A Highly Attenuated Mumps Virus Strain of Genotype F Generated by Passaging in Vero Cells.
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ABSTRACT:
SUBMITTER: Zhang Y
PROVIDER: S-EPMC8087723 | biostudies-literature | 2021 Apr
REPOSITORIES: biostudies-literature
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A Highly Attenuated Mumps Virus Strain of Genotype F Generated by Passaging in Vero Cells.
Zhang Yajing Y Xie Lixia L Chai Benjie B Ruan Juncheng J Gu Yulin Y Niu Biao B Zhang Yachun Y Fu Zhenfang Z An Qi Q Tian Dayong D
Virologica Sinica 20200929 2
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Project description:Recombinant mumps viruses (MuVs) based on established vaccine strains represent attractive vector candidates as they have known track records for high efficacy and the viral genome does not integrate in the host cells. We developed a rescue system based on the consensus sequence of the L-Zagreb vaccine and generated seven different recombinant MuVs by (a) insertion of one or two additional transcription units (ATUs), (b) lengthening of a noncoding region to the extent that the longest noncoding region in MuV genome is created, or (c) replacement of original L-Zagreb sequences with sequences rich in CG and AT dinucleotides. All viruses were successfully rescued and faithfully matched sequences of input plasmids. In primary rescued stocks, low percentages of heterogeneous positions were found (maximum 0.12%) and substitutions were predominantly obtained in minor variants, with maximally four substitutions seen in consensus. ATUs did not accumulate more mutations than the natural MuV genes. Six substitutions characteristic for recombinant viruses generated in our system were defined, as they repetitively occurred during rescue processes. In subsequent passaging of primary rescue stocks in Vero cells, different inconsistencies within quasispecies structures were observed. In order to assure that unwanted mutations did not emerge and accumulate, sub-consensus variability should be closely monitored. As we show for Pro408Leu mutation in L gene and a stop codon in one of ATUs, positively selected variants can rise to frequencies over 85% in only few passages.
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Project description:Recent mumps outbreaks have been observed in vaccinated young adults due to the mumps virus (MuV) of genotype G, whereas the current vaccine is a mixture of two genotype A strains. These outbreaks could be attributed to waning vaccine immunity or the antigenic differences between the HN and F glycoproteins in the vaccine and circulating MuV. These glycoproteins are essential targets for the immune system, and antigenic variations may reduce the recognition of mumps antibodies, rendering the population susceptible to the MuV. We established stable cell lines expressing the MuV glycoproteins to study cross-reactivity between genotype A and genotype G. Cross-reactivity between the genotypes was evaluated via immunofluorescence using patient sera from vaccinated individuals, infected individuals, and vaccinated individuals infected with genotype G. Titer ratios showed that the vaccinated individuals exhibited a titer 3.68 times higher for the HN protein and 2.3 times higher for the F protein when comparing genotype A with genotype G. In contrast, the infected individuals showed a lower titer for genotype A compared with genotype G, at 0.43 and 0.33 for the HN and F proteins, respectively. No difference in titer ratio was observed for individuals vaccinated and subsequently infected with mumps. These findings suggest that antigenic variations between the two genotypes may potentially result in immune escape of the circulating strain, resulting in individuals susceptible to the MuV.
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Project description:BackgroundPorcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains.MethodsIn this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability.ResultsPathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120.ConclusionsThus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.
