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PubMed1999

Effects of long terminal repeat sequence variation on equine infectious anemia virus replication in vitro and in vivo.

The long terminal repeat (LTR) is reported to be one of the most variable portions of the equine infectious anemia virus (EIAV) genome. To date, however, no information is available on the effects of observed sequence variations on viral replication properties, despite a widespread assumption of the biological importance of EIAV LTR variation. EIAV LTR sequence variability is confined mostly to a small portion of the enhancer within the U3 segment of the LTR. Analysis of published EIAV LTR sequences revealed six different types of LTR based on the pattern of putative transcription factor motifs within the variable region of the enhancer. To test directly the significance of LTR variation, the in vitro and in vivo replication properties of two variant LTR species were investigated using two isogenic viruses, EIAV(19-2) and EIAV(19-2-6A), differing only within the enhancer region. The results of these studies demonstrated that the two variants replicated with similar kinetics and to equal levels in cultured equine fibroblasts or in equine macrophage, the natural target cell of EIAV, even after prolonged serial passage in the latter cell type. Furthermore, EIAV(19-2) and EIAV(19-2-6A) variants demonstrated similar replication levels in experimentally infected ponies. However, ponies infected with EIAV(19-2-6A) exhibited a rapid switch in the prevalent LTR type, such that by 112 days postinfection, no original-LTR-type viruses were evident. This specific and rapid shift in LTR quasispecies indicates an in vivo selection that is not reflected in simple in vitro replication rates, suggesting undefined selection pressures in vivo that drive LTR variation during persistent EIAV infection.

Animals↗

PubMed2002

Strain characterization of natural sheep scrapie and comparison with BSE.

Scrapie was transmitted to mice from ten sheep, collected in the UK between 1985 and 1994. As in previous natural scrapie transmissions, the results varied between scrapie sources in terms of the incidence of disease, incubation periods and neuropathology in challenged mice. This contrasted with the uniformity seen in transmissions of BSE to mice. The scrapie and BSE isolates were characterized further by serial passage in mice. Different TSE strains were isolated from each source according to the Sinc or PrP genotype of the mouse used for passage. The same two mouse-passaged strains, 301C and 301V, were isolated from each of three BSE sources. Despite the variation seen in the primary transmissions of scrapie, relatively few mouse-passaged scrapie strains were isolated and these were distinct from the BSE-derived strains. The ME7 scrapie strain, which has often been isolated from independent sheep sources in the past, was identified in isolates from four of the sheep. However, a new distinct strain, 221C, was derived from a further four scrapie sheep. These results suggest that there is agent strain variation in natural scrapie in sheep and that the spectrum of strains present may have changed over the last 20 years. The tested sample is too small to come to any conclusions about whether the BSE strain is present in sheep, but the study provides a framework for further more extensive studies.

Animals↗

PubMed2000

Heteroclite subgenomic RNAs are produced in porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) was shown to produce atypical subgenomic RNAs that contain open reading frame la nucleotides and are present under a wide variety of culture conditions, including high and low multiplicities of infection, in simian and porcine host cells, and during infection with cell-adapted and wild-type PRIRSV strains. Sequence analysis demonstrated that they are heterogeneous in 5-3' junction sequence and size and may code for different predicted fusion proteins. This is the first report of these novel RNA5 in arteriviruses and we have termed them heteroclite (meaning 'deviating from common forms or rules") subgenomic RNAs. The unique properties of these subgenomic RNAs include (a) apparent association with normal virus infection and stability during serial passage, (b) packaging of heteroclite RNAs into virus-like particles, (c) short, heterogeneous sequences which may mediate the generation of these RNAs, (d) a primary structure which consists of the two genomic termini with one large internal deletion, and (eJ little apparent interference with parental virus replication. These subgenomic RNA5 may be critical to, or a necessary side product of, viral replication. The expression of these novel RNA species support the template-switching model of similarity-assisted RNA recombination. In summary, PRRSV readily undergoes nonhomologous RNA recombination to generate heteroclite sub-genomic RNA5.

