Evolution of a persistent aphthovirus in cytolytic infections: partial reversion of phenotypic traits accompanied by genetic diversification.

Foot-and-mouth disease virus (FMDV) shows a dual potential to be cytolytic or to establish persistent infections in cell culture. FMDV R100, a virus rescued after 100 passages of carrier BHK-21 cells persistently infected with FMDV clone C-S8c1, showed multiple genetic and phenotypic alterations relative to the parental clone C-S8c1. Several FMDV R100 populations have been subjected to 100 serial cytolytic infections in BHK-21 cells, and the reversion of phenotypic and genetic alterations has been analyzed. An extreme temperature sensitivity of R100 reverted totally or partially in some passage series but not in others. The small-plaque morphology reverted to normal size in all cases. The hypervirulence for BHK-21 cells did not revert, and even showed an increase, upon cytolytic passage. Most of the mutations that had been fixed in the R100 genome during persistence did not revert in the course of cytolytic passages, but the extended polyribocytidylate tract of R100 (about 460 residues, versus 290 in C-S8c1) decreased dramatically in length, to the range of 220 to 260 residues in all passage series examined. In passages involving very large viral populations, a variant with two amino acid substitutions (L-144-->V and A-145-->P) next to the highly conserved Arg-Gly-Asp (RGD motif; positions 141 to 143) within the G-H loop of capsid protein VP1 became dominant. A clonal analysis allowed isolation of a mutant with the single replacement A-145-->P. Viral production and growth competition experiments showed the two variants to have a fitness very close to that of the parental virus. The results provide evidence that the repertoire of variants that could potentially become dominant in viral quasispecies may be influenced by the population size of the evolving virus. The net results of a series of persistent-infection passages followed by a series of cytolytic passages was progressive genomic diversification despite reversion or stasis of phenotypic traits. Implications for the evolution of RNA viruses are discussed.     

In Vitro Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants with Increased Resistance to ABT-378, a Novel Protease Inhibitor

ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 μM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P₁′ residue Leu to Phe) and p7/p1 (P₂ residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 μM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 μM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763–3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662–6670, 1997).     

Characterization of a human immunodeficiency virus type 1 variant with reduced sensitivity to an aminodiol protease inhibitor.

Development of viral resistance to the aminodiol human immunodeficiency virus (HIV) protease inhibitor BMS 186,318 was studied by serial passage of HIV type 1 RF in MT-2 cells in the presence of increasing concentrations of compound. After 11 passages, an HIV variant that showed a 15-fold increase in 50% effective dose emerged. This HIV variant displays low-level cross-resistance to the C2 symmetric inhibitor A-77003 but remains sensitive to the protease inhibitors Ro 31-8959 and SC52151. Genetic analysis of the protease gene from a drug-resistant variant revealed an Ala-to-Thr change at amino acid residue 71 (A71T) and a Val-to-Ala change at residue 82 (V82A). To determine the effects of these mutations on protease and virus drug susceptibility, recombinant protease and proviral HIV type 1 clones containing the single mutations A71T and V82A or double mutation A71T/V82A were constructed. Subsequent drug sensitivity assays on the mutant proteases and viruses indicated that the V82A substitution was responsible for most of the resistance observed. Further genotypic analysis of the protease genes from earlier passages of virus indicated that the A71T mutation emerged prior to the V82A change. Finally, the level of resistance did not increase following continued passage in increasing concentrations of drug, and the resistant virus retained its drug susceptibility phenotype 34 days after drug withdrawal.     

Response of Schistosoma mansoni isolates having different drug sensitivity to praziquantel over several life cycle passages with and without therapeutic pressure

The stability of praziquantel (PZQ)-insusceptible S. mansoni isolates and the possible selection of PZQ-insusceptible parasites upon applying therapeutic pressure were examined over several life cycle passages (snails to mice). To test isolate stability, 3 PZQ-susceptible and 7 PZQ-insusceptible isolates were used to establish infection in mice, and they were passaged each for 2-5 life cycles. After each passage, 6 groups of mice were used to assess the PZQ dose at which the worm burden was decreased by 50% (ED50). Five of them were treated with doses of PZQ (12.5, 25, 50, 100, and 200 mg/kg for 5 days) 7 wk after infection; the last group represented infected, but untreated, controls. Possible selection of PZQ-insusceptible parasites under therapeutic pressure was examined by subjecting 1 PZQ-susceptible and 1 PZQ-insusceptible S. mansoni isolate to therapeutic pressure by PZQ for 8 passages. After the final passage, PZQ ED50 was estimated. All PZQ-susceptible S. mansoni isolates showed stable susceptibility to PZQ (mean PZQ ED50 = 85 mg/kg) over all passages. Two of the 7 PZQ-insusceptible S. mansoni isolates (847 and ER5) showed normal sensitivity to PZQ in 1-2 passages (although not the last passage, and without a declining ED50 profile), whereas the remaining passages kept a sustained insusceptibility to the drug (mean PZQ ED50 = 217 mg/kg). Worm maturity and sex were irrelevant to variability in drug ED50 within an individual isolate over different passages, revealing the heterogeneous nature of the parasite. Therapeutic pressure for limited life cycle passages did not result in a significant increase in drug ED50. The fact that reversion of some of the PZQ-insusceptible S. mansoni isolates to normal drug-sensitive state is not long lasting and that the therapeutic pressure by PZQ in the field is not comparable with that in the laboratory (unlimited), make monitoring the response of patients to the drug in the field an integral part of schistosomiasis control measures.     

