Serial Passage In BHK-21 Cells Research Articles

Sequence duplication in 3′ UTR modulates virus replication and virulence of Japanese encephalitis virus

ABSTRACT Japanese encephalitis virus (JEV), an important neurotropic pathogen, belongs to the genus Flavivirus of the family Flaviviridae and has caused huge threat to public health. It is still obscure regarding the functions of stem-loop (SL) and dumbbell (DB) domains of JEV 3′ UTR in viral replication and virulence. In the current study, using the infectious clone of JEV SA14 strain as a backbone, we constructed a series of deletion mutants of 3′ UTR to investigate their effects on virus replication. The results showed that partial deletions within SL or DB domain had no apparent effects on virus replication in both mammalian (BHK-21) and mosquito (C6/36) cells, suggesting that they were not involved in viral host-specific replication. However, the entire SL domain deletion (ΔVR) significantly reduced virus replication in both cell lines, indicating the important role of the complete SL domain in virus replication. The revertant of ΔVR mutant virus was obtained by serial passage in BHK-21 cells that acquired a duplication of DB domain (DB-dup) in the 3′ UTR, which greatly restored virus replication as well as the capability to produce the subgenomic flavivirus RNAs (sfRNAs). Interestingly, the DB-dup mutant virus was highly attenuated in C57BL/6 mice despite replicating similar to WT JEV. These findings demonstrate the significant roles of the duplicated structures in 3′ UTR in JEV replication and provide a novel strategy for the design of live-attenuated vaccines.

The Neutralizing Antibody Response Elicited by Tembusu Virus Is Affected Dramatically by a Single Mutation in the Stem Region of the Envelope Protein.

Tembusu virus (TMUV) is a mosquito-borne flavivirus that most commonly affects adult breeder and layer ducks. However, a TMUV-caused neurological disease has also been found in ducklings below 7 weeks of age, highlighting the need to develop a safe vaccine for young ducklings. In this study, a plaque-purified PS TMUV strain was attenuated by serial passage in BHK-21 cells. Using 1-day-old Pekin ducklings as a model, the virus was confirmed to be attenuated sufficiently after 180 passages, whereas the neutralizing antibody response elicited by the 180th passage virus (PS180) was substantially impaired compared with PS. The findings suggest that sufficient attenuation results in loss of immunogenicity in the development of the live-attenuated TMUV vaccine. Comparative sequence analysis revealed that PS180 acquired one mutation (V41M) in prM and four mutations (T70A, Y176H, K313R, and F408L) in the envelope (E) protein. To identify the amino acid substitution(s) associated with loss of immunogenicity of PS180, we rescued parental viruses, rPS and rPS180, and produced mutant viruses, rPS180-M41V, rPS180-A70T, rPS180-H176Y, rPS180-R313K, rPS180-L408F, and rPS180-M5, which contained residue 41V in prM, residues 70T, 176Y, 313K, and 408F in E, and combination of the five residues, respectively, of PS in the backbone of the rPS180 genome. The neutralizing antibody response elicited by rPS180-L408F and rPS180-M5 was significantly higher than those by other mutant viruses and comparable to that by rPS. Furthermore, we produced mutant virus rPS-F408L, which contained residue 408L of PS180 in the backbone of the rPS genome. The F408L mutation conferred significantly decreased neutralizing antibody response to rPS-F408L, which was comparable to that elicited by rPS180. Based on homologous modeling, residue 408 was predicted to be located within the first helical domain of the stem region of the E protein (EH1). Together, these data demonstrate that a single mutation within the EH1 domain exerts a dramatical impact on the TMUV neutralizing antibody response. The present work may enhance our understanding of molecular basis of the TMUV neutralizing antibody response, and provides an important step for the development of a safe and efficient live-attenuated TMUV vaccine.

Attenuation of duck Tembusu virus ZJSBL01 strain following serial passage in BHK-21 cells supplied with 5-Fluorouracil

Duck Tembusu virus (DTMUV) is a new pathogen that produces an acute and potent disease in ducks which has caused serious economic losses in China. In this study, a virulent strain of DTMUV, designated as ZJSBL01, was attenuated by serial passages in BHK-21 cells supplied with 5-Fluorouracil (5-FU) for 50 passages to induce mutation and attenuation. Growth kinetics of different passages of ZJSBL01 strain in BHK-21 cells show that these viruses have similar replication characteristics. The virus was highly attenuated after 40 passages in BHK-21 cells supplied with 5-FU, based on mortality, morbidity, and viral load in inoculated Sheldrake ducklings. In addition, all of the ducklings immunized with ZJSBL01-P40, the virus obtained at passage 40 of ZJSBL01, showed seroconversion on day 14 post inoculation. Moreover, P40 did not cause clinical symptom for layding ducks. Immunization with ZJSBL01-P40 could provide effective protection against the virulent parental ZJSBL01 strain. Seventeen amino acid substitutions were observed in the polyprotein of ZJSBL01-P40 compared with parental ZJSBL01. These results indicate that ZJSBL01-P40 may be a live vaccine candidate for prevention of DTMUV-disease.

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94–101, 93–101, and 93–102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93–102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93–102 residues to 8 aa residues at positions 94–101 in 3A protein.

Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M<NS4A-R79K<NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

Expression and Stability of Foreign Epitopes Introduced into 3A Nonstructural Protein of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV) is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa) HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags.

Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

BackgroundSuccess of a viral infection requires that each infected cell delivers a sufficient number of infectious particles to allow new rounds of infection. In picornaviruses, viral replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown.Methodology and Principal FindingsWe have constructed four mutant FMDVs that encode only one VPg: either VPg1, VPg3, or two chimeric versions containing part of VPg1 and VPg3. All mutants, except that encoding only VPg1, were replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21 cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with restoration of plaque formation. Specifically, replacement R55W in 2C was repeatedly found in several mutant viruses that had regained competence in plaque development. The effect of R55W in 2C was to mediate an increase in the extracellular viral RNA release without a detectable increase of total viral RNA that correlated with an enhanced capacity to alter and detach BHK-21 cells from the monolayer, the first stage of cell killing.ConclusionsThe results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. Implications for picornaviruses pathogenesis are discussed.

Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Muller’s ratchet

Repeated bottleneck passages of RNA viruses result in fitness losses due to accumulation of deleterious mutations. We have analysed the molecular events underlying fitness recovery of a highly debilitated foot- and-mouth disease virus (FMDV) clone, upon serial passage in BHK-21 cells. The debilitated clone included an unusual, internal polyadenylate extension preceding the second functional AUG initiation codon, and a number of additional mutations scattered throughout the genome. Comparison of entire genomic nucleotide sequences in the course of passaging documented that loss of the internal polyadenylate was the first event in the process of fitness recovery. Further increases of fitness were associated with very few true reversions and with the accumulation of additional mutations affecting non-coding and coding regions. Remarkably, four biological subclones of the same debilitated FMDV clone gained fitness through three separate molecular pathways regarding correction of the internal polyadenylate: (i) a true reversion to yield the wild-type sequence at the second functional AUG; (ii) a shortening of the internal polyadenylate tract; or (iii) a deletion of 69 residues spanning the site of the polyadenylate extension. The results document that an RNA virus can find multiple pathways to reach alternative high fitness peaks on the fitness landscape.

Antigenic variation of avian infectious bronchitis virus during replication in BHK-21 cells.

Avian infectious bronchitis virus Beaudette-42 strain showed antigenic variation during serial passages in BHK-21 cells. The same strain serial passaged in chicken cells or five times in the presence of antibody did not change its antigenicity.