3' Untranslated Regions↗

PubMed1990

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. I. Pre-clinical studies.

Dengue 4 (DEN-4) virus strain 341750 Carib was modified by serial passage in primary canine kidney (PCK) cell cultures. By the 15th PCK passage, this virus was less infectious for monkeys and resulted in a significantly reduced viremia as compared to the parent DEN-4 virus. The 30th PCK passage of DEN-4 341750 Carib was non-infectious for monkeys. A vaccine prepared at the 20th PCK passage in DBS-FRhL-2 cells stimulated the production of both neutralizing and hemagglutination inhibition antibodies in monkeys; these animals were also protected against challenge with the homologous strain as well as a heterologous strain of DEN-4. An ID50 titration in monkeys resulted in a titer of greater than 10(4) plaque-forming units (PFU) for the vaccine virus and 0.5 PFU for the parent virus. Reduced monkey infectivity of this magnitude has been correlated with human attenuation in previous dengue vaccine candidates. The DEN-4 strain 341750 Carib PCK-20/FRhL-4 vaccine has been characterized and sufficiently tested to be considered for safety and immunogenicity trials in humans.

Animals↗

PubMed1991

Long term maintenance of Toxoplasma gondii (Rh strain) in Vero cell line and use of harvested antigens for immunodiagnosis.

Thirty in vitro serial passages of Toxoplasman gondii cultures in Vero cell line performed once in every five days had a mean increase in parasite count of 74.4 +/- 14.8 times from that of initial counts. Long term cultures in Vero cell line did not alter the virulence of the parasite. The good correlation (r = 0.99) between the IFA titer and ELISA OD values using the parasite antigens from in vitro sources indicates that long term maintenance of T. gondii in culture does not affect significantly the ability to recognize antibodies to surface and soluble antigens. The results also show that soluble antigens containing host cells can be directly used for immunodiagnostic purposes without purification. The in vitro maintenance of T. gondii is safer and cheaper when compared to the in vivo method.

Animals↗

PubMed2005

Production, characterization and evaluation of virulence of an adhesion defective mutant of Flavobacterium columnare produced by beta-lactam selection.

AIMS: The aim of this study was to develop and evaluate mutants of Flavobacterium columnare, the causative agent of columnaris disease in fish. METHODS AND RESULTS: Serial passage on ampicillin (beta-lactam) enriched modified Hsu-Shotts medium resulted in a F. columnare mutant that differed from the parent strain in colony morphology, whole cell proteins, adhesion and virulence. The mutant differed from its parent in virulence during immersion challenge, but not during injection challenge or generation of antibodies. CONCLUSION: Flavobacterium columnare exposure to ampicillin produces both resistance to that antibiotic and produces mutants that lack or have reduced adhesion characteristics and modified ability to adhere to fish tissue. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description of an adhesion-defective mutant of F. columnare and the effects of altered adhesion on columnaris disease. This mutant has considerable potential as a tool to study the role of adhesion in columnaris disease.

Ampicillin↗

PubMedDate not supplied

Isolation and molecular characterization of a mouse renal microvascular endothelial cell line.

Murine endothelial cells (ECs) have proven difficult to obtain and maintain in culture. Long-term maintenance of normal ECs remains a difficult task. In this article we report the establishment of the first cellular line of renal microvascular endothelium obtained from normal tissue. Cells were isolated, cloned, and maintained by serial passages for longer than 24 mo, using endothelial cell growth supplement (ECGS) and gelatin-coated plates. Their morphology and ultrastructure, expression of von Willebrand factor, presence of smooth muscle alpha-actin, vimentin, cytokeratin filaments, capillary structures formed on Matrigel, and some typical ECs surface molecules were the criteria used to characterize cultured ECs. When examined for responsiveness to Shiga toxin-1, 13-20% of cytotoxicity was observed when coincubated with lipopolysaccharides. This cytotoxicity was not observed for normal lung ECs (1G11). Consequently, REC-A4 line retains characteristics of resting microvascular ECs and represents a useful in vitro model to study biological and physiopathological properties of renal endothelium.

