Passages In Vero Cells Research Articles

Comparison of biological and genetic background of CA16 between different epidemic strains

Objective To study the biological characterization and the genetic background of circulating CA16 strains in mainland of China for the purpose of CA16 vaccine development in the future.Methods CA16 strains were isolated from throat swabs of patients with hand-foot-mouth disease and identified by neutralization assay and RT-PCR.The genotype of these isolates were determined by sequence alignment and phylogenetic analysis of VP1 gene.The proliferation dynamics and the plaque morphology were observed when propagated in Vero cells.The pathogenicity of these CA16 isolates was evaluated by challenging newborn mice.Results In this study,six CA16 circulating isolates,BJ-1-6 were obtained.The RT-PCR products were 150 bp amplified with the general enterovirus primers and 210 bp with CA16 primers respectively,which cannot be amplified by EV71 primers.Additionally,these isolates were identified to display some obvious proliferation dynamics and plaque morphology when propagated for 96 h in Vero cells.The diameter of plaques were about 1.5 to 2 mm for BJ-1,BJ-2,BJ-4,BJ-6,4-5 mm for BJ-3 and 3 mm for BJ5,the plaques were regular except BJ-3.All the six isolates can be neutralized by the convalescent serum of patient infected with CA16.The virus titer of different isolates propagated for five passages in Vero cells was 7.0LgCCID50/ml.The sequence alignment of VP1 gene demonstrated that the genotypes of BJ-2,BJ-4,BJ5 were C1 and BJ-1,BJ-3,B J-6 were C,3 comparatively.The genetic distance of the VPI gene from theseisolates suggested that they were highly genetic identity with the homology of 90％ in nucleotide and 99％ in dedicated amino acid respectively.However,a distinctive difference in pathogenic ability in neonatal mice was found that the suckling mice challenged with BJ-3 & BJ-5 were paralyzed 4-5 d and dead 6-7d postchallenge,compared with the control group without any abnormality in the during of 14 d.Conclusion The circulating CA16 isolates in China have different biological characteristics,different pathogenic ability and similar genetic backgrounds,which is helpful for the development of a CA16 vaccine in the future. Key words: Coxsachievirus 16(CA16); Epidemic strain; Hand, foot and mouth disease (HFMD)

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-Q438H, E-V463L, NS2B-Q78H, and NS2B-A113T 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.

Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo

Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.

Biological and immunological characterization of recombinant Yellow Fever 17D Viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+T cell epitope at two distinct regions of the genome

BackgroundThe attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.ResultsRecombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi.ConclusionsWe conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.

Genetic characterization of the Korean LATC06 rinderpest vaccine strain

We sequenced the genome of LATC06 generated by in vitro passage in Vero cells of the lapinized-avianized (LA) strain and compared its sequence to those of other rinderpest viruses. The LATC06 genome consists of 15882 nucleotides. Its transcriptional regulatory control sequences (TRSs) at gene boundaries are identical to those of the Kabete O strain. Cleavage sites for generating F1/F2 proteins were identified in the same amino acid position (aa 108) as F proteins in LATC06, L13, RBT1, Kabete O, and RBOK strains. There are three predicted N-glycosylation sites of H proteins in LA (Japan) and LATC06 strains. The six epitopes of H protein in the LA (Japan) strain that elicit immunodominant humoral responses are also found in the LATC06 strain.

Stability of the Glycoprotein Gene of Avian Metapneumovirus (Canada Goose Isolate 15a/01) After Serial Passages in Cell Cultures

The glycoprotein (G) gene sequences of avian metapneumovirus (aMPV) subtypes A, B, C, and D are variable in size and number of nucleotides. The G gene of early U.S. turkey isolates of aMPV-C have been reported to be 1798 nucleotides (nt) (585 aa) in length, whereas the G genes of more recent turkey isolates have been reported to be 783 nucleotides. In some studies, the G gene of aMPV-C turkey isolates was found to be truncated to a smaller G gene of 783 nt (261 aa) upon serial passages in Vero cells. This is believed to be due to the deletion of 1015 nt near the end of the open reading frame. The purpose of this study was to determine variation, if any, in the G gene of an aMPV-C isolated from a wild bird (Canada goose [Branta canadensis]) following serial passages in Vero cells. No size variation was observed for up to 50 passages, except for a few amino acid changes in the extracellular domain at the 50th passage level. The G gene of this wild bird isolate appears to be unique from subtype C metapneumoviruses of turkeys.

