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  1. Viability of dengue virus in culture stocks is efficiently preserved by storage in diluted forms
    Suppiah J, Nadaraju S, Hamzah S, Chee HY
    Trop Biomed, 2020 Jun 01;37(2):282-287.
    PMID: 33612798
    Storage of dengue virus (DENV) culture stocks in -80°C is a common laboratory practice to maintain the viability of the virus for long-term usage. However, the efficiency of this method could still be hindered by multiple factors. In our laboratory, we observed a constant and substantial deterioration in the titer of DENV in Vero culture supernatant stored in -80°C. Such incident had badly hampered the laboratory work and prompted an investigation to determine the cause. DENV isolates representing all four serotypes were propagated and the culture supernatants were harvested and stored in aliquots of original stock and 10 fold dilutions (10-1 -10-4). DENV titer in these stocks was determined prior to storage and reassessed on the third and sixth month of storage by focus forming unit assay (FFUA). The result demonstrated a constant preservation of titer ranging from 104 ffu/ml to 105 ffu/ml in the diluted DENV virus culture stocks of 10-1, and 10-2 of DENV1-4, a minor reduction of titer from 103 ffu/ml to 102 ffu/ml at dilution 10-3 for DENV4 only and complete deterioration in undiluted culture stock and lower dilution (10-4) within 6 months of storage in -80°C for all serotypes. It is recommended that propagated DENV in Vero cells are stored in 10 fold dilutions as compared to the original form to preserve the titer for long-term usage.
    Matched MeSH terms: Virus Cultivation*
  2. A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor
    Lohr V, Genzel Y, Behrendt I, Scharfenberg K, Reichl U
    Vaccine, 2010 Aug 31;28(38):6256-64.
    PMID: 20638458 DOI: 10.1016/j.vaccine.2010.07.004
    An adherently growing MDCK cell line was adapted in a two-step process in a fully defined medium and in suspension. The resulting MDCK.SUS2 cells were subsequently evaluated for their potential as host cells for influenza vaccine production in two lab-scale bioreactors (wave and stirred-tank). Cell concentrations up to 2.3 x 10(6)cells/mL were obtained after 96 h, which is slightly higher than cell concentrations obtained with adherent MDCK cells cultivated on microcarriers (2g/L). Infections with influenza A/PR/8/34 and B/Malaysia resulted in high virus titers (2.90 and 2.75 log HA units/100 microL, respectively). The monitoring of extracellular metabolites, including amino acids, revealed a change in some of the metabolite consumption or release profiles, which indicates changes in metabolism during the adaptation process. Overall, the MDCK.SUS2 cell line represents a new cell substrate for a robust influenza vaccine production in a fully defined process.
    Matched MeSH terms: Virus Cultivation
  3. Plaque formation in vero cell cultures by some Malaysian viruses
    Rudnick A, Dewey RW
    Southeast Asian J. Trop. Med. Public Health, 1973 Jun;4(2):272-3.
    PMID: 4201359
    Matched MeSH terms: Virus Cultivation*
  4. Identification of molecular determinants of cell culture growth characteristics of Enterovirus 71
    Yee PT, Tan KO, Othman I, Poh CL
    Virol J, 2016 11 28;13(1):194.
    PMID: 27894305
    BACKGROUND: Hand, foot and mouth disease is caused by Enterovirus 71 (EV-A71) and Coxsackieviruses. EV-A71 infection is associated with high fever, rashes and ulcers but more severe symptoms such as cardiopulmonary failure and death have been reported. The lack of vaccines highlighted the urgency of developing preventive agents against EV-A71. The molecular determinants of virulent phenotypes of EV-A71 is unclear. It remains to be investigated if specific molecular determinants would affect the cell culture growth characteristics of the EV-A71 fatal strain in Rhabdomyosarcoma (RD) cells.

    RESULTS: In this study, several genetically modified sub-genotype B4 EV-A71 mutants were constructed by site-directed mutations at positions 158, 475, 486, 487 and 5262 or through partial deletion of the 5'-NTR region (∆ 11 bp from nt 475 to 486) to generate a deletion mutant (PD). EV-A71 mutants 475 and PD caused minimal cytopathic effects, produced lowest viral RNA copy number, viral particles as well as minimal amount of viral protein (VP1) in RD cells when compared to mutants 158, 486, 487 and 5262.