Actins↗

PubMed1956

Antigenic variants of influenza A virus (PR8 strain). II. Serological and immunological characteristics of variants derived from variants.

Four successive generations of antigenic variants of influenza PR8-S virus, each derived from the previous one by serial passage in the lungs of mice immunized with the homologous agent, were compared with the original parent PR8-S virus with respect to their serological and immunological character. It was demonstrated by means of H.I., complement-fixation and in ovo-neutralization tests that the variants exhibited a progressively decreasing reactivity with the parent PR8-S antiserum while retaining the ability to elicit antibody to PR8-S influenza virus and to their respective predecessors. Accompanying these changes was a progressive reduction in antigenicity without any significant changes in pathogenicity for mice. Experimental evidence was presented which indicates that the serological changes observed with the variants are not related to the P-Q phenomenon. Antibody absorption tests showed that the variants share antigens with PR8-S virus but differ from it by the presence of specific antigenic components; these increase in quantity with each successive variant while the amount of related antigens shows a progressive decrease. The importance of evaluating the significance of antigenic changes of influenza viruses with active immunity tests was emphasized by the fact that PR8-S vaccine protected mice against fatal infection with lethal doses of the variant strains although the latter had a progressively decreasing serological reactivity with PR8-S antiserum. The inheritable character of the new antigenic properties of the variant strains was demonstrated by their persistence in the absence of thea selective environment following 18 to 24 serial intranasal passages with large inocula in normal mice and following limiting dilution passage in fertile eggs.

Animals↗

PubMed2003

A pseudorabies virus recombinant simultaneously lacking the major tegument proteins encoded by the UL46, UL47, UL48, and UL49 genes is viable in cultured cells.

The UL46, UL47, UL48, and UL49 genes, which encode major tegument proteins, are conserved in most alphaherpesvirus genomes. However, the relative importance of each of these proteins for replication of individual alphaherpesviruses appears to be different. Recently, we demonstrated that single deletions of UL47 or UL48 impair maturation and egress of pseudorabies virus (PrV) particles to different extents, whereas deletions of UL46 or UL49 have no significant effects on virus replication in cell culture (W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp, and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002; M. Kopp, B. G. Klupp, H. Granzow, W. Fuchs, and T. C. Mettenleiter, J. Virol. 76:8820-8833, 2002). To test for possible functional redundancy between the four tegument proteins, a quadruple gene deletion mutant (PrV-DeltaUL46-49) was generated and characterized in vitro. Although plaque formation by this mutant was almost abolished and maximum titers were reduced more than 100-fold compared to those of parental wild-type virus, PrV-DeltaUL46-49 could be propagated and serially passaged in noncomplementing porcine and rabbit kidney cells. Electron-microscopic studies revealed that nucleocapsid formation and egress of PrV-DeltaUL46-49 from the host cell nucleus were not affected, but secondary envelopment of nucleocapsids in the cytoplasm was only rarely observed. The replication defect of PrV-DeltaUL46-49 could be fully corrected by reinsertion of the UL46-to-UL49 gene cluster. Plaque sizes and virus titers were only slightly increased after restoration of only UL47 expression, whereas repair of only UL48 resulted in a significant increase in replication capacity to the level of a UL47 deletion mutant. In conclusion, we show that none of the UL46 to UL49 tegument proteins is absolutely required for productive replication of PrV. Moreover, our data indicate that the UL47 and UL48 proteins function independently during cell-to-cell spread and virus egress.

Animals↗

PubMed2005

A segmented form of foot-and-mouth disease virus interferes with standard virus: a link between interference and competitive fitness.