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Project description:In reverse genetic experiments we have isolated recombinant mumps viruses (rMuV) that carry large numbers of mutations clustered in small parts of their genome, which are not caused by biased hyper-mutation. In two separate experiments we obtained such recombinant viruses: one virus had 11 mutations in the V/P region of the genome; the other, which also contained an extra transcription unit encoding green fluorescent protein (EGFP), had 32 mutations in the N gene. These specific sets of mutations have not been observed in naturally occurring MuV isolates. Unusually, the vast majority of the mutations (48/51) were synonymous. On passage in Vero cells and human B-LCL cells, a B lymphocyte-like cell line, these mutations appear stable as no reversion occurred to the original consensus sequence, although mutations in other parts of the genome occurred and changed in frequency during passage. Defective interfering RNAs accumulate in passage in Vero cells but not in B-LCL cells. Interestingly, in all passaged samples the level of variation in the EGFP gene is the same as in the viral genes, though it is unlikely that this gene is under any functionality constraint. What mechanism gave rise to these viruses with clustered mutations and their stability remains an open question, which is likely of interest to a wider field than mumps reverse genetics.
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Project description:Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1?4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells.
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Project description:BackgroundIn October 2016, a mumps outbreak occurred among the students living in the on-campus dormitory of a public university located in Kota Kinabalu, Sabah, Malaysia. This study aimed to investigate the outbreak and identify the genotype of the mumps virus (MuV) strain that was involved in the outbreak.Main bodyDuring the outbreak, one 21-year-old and four 20-year-old males staying in the same dormitory building were reported to have developed symptoms of mumps. Of these, two students were available during the investigation for sample collection to detect MuV by reverse transcription polymerase chain reaction (RT-PCR) of the 639-bp fragment encompassing the entire small hydrophobic (SH) gene. Nucleotide sequencing of the amplicon and phylogenetic analysis using the neighbor-joining method was performed to determine the MuV genotype. Of the two buccal swab samples, one was positive for MuV. The MuV strain in this sample belonged to genotype F and it was clustered together with genotype F strains from China with 96.84-99.68% nucleotide identity.ConclusionsGenotype F has limited circulation and is endemic in mainland China. Genotype F strains occasionally reported from other countries were epidemiologically linked to China. This study is the first to report a case of genotype F MuV in Malaysia and no epidemiological link could be established with mainland China. The results provide important information that can assist in strategic planning to improve the prevention and control of mumps infection in Malaysia.
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Project description:With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.
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Project description:PurposeHepatitis A virus (HAV) production has been limited by its slow replication rate and reliance on diploid cell lines like MRC-5, which present challenges in scalability, passage limitations, and serum-free culture conditions. This study aimed to develop an HAV vaccine strain with enhanced replication capacity.Materials and methodsWe generated a reverse genetically modified HAV vaccine strain (RG-HAV) and adapted it to Vero cells through sequential culturing. Replication rates of RG-HAV and a commercially used strain, HM-175, were compared in Vero and MRC-5 cells. Nucleotide sequences, including coding and non-coding regions like the internal ribosomal entry site (IRES), were analyzed. Structural assessments included 3-dimensional modeling of IRES and relative codon deoptimization analysis of the capsid. Immunogenicity was evaluated by measuring HAV-specific antibody responses in mice.ResultsVero-adapted RG-HAV achieved a 30-fold increase in production yield compared to initial transfection. In Vero cells, RG-HAV peaked at 15 days post-infection, compared to 20 days for HM-175. In MRC-5 cells, RG-HAV and HM-175 reached peak production at 10 and 15 days, respectively. RG-HAV produced over 5-fold more HAV in Vero cells and 8-fold more in MRC-5 cells than HM-175. Sequence analysis revealed nine amino acid differences in RG-HAV structural proteins and five nucleotide changes in the type III IRES region, potentially enhancing IRES functionality. Immunization with inactivated RG-HAV with alum hydroxide induced HAV-specific antibody responses in mice.ConclusionRG-HAV offers enhanced replication and production yields, supporting its potential in advancing HAV vaccine development.
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Project description:During May-August 2016, mumps virus genotype K was detected in 12 Vietnam citizens who entered China at the Shuikou border crossing and 1 girl from China. We provide evidence that mumps genotype K is circulating in Vietnam and was imported to China from Vietnam.
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Project description:BackgroundThe most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous.ResultsWe identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo.ConclusionL-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.
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