Molecular characterization of human rotavirus vaccine strain CDC-9 during sequential passages in vero cells

We have developed several candidate human rotavirus vaccine strains with common serotypes via adaptation in Vero cells, adhering to the Good Laboratory Practice (GLP) guidelines. We sequenced the entire genome of a G1P[8] strain CDC-9 in original stool and passaged materials from Vero cells and examined its genetic relatedness to the prototype human rotavirus KU and other strains. With the exception of VP3 gene which was closely related to that of strain DS-1, the culture-adapted CDC-9 strain shared moderate to high nt (range 83.4%-95.1%) and deduced aa (range 81.3%-97.9%) sequence identities with the KU and other G1P[8] strains. Alignments of the deduced aa sequences of 11 gene segments of the wild-type and culture-adapted CDC-9 showed complete sequence identity in genes encoding NSP2, NSP3, NSP4, VP1, VP2, VP3, and VP7, a single aa change in genes coding for NSP1, NSP5, and VP6 and several scattered aa changes in the VP4 gene during the passage in Vero cells. Two of the VP4 aa substitutions (385 and 388) are within sites associated with neutralization resistant mutants selected by cross-reactive monoclonal antibodies. Although some sequence changes were evident, we do not know if these changes contribute to the possible attenuation of this strain. Further testing of this vaccine strain in clinical trials is justified.

Screening of a high growth influenza B virus strain in Vero cells

Due to the insufficient supply of embryonated chicken eggs, the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus. The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines. However, most of the influenza viruses can not grow well in Vero cells. To develop a new influenza vaccine with Vero cells as a substrate, the virus needs to adapt to this cell substrate to maintain high growth characteristics. By serial passages in Vero cells, the B/Yunnan/2/2005va (B) strain was successfully adapted to Vero cells, with the hemagglutination titer (HAT) of the virus reaching 1:512. The high growth characteristic of this strain is stable up to 21 passages. The strain was identified by hemagglutination inhibition (HAI) test and sequencing respectively; the HA₁ gene sequence of the virus was cloned and analyzed. The screening and establishment of high growth B virus provides an important tool for influenza vaccine production in Vero cells.

Isolation and characterization of avian metapneumovirus from chickens in Korea

Avian metapneumovirus (aMPV) causes upper respiratory tract infections in chickens and turkeys. Although the swollen head syndrome (SHS) associated with aMPV in chickens has been reported in Korea since 1992, this is the study isolating aMPV from chickens in this country. We examined 780 oropharyngeal swab or nasal turbinate samples collected from 130 chicken flocks to investigate the prevalence of aMPV and to isolate aMPV from chickens from 2004-2008. Twelve aMPV subtype A and 13 subtype B strains were detected from clinical samples by the aMPV subtype A and B multiplex real-time reverse transcription polymerase chain reaction (RRT-PCR). Partial sequence analysis of the G glycoprotein gene confirmed that the detected aMPVs belonged to subtypes A and B. Two aMPVs subtype A out of the 25 detected aMPVs were isolated by Vero cell passage. In animal experiments with an aMPV isolate, viral RNA was detected in nasal discharge, although no clinical signs of SHS were observed in chickens. In contrast to chickens, turkeys showed severe nasal discharge and a relatively higher titer of viral excretion than chickens. Here, we reveal the co-circulation of aMPV subtypes A and B, and isolate aMPVs from chicken flocks in Korea.

Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

Sequence Variation in Measles Virus F Gene of AIK-C Vaccines Strain after Passage in Vero Cells

Sequence Variation in Measles Virus F Gene of AIK-C Vaccines Strain after Passage in Vero Cells

Wild type measles virus attenuation independent of type I IFN

BackgroundMeasles virus attenuation has been historically performed by adaptation to cell culture. The current dogma is that attenuated virus strains induce more type I IFN and are more resistant to IFN-induced protection than wild type (wt).ResultsThe adaptation of a measles virus isolate (G954-PBL) by 13 passages in Vero cells induced a strong attenuation of this strain in vivo. The adapted virus (G954-V13) differs from its parental strain by only 5 amino acids (4 in P/V/C and 1 in the M gene). While a vaccine strain, Edmonston Zagreb, could replicate equally well in various primate cells, both G954 strains exhibited restriction to the specific cell type used initially for their propagation. Surprisingly, we observed that both G954 strains induced type I IFN, the wt strain inducing even more than the attenuated ones, particularly in human plasmacytoid Dendritic Cells. Type I IFN-induced protection from the infection of both G954 strains depended on the cell type analyzed, being less efficient in the cells used to grow the viral strain.ConclusionThus, mutations in M and P/V/C proteins can critically affect MV pathogenicity, cellular tropism and lead to virus attenuation without interfering with the α/β IFN system.