Response of Hepatitis C Virus to Long-Term Passage in the Presence of Alpha Interferon: Multiple Mutations and a Common Phenotype

Cell culture-produced hepatitis C virus (HCV) has been subjected to up to 100 serial passages in human hepatoma cells in the absence or presence of different doses of alpha interferon (IFN-α). Virus survival, genetic changes, fitness levels, and phenotypic traits have been examined. While high initial IFN-α doses (increasing from 1 to 4 IU/ml) did not allow HCV survival beyond passage 40, a gradual exposure (from 0.25 to 10 IU/ml) allowed the virus to survive for at least 100 passages. The virus passaged in the presence of IFN-α acquired IFN-α resistance as evidenced by enhanced progeny production and viral protein expression in an IFN-α environment. A partial IFN-α resistance was also noted in populations passaged in the absence of IFN-α. All lineages acquired adaptative mutations, and multiple, nonsynonymous mutations scattered throughout the genome were present in IFN-α-selected populations. Comparison of consensus sequences indicates a dominance of synonymous versus nonsynonymous substitutions. IFN-α-resistant populations displayed decreased sensitivity to a combination of IFN-α and ribavirin. A phenotypic trait common to all assayed viral populations is the ability to increase shutoff host cell protein synthesis, accentuated in infections with IFN-α-selected populations carried out in the presence of IFN-α. The trait was associated with enhanced phosphorylation of protein kinase R (PKR) and eIF2α, although other contributing factors are likely. The results suggest that multiple, independent mutational pathways can confer IFN-α resistance to HCV and might explain why no unified picture has been obtained regarding IFN-α resistance in vivo.     

Evolution of the Influenza A Virus Genome during Development of Oseltamivir Resistance In Vitro

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA II. Further Studies on the Serial Passage of Virus Clones

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.     

Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits’ corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.     

Equine infectious anemia virus envelope evolution in vivo during persistent infection progressively increases resistance to in vitro serum antibody neutralization as a dominant phenotype

Equine infectious anemia virus (EIAV) infection of horses is characterized by well-defined waves of viremia associated with the sequential evolution of distinct viral populations displaying extensive envelope gp90 variation; however, a correlation of in vivo envelope evolution with in vitro serum neutralization phenotype remains undefined. Therefore, the goal of the present study was to utilize a previously defined panel of natural variant EIAV envelope isolates from sequential febrile episodes to characterize the effects of envelope variation during persistent infection on viral neutralization phenotypes and to define the determinants of EIAV envelope neutralization specificity. To assess the neutralization phenotypes of the sequential EIAV envelope variants, we determined the sensitivity of five variant envelopes to neutralization by a longitudinal panel of immune serum from the source infected pony. The results indicated that the evolution of the EIAV envelope sequences observed during sequential febrile episodes produced an increasingly neutralization-resistant phenotype. To further define the envelope determinants of EIAV neutralization specificity, we examined the neutralization properties of a panel of chimeric envelope constructs derived from reciprocal envelope domain exchanges between selected neutralization-sensitive and neutralization-resistant envelope variants. These results indicated that the EIAV gp90 V3 and V4 domains individually conferred serum neutralization resistance while other envelope segments in addition to V3 and V4 were evidently required for conferring total serum neutralization sensitivity. These data clearly demonstrate for the first time the influence of sequential gp90 variation during persistent infection in increasing envelope neutralization resistance, identify the gp90 V3 and V4 domains as the principal determinants of antibody neutralization resistance, and indicate distinct complex cooperative envelope domain interactions in defining sensitivity to serum antibody neutralization.     

SHIV-XJ02170感染恒河猴后期的传代特点和env基因变异

目的研究SHIV-XJ02170在中国恒河猴感染后期传代过程中病毒和宿主的变化特点,并分析env基因的序列变异。方法将感染中国恒河猴G0401V后期（5年）的SHIV-XJ02170病毒垂直传代2只猴（G0401V→G0402V→0403V）,同时,剔除G0401V猴CD8＋T细胞使潜伏的病毒大量复制后传代1只猴（G0401V→G0404V）,应用流式细胞术、病毒载量测定、序列分析等方法研究该病毒在猴体内长期适应后的病毒和免疫学指标及序列变异特点。结果 G0401V在感染后期仍能稳定传代,且表现出毒力增强的特点。其传代猴G0402V在传代后41 d死亡,外周血CD4＋T淋巴细胞衰竭,仅为43个/mL,符合艾滋病感染猴快速进展型的特征。剔除体内CD8＋T细胞之后的传代猴G0404V的表现类似G0401V,即长期低水平的病毒血症水平。env基因序列分析发现SHIV-XJ02170在G0401V体内长期适应后发生了可遗传的序列变异,并引起糖基化位点的改变。结论 SHIV-XJ02170在猴体长期适应后的传代过程中表现出向强毒株过渡的特征,为进行SHIV-XJ02170感染性克隆的构建奠定了良好的实验基础。     