Serial passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells at high multiplicity of infection resulted in dominance of particles containing defective RNAs that were infectious by complementation in the absence of standard viral RNA. In the present study, we show that the defective FMDV particles interfere with replication of the cognate standard virus. Coinfections of defective FMDV with standard FMDV mutants that differ up to 151-fold in relative fitness have documented that the degree of interference is higher for low fitness than for high fitness standard virus. These comparisons suggest a likely overlap between those mechanisms of intracellular competition that underlie viral interference and those expressed as fitness differences between two viruses when they coinfect the same cells. Interference may contribute to the selective pressures that help maintain dominance of segmented defective RNAs over the standard FMDV genome.

Animals↗

PubMed1997

Development of resistance to metronidazole and minocycline in vitro.

By local delivery of antibiotics to periodontal pockets, very high initial concentrations are often quickly succeeded by subinhibitory concentrations, which may facilitate development of bacterial resistance. The purpose of the present study was to investigate possible development of resistance in suspected periodontal pathogens after exposure to subinhibitory concentrations of metronidazole and minocycline. The minimal inhibitory concentration (MIC) of 18 reference strains and 12 clinical isolates was determined by a broth dilution method. Subsequently, all strains with MIC < 8 micrograms/ml were exposed to serial passage on plates containing subinhibitory and gradually increasing concentrations of antibiotics, until growth was inhibited. Initially, most strains were inhibited at < or = 0.250 microgram/ml of minocycline and < or = 0.5 microgram/ml of metronidazole, though A. actinomycetemcomitans was resistant to metronidazole. After growth at subinhibitory concentrations, 8 strains survived 1-2 x and 11 stains survived 8-32 x their initial MIC of metronidazole, growing at up to 8 micrograms/ml. All A. actinomycetemcomitans survived 8-64 x their initial MIC of minocycline, growing at > or = 2 micrograms/ml, while all other strains were inhibited at < or = 0.250 microgram/ml, corresponding to a 1-8 x increase in their initial MIC. Thus, development of resistance was observed for periodontal bacteria growing at up to 64 x their initial MIC, but the final level of resistance was moderate.

Aggregatibacter actinomycetemcomitans↗

PubMed2026

Development and protective efficacy of a live attenuated vaccine candidate against goose astrovirus.

Goose astrovirus (GAstV) is a significant pathogen affecting goslings by inducing visceral gout, yet no commercial vaccine is currently available. This study involved the serial passaging of the GAstV-GXNN strain in LMH cells to investigate alterations in viral replication, genomic stability, and pathogenicity, as well as to assess the potential of a vaccine candidate. The findings indicated that the viral titer increased progressively with each passage, reaching 107.35 TCID50/mL by the 120th passage (GAstV-GXNNP120). Whole-genome sequencing revealed the presence of 6, 19, 26, and 28 nucleotide mutations at the 30th, 60th, 90th, and 120th passages, respectively. Pathogenicity assays demonstrated a reduction in virulence with successive passages, culminating in the complete attenuation of GAstV-GXNNP120, which did not induce clinical signs or lesions in one-day-old goslings. Following five successive passages in goslings, the attenuated strain exhibited stable genetic characteristics without any reversion to virulence. Goslings aged one day, inoculated with GAstV-GXNNP120 at dosages ranging from 102.0 to 105.0 TCID50, developed neutralizing antibodies by the third day post-vaccination. Antibody levels increased in a dose-dependent manner, peaking at day 21 and remaining elevated through day 42. Challenge experiments utilizing the virulent GAstV-GXNN strain revealed that groups vaccinated with doses of 103.0 TCID50 and above achieved complete protection. These groups exhibited no clinical symptoms or pathological damage post-challenge, and both tissue viral loads and virus shedding levels were significantly reduced compared to the control group. Consequently, the minimum effective vaccination dose was established at 103.0 TCID50. These results provide a crucial foundation for the development of a live attenuated GAstV vaccine.