The attenuation of vaccinia Tian Tan strain by the removal of the viral M1L-K2L genes

To generate a safe vaccinia Tian Tan (VTT)-based vaccine vector, it is necessary to develop a method to attenuate the virus. A modified VTT (MVTT 2-GFP) was constructed by replacing the viral M1L-K2L genes with a GFP gene. In comparison to the parental VTT, MVTT 2-GFP lost its replication capacity in rabbit RK13 and human HeLa cell lines. The life cycle of viral replication was blocked at different stages in these two cell lines as determined by electron microscope examination. MVTT 2-GFP was less virulent than VTT for 100-fold by measuring mouse body weight loss after intranasal viral inoculation and for 340-fold by determining the intracranial LD 50 value in mice. The foreign GFP gene was stable genetically after 10 rounds of passage in Vero cells. Importantly, MVTT 2-GFP elicited both humoral and cell-mediated immune responses to the GFP gene in mice. With two intramuscular inoculations of 10 5 PFU virus, the anti-GFP antibody reciprocal endpoint titer reached over 700 as determined by an ELISA. The number of IFN-γ secreting T cells reached over 350 SFU per million splenocytes against a CD8+ T cell-specific epitope of GFP. Collectively, the removal of the M1L-K2L genes is a useful method to generate an attenuated vaccinia Tian Tan vaccine vector.

Isolation of Mycoplasma genitalium from First-Void Urine Specimens by Coculture with Vero Cells

Isolation of Mycoplasma genitalium from clinical specimens remains difficult. We describe an improvement of the Vero cell coculture method in which the growth of M. genitalium was monitored by quantitative real-time PCR. Four new M. genitalium strains were isolated from six first-void urine specimens of male Japanese patients with urethritis. In two of them, only M. genitalium was detected: one also contained Ureaplasma urealyticum, and one contained Chlamydia trachomatis, Neisseria gonorrhoeae, U. urealyticum, and Ureaplasma parvum. In the specimens yielding isolates of M. genitalium, growth was documented by quantitative PCR after two to five passages in Vero cells. The complete isolation procedure from the initial inoculation to completion of single-colony cloning took about 1 year. Isolation of M. genitalium from urine specimens proved to be more difficult than from swab specimens. Due to the cytotoxic effect of urine, a procedure involving washing of the urinary sediment was introduced. Furthermore, prolonged storage of the urine specimens before culture was shown to be detrimental to the success of isolation, as shown by the lack of success in attempts to isolate M. genitalium from mailed urine specimens as well as by simulation experiments. High concentrations of penicillin G and amphotericin B were surprisingly inhibitory to the growth of wild-type M. genitalium strains, but penicillin G at 200 IU/ml and polymyxin B at 500 microg/ml could be used as selective antibiotics to avoid bacterial overgrowth in the Vero cell cultures.

Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

BackgroundThe yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.ResultsYF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.ConclusionThis system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.

Stability of canine distemper virus (CDV) after 20 passages in Vero-DST cells expressing the receptor protein for CDV

Isolates 007Lm, S124C and Ac96I and a Vero cell-adapted Onderstepoort strain of canine distemper viruses (CDV) were examined for stability after passages in Vero cells expressing the canine signaling lymphocyte activation molecule (dogSLAM, the intrinsic receptor to CDV). These viruses passage once in Vero cells expressing dogSLAM (Vero-DST) cells (original) and after 20 passages (20p) were compared by using sequence analyses and growth characteristics. All four strains of 20p grew well and were slightly better than their originals. The 20p viruses developed a cytopathic effect slightly lower than the original strains. A few changes in amino acids in the H gene were between the 20p and the original viruses, but the sites of changes were not specific. Fragments of P, M and L genes of all strains showed no nucleotide changes after the passages. These results showed that: (1) passages of CDVs in Vero-DST cells induced amino acid changes only in the H gene, not in the P, M and L genes, unlike in a previous study with Vero cells; (2) passages did not markedly affect the growth characteristics of every viral strain. These results indicate that Vero cells expressing canine SLAM allow the isolation and passaging of CDV without major changes in viral genes.

