent potential, particularly in assays of live attenuated oral poliovirus vaccine lots. Virulence may be masked by adverse species-specific conditions in mouse cells that may not allow accurate prediction of neurovirulence in the human host. Thus, novel poliovirus variants in line for possible development of human vaccines must be tested in nonhuman primates.