Animals↗

PubMed1992

A novel variant of Sindbis virus is both neurovirulent and neuroinvasive in adult mice.

A strain of Sindbis virus (SV), recently isolated from mosquitoes in Israel, was used as a source for variants which differ in neuroinvasiveness and virulence that were generated by serial passage of SV in suckling and weanling mouse brain. At the 15th passage a neurovirulent variant was observed and designated SVN (neurovirulent). After 7 more passages in weanling mouse brains, another variant was observed and designated SVNI (neuroinvasive) and both were isolated and purified. All strains caused similar viremia after intraperitoneal (I.P.) injection of weanling mice, but whereas SV was neuroinvasive but nonvirulent, SVN was neurovirulent but noninvasive and SVNI was both virulent and invasive. SVNI is the first SV variant which is both neurovirulent and neuroinvasive in weanling mice. Co-injection I.P. of SV + SVN resulted in presence of SV alone in the mouse brain; co-injection of SVNI + SVN resulted in full-titered replication of both strains in the brain. We assume that this is achieved through a breach of the blood brain barrier effected by SVNI replication and used by SVN for co-invasion. SV probably invades the brain by a different mechanism. I.P. infection with SVNI of inbred BALB/c mice gave rise to clinical signs only in a few mice even though substantial viremia was demonstrated.

Animals↗

PubMed2003

Evolution of Soybean mosaic virus-G7 molecularly cloned genome in Rsv1-genotype soybean results in emergence of a mutant capable of evading Rsv1-mediated recognition.

Plant resistance (R) genes direct recognition of pathogens harboring matching avirluent signals leading to activation of defense responses. It has long been hypothesized that under selection pressure the infidelity of RNA virus replication together with large population size and short generation times results in emergence of mutants capable of evading R-mediated recognition. In this study, the Rsv1/Soybean mosaic virus (SMV) pathosystem was used to investigate this hypothesis. In soybean line PI 96983 (Rsv1), the progeny of molecularly cloned SMV strain G7 (pSMV-G7) provokes a lethal systemic hypersensitive response (LSHR) with up regulation of a defense-associated gene transcript (PR-1). Serial passages of a large population of the progeny in PI 96983 resulted in emergence of a mutant population (vSMV-G7d), incapable of provoking either Rsv1-mediated LSHR or PR-1 protein gene transcript up regulation. An infectious clone of the mutant (pSMV-G7d) was synthesized whose sequences were very similar but not identical to the vSMV-G7d population; however, it displayed a similar phenotype. The genome of pSMV-G7d differs from parental pSMV-G7 by 17 substitutions, of which 10 are translationally silent. The seven amino acid substitutions in deduced sequences of pSMV-G7d differ from that of pSMV-G7 by one each in P1 proteinase, helper component-proteinase, and coat protein, respectively, and by four in P3. To the best of our knowledge, this is the first demonstration in which experimental evolution of a molecularly cloned plant RNA virus resulted in emergence of a mutant capable of evading an R-mediated recognition.

Cloning, Molecular↗

PubMed2006

Characterization of homologous defective interfering RNA during persistent infection of Vero cells with Japanese encephalitis virus.

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese en-cephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homolo-gous DI virus intimately associated with JEV persis-tence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a per-sistent JEV infection in which the DI RNA co-replicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated dur-ing its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identifi-cation of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules re-tained their open reading frames despite a large dele-tion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these ob-servations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

Amino Acid Sequence↗

PubMedDate not supplied

Comparison of heterophil phagocytosis for heterophil-adapted Salmonella enteritidis (HASE) and wild-type Salmonella enteritidis (SE).