Passage in Vero cells alters the characteristics of measles AIK-C vaccine strain

Objective of this study is to evaluate the feasibility of measles vaccine production in Vero cell culture. We constructed the full-length cDNA, pIC-MVAIK-F278Leu (small plaque-type) and pIC-MVAIK-F278Phe (large plaque-type) from the AIK-C measles vaccine strain attenuated from the Edmonston wild-type. MVAIK-S/B2 was rescued from pIC-MVAIK-F278Leu after two passages in B95a cells and MVAIK-SL/B2V1 was obtained through large plaque cloning in Vero cells. MVAIK-SL/B2V8 was obtained after eight passages in Vero cells. It produced large plaques in Vero cells, grew well at 39 °C, and thus the characteristics of the AIK-C vaccine strain were lost. Thirteen amino acid changes were observed; one in the N, two in the P, one in the C, three in the F, one in the H, and five in the L protein regions. Twelve of these changes excluding one in the L gene were back mutated to the Edmonston strain. Change from Leu to Phe at position 278 of the F protein was an early event during adaptation to Vero cells and the P gene was back-mutated to the Edmonston wild-type. As for the control, MVAIK-L/B9 strain was obtained after passages in B95a cells from pIC-MVAIK-F278Phe (large plaque-type). It maintained the same temperature sensitivity as the AIK-C vaccine strain and only four amino acid changes, one in the N and three in the L protein region, were observed without any mutations in the P, C, M, F, and H genes. The passage of the measles vaccine AIK-C strain in Vero cells lost the characteristics of small plaque inducibility and temperature sensitivity ( ts) phenotype.

Determining genetic stabilities of chimeric dengue vaccine candidates based on dengue 2 PDK-53 virus by sequencing and quantitative TaqMAMA

The genetic stabilities of the three attenuation loci of the candidate dengue 2 (D2) PDK-53 vaccine virus were evaluated for the PDK-53 virus and PDK-53-vectored chimeric D2/1, D2/3, and D2/4 viruses following 10 sequential passages in Vero cells. Sequencing revealed that the dominant NS1–53-Asp and the NS3–250-Val attenuation loci were extremely stable, whereas reversion occurred at the 5′NCR-57-U locus in 10 of the 18 viral lineages tested. A more sensitive and quantitative assay, the TaqMan mismatch amplification mutation assay (TaqMAMA), was employed to more finely discriminate the level of reversion at the 5′NCR-57 locus. This rapid genetic assay permitted detection of ≤1% reversion of 5′NCR-57 U-to-C in viral populations. By TaqMAMA, various levels of reversion at 5′NCR-57 were detected in all 18 of the PDK-53-based viral lineages tested at Vero passage 10, but only 3 lineages had reversion levels >80% in the viral population. Chimeric viruses based on the PDK-53-V (all three mutations present) genetic background were more stable than those developed in the PDK-53-E (5′NCR and NS1 mutations present) background. The TaqMAMA can be applied in quality control analyses to ensure that attenuated vaccine seeds contain undetectable or minimal levels of reversion at a given attenuation locus.

Molecular Basis for Adaptation of a Chimeric Dengue Type‐4/Japanese Encephalitis Virus to Vero Cells

The premembrane and envelope (E) genes of a full-length cDNA clone of the dengue type-4 (DEN4) virus 814669 strain were replaced with those of the Japanese encephalitis (JE) virus JaOH0566 strain. The in vitro-synthesized RNA transcripts prepared from chimeric cDNA were used to transfect mosquito C6/36 cells. A viable chimeric virus (designated DEN4/JE) was recovered. Unexpectedly, DEN4/JE exhibited restricted growth in Vero cells. After a serial passage in Vero cells, the Vero-adapted chimeras were obtained (two clones, designated Strain I and Strain II, respectively). The entire genomes of DEN4/JE, Strain I, and Strain II were sequenced and compared. There were multiple mutations, but amino acid substitutions occurred only in E and nonstructural (NS) protein NS4B. Our findings in this study indicate that the 5' nontranslated region, E, and NS4B may be involved in Vero cell adaptation in this chimeric system.

Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003.

BackgroundThe SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations.MethodsWe sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates.ResultsClose examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes.ConclusionsOur results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.