    CONCLUSIONS: The molecular determinants of virulent phenotypes of EV-A71 sub-genotype B4 strain 41 (5865/Sin/000009) were found to differ from the C158 molecular determinant reported for the fatal EV-A71 sub-genotype B1 strain (clinical isolate 237). The site-directed mutations (SDM) introduced at various sites of the cDNA affected growth of the various mutants when compared to the wild type. Lowest viral RNA copy number, minimal number of plaques formed, higher infectious doses required for 50% lethality of RD cells and much reduced VP1 of the EV-A71 sub-genotype B4 strain 41 genome was attained in mutants carrying SDM at position 475 and through partial deletion of 11 bp at the 5'-NTR region.

    Matched MeSH terms: Virus Cultivation*
  5. Fulltext Production of Newcastle disease virus by Vero cells grown on cytodex 1 microcarriers in a 2-litre stirred tank bioreactor
    Arifin MA, Mel M, Abdul Karim MI, Ideris A
    J Biomed Biotechnol, 2010;2010:586363.
    PMID: 20625497 DOI: 10.1155/2010/586363
    The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3( * *)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.
    Matched MeSH terms: Virus Cultivation/instrumentation*; Virus Cultivation/methods
  6. Lanjan virus, a new agent isolated from Dermacentor auratus in Malaya
    Tan DS, Smith CE, McMahon DA, Bowen ET
    Nature, 1967 Jun 10;214(5093):1154-5.
    PMID: 4964058
    Matched MeSH terms: Virus Cultivation
  7. Isolation of cytopathic small round virus (Aichi virus) from Pakistani children and Japanese travelers from Southeast Asia
    Yamashita T, Sakae K, Kobayashi S, Ishihara Y, Miyake T, Mubina A, et al.
    Microbiol. Immunol., 1995;39(6):433-5.
    PMID: 8551977
    Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.
    Matched MeSH terms: Virus Cultivation/methods
  8. Mouse fibroblast L929 cells are less permissive to infection by Nelson Bay orthoreovirus compared to other mammalian cell lines
    Mok L, Wynne JW, Grimley S, Shiell B, Green D, Monaghan P, et al.
    J Gen Virol, 2015 Jul;96(Pt 7):1787-94.
    PMID: 25748429 DOI: 10.1099/vir.0.000112
    In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.
    Matched MeSH terms: Virus Cultivation
  9. Phenotypic and genotypic characterization of induced acyclovir-resistant clinical isolates of herpes simplex virus type 1
    Hussin A, Md Nor NS, Ibrahim N
    Antiviral Res, 2013 Nov;100(2):306-13.
    PMID: 24055837 DOI: 10.1016/j.antiviral.2013.09.008
    Eleven strains of acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) were generated from HSV-1 clinical isolates by exposure to ACV. Genotype of the thymidine kinase (TK) and DNA polymerase (pol) genes from these mutants were further analyzed. Genotypic analysis revealed four non-synonymous mutations in TK gene associated with gene polymorphism and two to three non-synonymous mutations in DNA pol gene. Seven and six strains contained at least one resistance-associated mutation at TK and DNA pol gene, respectively. Resistance-associated mutations within the TK gene consisted of 64% of non-synonymous frameshift mutations within the homopolymer region of G's and C's, and 36% of non-synonymous nucleotide substitutions of the conserved gene region (C336Y, R51W and R222H), nucleotide that produced stop codon (L288Stop) and two amino acid substitutions outside the conserved region (E39G & L208F). There were 10 non-synonymous amino acid substitutions located outside the conserved region with the unclear significance to confer resistance observed. Resistance-associated mutations in DNA pol gene include insertion of G at the homopolymer region of G's (794-797) and amino acid substitutions inside (V621S) or outside (H1228D) the conserved region. In silico analysis of the mutated TK (C336Y, R51W and L208F), and DNA pol (V621S and H1228D) suggested structural changes that might alter the stability of these proteins. However, there were several mutations with unclear significance to confer ACV-resistance identified, especially mutations outside the conserved region.