Serial passage of Salmonella enteritidis (SE) yields heterophil-adapted SE (HASE) strains that have resulted in decreased shedding of SE in feces and reduced egg contamination. Additionally, increasing the number of heterophil passages further reduced the number and frequency of fecal shedding. To evaluate SE and heterophil interaction, nine SE strains were fluorescein isothiocyanate-labeled when viable. There were six wild-types: SE TK 474, SE TK 584, SE TK 599, SE TK 600, SE TK 655, and SE TK 657; and three HASE strains: TK 499 heterophil adapted five times, TK 598 heterophil adapted six times, and TK 605 heterophil adapted 11 times. Trials were repeated seven times in duplicate with heterophils isolated from seven healthy chickens. Heterophils were incubated with the bacterial strains at 41 C for 15 min, and 10,000 heterophils were analyzed by flow cytometry. Percentage of phagocytosis and mean channel number of fluorescence were compared. Both parameters were significantly increased for all HASE-type strains compared with wild-type, nonadapted SE strains. Increased phagocytosis of HASE bacterial strains may be significant in processing and elimination of the HASE strains and may be related to the protective effect of HASE by decreased shedding of wild-type SE challenge strains.

Animals↗

PubMed1996

In vitro selection and molecular characterization of human immunodeficiency virus type 1 with reduced sensitivity to 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA).

9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA) is an acyclic nucleotide with potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). The present study was undertaken to determine whether HIV-1 resistance to PMEA could be generated by in vitro selection and if so, to determine which mutations in reverse transcriptase (RT) were responsible. HIV-1LAI was serially passaged for 10 months in the presence of increasing concentrations of PMEA up to a maximum of 40 microM. After 40 passages, the 50% inhibitory concentration (IC50) of PMEA had increased almost 7-fold from 4.45 to 30.5 microM. Some cross-resistance to 2',3'-dideoxycytidine (ddC, zalcitabine), 2',3-dideoxyinosine (ddI, didanosine), and 3'-thiacytidine (3TC, lamivudine) was also observed, but no cross-reactive resistance to 3'-azido-3'-thymidine (AZT, zidovudine). Sequencing of the RT encoding region of each of eight pol clones from resistant isolates revealed a Lys-65-->Arg (K65R) substitution. HIV with the K65R mutation inserted by site-directed mutagenesis also had decreased sensitivity to PMEA in H9 cells and a similar cross-resistance profile. Thus, HIV can develop decreased sensitivity to PMEA after long-term in vitro exposure and this change is associated with a K65R substitution. Additional studies will be needed to determine whether a similar mutation in HIV RT develops in patients receiving PMEA or its orally bioavailable prodrug adefovir dipivoxil (bis-POM PMEA).

Adenine↗

PubMed2000

Transient resistance of influenza virus to interferon action attributed to random multiple packaging and activity of NS genes.

Interferon (IFN) action survival curves for an avian influenza virus (AIV) in chicken or quail cells showed that 40-60% of the virions in a stock of virus were highly sensitive to the inhibitory effects of chicken IFN-alpha (ChIFN-alpha), whereas the rest were up to 100 times less sensitive. This greater resistance to IFN was transient, that is, was not a stable characteristic, in that virus stocks grown from plaques that formed in the presence of 50-800 U/ml IFN gave rise to virus populations that contained both sensitive and resistant virions. If AIV was serially passaged several times in the presence of IFN, the proportion of transiently IFN-resistant virus was greater. We propose a model to account for this transient resistance of AIV to IFN action based on the reported inactivation of the dsRNA-dependent protein kinase (PKR) and its activator dsRNA by the NS1 protein of influenza virus and also on the increase in the survival of AIV in IFN-treated cells exposed to 2-aminopurine, a known inhibitor of PKR. We suggest that IFN-resistant AIV is generated from a random packaging event that results in virions that contain two or more copies of RNA segment 8, the gene segment that encodes the NS1 protein of AIV, and that these virions will produce correspondingly elevated levels of NS1. The experimental data fit well to theoretical curves based on this model and constructed from the fraction of virus in the population expected by chance to contain one, two, or three copies of the NS gene when packaging an average of 12 influenza gene segments that include the 8 segments essential for infectivity.

2-Aminopurine↗