Isolation of drug resistant mutants of varicella-zoster virus: cross resistance of acyclovir resistant mutants with phosphonoacetic acid and bromodeoxyuridine

Mutants of Varicella-Zoster Virus (VZV) which are resistant to phosphonoacetic acid (PAA), bromodeoxyuridine (BuDR), and acyclovir (ACV) were obtained by serial passages of VZV with increasing concentrations of these drugs. A PAA-resistant mutant and a BuDR-resistant mutant were found also to be resistant to ACV. Five of 8 ACV-resistant mutants acquired resistance to PAA, but none acquired resistance to BuDR. The BuDR-resistant mutant did not induce viral thymidine kinase (TK) activity, but all the ACV-resistant mutants selected in ACV showed viral TK activity which was suppressed with anti-VZV serum and had almost the same electrophoretic mobility as that of the parent strain on polyacrylamide gel electrophoresis in non-denaturing conditions. However, in competitive TK assay with ACV, 2 of 8 ACV-resistant mutants showed no change of phosphorylation of radioactive thymidine, while the other 6 showed decreased phosphorylation of radioactive thymidine. It was suggested that TK induced by the former 2 ACV-resistant mutants had lost affinity to ACV, and so the mutants could grow in the presence of ACV. Thus of the 8 ACV-resistant mutants selected in ACV, 2 were sensitive to PAA with altered TK activity, 5 were resistant to PAA with unaltered TK activity, and 1 was sensitive to PAA with unaltered TK activity, and may have altered DNA polymerase activity to ACV, retaining sensitivity to PAA. These results suggest that resistance of VZV to ACV results from alterations in the virus-specified TK or DNA polymerase, as demonstrated in HSV resistant to ACV.     

A rapid and automated colorimetric assay for evaluating the sensitivity of herpes simplex strains to antiviral drugs

A rapid and sensitive colorimetric assay has been developed to evaluate the sensitivity of herpes simplex viruses (HSV) to antiviral agents. The chessboard titration of viruses and antiviral drugs and the automatic reading and analysis of the data allows objective and accurate results to be rapidly obtained. Virus sensitivity was expressed as an ED50 value which was the concentration of drug (micrograms/ml) reducing viral cpe by 50%. The ED50 values of antiviral drugs [acetylguanosine (ACV), idoxuridine (IDU), deoxycytidine (IDC) and bromovinyl deoxyuridine] for several HSV reference strains were determined and the method was then applied to clinical specimens. The selection of ACV and IDU resistant mutants was performed on a cloned sensitive HSV 1 ocular strain. We observed cross-resistance between ACV and IDU for the mutants isolated. The resistance to thymidine-kinase-dependent antiviral agents varied in inverse ratio to the thymidine kinase activity induced by HSV strains.     

Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1'F) and P453L (p1/p6 PP5'L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity approximately 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.     

Eimeria tenella: experimental studies on the development of resistance to amprolium, clopidol and methyl benzoquate.

The development of resistance by the Houghton strain of Eimeria tenella to the anticoccidial drugs amprolium, clopidol and methyl benzoquate has been studied. Resistance to amprolium and clopidol developed more readily in experiments where a large number of coccidia were exposed to the drug, either by increasing the number of oocysts in the inoculum or by increasing the number of birds in the group. When 45 birds were given 2.0 X 10(6) oocysts, resistance to amprolium and clopidol appeared after 6 and 7 passages respectively. In previous experiments, under similar conditions, resistance to robenidine developed after 6 passages, suggesting little difference between these three drugs. Resistance to amprolium and clopidol arose gradually as the concentration of drug was increased, but resistance to methyl benzoquate appeared in a single step from sensitivity to high-level resistance. Both amprolium and clopidol-resistant lines showed an 8-fold reduction in drug sensitivity. Attempts to measure the degree of resistance by calculation of the ED50 were unsuccessful.     

Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Antigenic stability of foot-and-mouth disease virus variants on serial passage in cell culture.

Two neutralizing monoclonal antibody (MAb)-resistant variants selected from an isolate of foot-and-mouth disease virus (FMDV) type A5 were repeatedly passaged in cell culture and monitored for susceptibility to neutralization by the selecting MAb. A variant isolated with a MAb to a conformational epitope (1-OG2) lost resistance in 20 passages, while a variant isolated with a MAb to a linear epitope (1-HA6) persisted for 30 passages. In both cases, the virus population emerging after passage was antigenically and genetically indistinguishable from the original wild-type parental virus (FMDV A5 Spain-86). Coinfection assays with the wild type and each variant, and between the variants, showed rapid conversion to a homogeneous population. Wild-type virus prevailed over the variants and for coinfection between the variants, the linear epitope variant 1-HA6. While both variants arose from a single nucleotide substitution and reversion to wild type occurred for each, it appears that the variant based on the continuous epitope (1-HA6) was more stable. We discuss the implications of these results for the antigenic diversity of FMDV and its relationship to virus evolution.