    Matched MeSH terms: Virus Cultivation
  10. Fulltext Comparison of the pathogenicity of Nipah virus isolates from Bangladesh and Malaysia in the Syrian hamster
    DeBuysscher BL, de Wit E, Munster VJ, Scott D, Feldmann H, Prescott J
    PLoS Negl Trop Dis, 2013;7(1):e2024.
    PMID: 23342177 DOI: 10.1371/journal.pntd.0002024
    Nipah virus is a zoonotic pathogen that causes severe disease in humans. The mechanisms of pathogenesis are not well described. The first Nipah virus outbreak occurred in Malaysia, where human disease had a strong neurological component. Subsequent outbreaks have occurred in Bangladesh and India and transmission and disease processes in these outbreaks appear to be different from those of the Malaysian outbreak. Until this point, virtually all Nipah virus studies in vitro and in vivo, including vaccine and pathogenesis studies, have utilized a virus isolate from the original Malaysian outbreak (NiV-M). To investigate potential differences between NiV-M and a Nipah virus isolate from Bangladesh (NiV-B), we compared NiV-M and NiV-B infection in vitro and in vivo. In hamster kidney cells, NiV-M-infection resulted in extensive syncytia formation and cytopathic effects, whereas NiV-B-infection resulted in little to no morphological changes. In vivo, NiV-M-infected Syrian hamsters had accelerated virus replication, pathology and death when compared to NiV-B-infected animals. NiV-M infection also resulted in the activation of host immune response genes at an earlier time point. Pathogenicity was not only a result of direct effects of virus replication, but likely also had an immunopathogenic component. The differences observed between NiV-M and NiV-B pathogeneis in hamsters may relate to differences observed in human cases. Characterization of the hamster model for NiV-B infection allows for further research of the strain of Nipah virus responsible for the more recent outbreaks in humans. This model can be used to study NiV-B pathogenesis, transmission, and countermeasures that could be used to control outbreaks.
    Matched MeSH terms: Virus Cultivation
  11. [The biological and hemagglutinating properties of Ghetah arbovirus]
    Leont'eva NA, Fadeeva LL
    Vopr. Virusol., 1969 Jul-Aug;14(4):464-8.
    PMID: 4982330
    Matched MeSH terms: Virus Cultivation
  12. Fulltext Nipah Virus Matrix Protein Influences Fusogenicity and Is Essential for Particle Infectivity and Stability
    Dietzel E, Kolesnikova L, Sawatsky B, Heiner A, Weis M, Kobinger GP, et al.
    J Virol, 2016 Mar;90(5):2514-22.
    PMID: 26676785 DOI: 10.1128/JVI.02920-15
    Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on NiV strain Malaysia. Using an enhanced green fluorescent protein (eGFP)-expressing M protein-deleted NiV, we observed a slightly increased cell-cell fusion, slow replication kinetics, and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV but is necessary for proper assembly and release of stable infectious NiV particles.
    Matched MeSH terms: Virus Cultivation
  13. Cold adaptation improves the growth of seasonal influenza B vaccine viruses
    Kim H, Schoofs P, Anderson DA, Tannock GA, Rockman SP
    Vaccine, 2014 May 1;32(21):2474-9.
    PMID: 24631096 DOI: 10.1016/j.vaccine.2014.02.079
    Gene reassortment has proved useful in improving yields of influenza A antigens of egg-based inactivated vaccines, but similar approaches have been difficult with influenza B antigens. Current regulations for influenza vaccine seed viruses limit the number of egg passages and as a result resultant yields from influenza B vaccine seed viruses are frequently inconsistent. Therefore, reliable approaches to enhance yields of influenza B vaccine seed viruses are required for efficient vaccine manufacture. In the present study three stable cold-adapted (ca) mutants, caF, caM and caB derived from seasonal epidemic strains, B/Florida/4/2006, B/Malaysia/2506/2004 and B/Brisbane/60/2008 were prepared, which produced high hemagglutinin antigen yields and also increased viral yields of reassortants possessing the desired 6:2 gene constellation. The results demonstrate that consistent improvements in yields of influenza B viruses can be obtained by cold adaptation following extended passage. Taken together, the three ca viruses were shown to have potential as donor viruses for the preparation of high-yielding influenza B vaccine viruses by reassortment.
    Matched MeSH terms: Virus Cultivation
  14. Genomic variations among wildtype and mutant strains of pseudorabies virus
    Zeenathul NA, Mohd-Azmi ML, Ali AS, Aini I, Sheik-Omar AR, Abdul-Rahim AM, et al.
    Rev. Argent. Microbiol., 2002 Jan-Mar;34(1):7-14.
    PMID: 11942085
    Both wild-type virulent and mutant strains of pseudorabies virus (PrV) were used in this study. Mutants used were derived from the plaque purified strain PrVmAIP. A total of six drug resistant mutants, three bromodeoxyuridine (BUdR) resistant and three iododeoxyuridine (IUdR) resistant, respectively, were isolated and passaged in chicken embryo fibroblast (CEF) cells. The DNA of these PrVs were compared with the wild-type isolates by means of the restriction fragment pattern (RFP) findings produced with Bam HI, Kpn I, Hind III and Bgl II restriction enzymes (RE). Compared to the wild-type PrVs (PrV-VBA1-parental strain of PrVmAIP; PrV-VBA2; PrV-VBA3), the RFP of PrVmAIP showed the presence of mutations within the RE sites studied. Both PrV-VBA1 and PrV-VBA2 appeared to be closely related but their RFPs differed from PrV-VBA3. Significant differences either in the number, size or migrations of the DNA fragments could also be detected in the BUdR resistant strains. Even though different features of cytopathic effect (GPE) were observed in the IUdR resistant PrVs, the RFP findings remained identical. The PrVs studied showed considerable differences from the reference PrV (PrV-CD).
    Matched MeSH terms: Virus Cultivation
  15. Evaluation of different clinical sample types in diagnosis of human enterovirus 71-associated hand-foot-and-mouth disease
    Ooi MH, Solomon T, Podin Y, Mohan A, Akin W, Yusuf MA, et al.
    J Clin Microbiol, 2007 Jun;45(6):1858-66.
    PMID: 17446325
    Human enterovirus 71 and coxsackievirus A16 are important causes of hand-foot-and-mouth disease (HFMD). Like other enteroviruses, they can be isolated from a range of sterile and nonsterile sites, but which clinical sample, or combination of samples, is the most useful for laboratory diagnosis of HFMD is not clear. We attempted virus culture for 2,916 samples from 628 of 725 children with HFMD studied over a 3 1/2-year period, which included two large outbreaks. Overall, throat swabs were the single most useful specimen, being positive for any enterovirus for 288 (49%) of 592 patients with a full set of samples. Vesicle swabs were positive for 169 (48%) of 333 patients with vesicles, the yield being greater if two or more vesicles were swabbed. The combination of throat plus vesicle swabs enabled the identification of virus for 224 (67%) of the 333 patients with vesicles; for this patient group, just 27 (8%) extra patients were diagnosed when rectal and ulcer swabs were added. Of 259 patients without vesicles, use of the combination of throat plus rectal swab identified virus for 138 (53%). For 60 patients, virus was isolated from both vesicle and rectal swabs, but for 12 (20%) of these, the isolates differed. Such discordance occurred for just 11 (10%) of 112 patients with virus isolated from vesicle and throat swabs. During large HFMD outbreaks, we suggest collecting swabs from the throat plus one other site: vesicles, if these are present (at least two should be swabbed), or the rectum if there are no vesicles. Vesicle swabs give a high diagnostic yield, with the added advantage of being from a sterile site.
    Matched MeSH terms: Virus Cultivation
  16. Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses
    Cardosa MJ, Wang SM, Sum MS, Tio PH
    BMC Microbiol, 2002 May 5;2:9.
    PMID: 12019028
    In Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.
    Matched MeSH terms: Virus Cultivation
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