Production Of Cytopathic Effect Research Articles

Cell culture adaptation of Avipox viruses isolated from different species of birds

Avianpox (AP) is an infectious, slow spreading viral disease that has been reported to affect numerous species of birds including poultry. There is very limited information available regarding the molecular and biological characteristics of the avipox viruses (APVs) circulating in India. In the present study, APVs from fowl [FP/As- K(R)], pigeon [P2/ As- K9(R)] and duck [D2/ As- N] origin isolated from natural outbreaks of the disease in different areas of Assam were selected for adaptation in different cell culture. All the three isolates were propagated in chicken embryo fibroblast (CEF) primary cell culture and Vero cell line and their propagation was confirmed by observing virus specific cytopathic effects (CPE) and by performing PCR targeting the 4b gene of APV. All three isolates were adapted in the CEF primary culture with production of virus specific CPE. However, a high degree of CPE was observed in the cultures infected with the fowl isolate from an earlier passage (P-11) in comparison to the other two isolates. Even the time required for completion of CPE was considerably less in case of the fowl isolate. The log TCID50 of the fowl isolate was found to be 4.18 ± 0.11, 5.29 ± 0.06 and 6.29 ± 0.13, respectively in the 5th, 15th and 20th passage, which was higher in comparison to the other two isolates. Interestingly, none of the isolates showed any virus specific CPE in vero cell line on propagation till the 10th passage. Moreover, all the passages were found to be negative by PCR.

Evaluation of Monkeypox- and Vaccinia virus-neutralizing antibodies in human serum samples after vaccination and natural infection.

In early to mid-2022, an unexpected outbreak of Monkeypox virus infections occurred outside the African endemic regions. Vaccines originally developed in the past to protect against smallpox are one of the available countermeasures to prevent and protect against Orthopoxvirus infections. To date, there are few studies on the cross-reactivity of neutralizing antibodies elicited by previous vaccinia virus-based vaccination and/or Monkeypox virus infection. The aim of this study was to evaluate a possible approach to performing Monkeypox and vaccinia live-virus microneutralization assays in which the read-out is based on the production of cytopathic effect in the cell monolayer. Given the complexity of Orthopoxviruses, the microneutralization assay was performed in such a way as to uncover a potential role of complement, with and without the addition of an external source of Baby Rabbit Complement. A set of human serum samples from individuals who had been naturally infected with Monkeypox virus and individuals who may have and not have undergone vaccinia virus vaccinations, was used to evaluate the performance, sensitivity, and specificity of the assay. The results of the present study confirm the presence and cross-reactivity of antibodies elicited by vaccinia-based vaccines, which proved able to neutralize the Monkeypox virus in the presence of an external source of complement.

Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1–5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1–5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1–5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

Isolation, molecular characterization, and phylogenetic analysis of encephalomyocarditis virus from South China tigers in China

Although encephalomyocarditis virus (EMCV) can infect many host species and cause myocarditis and sudden death in many species, little is known about EMCV infection in tigers. A virus was isolated from organs of dead South China tigers with sudden death in southern China. The production of cytopathic effect on BHK cells, and the results of PCR, electron microscopy (EM), and whole genome sequencing indicated that the pathogen was EMCV, the strain was named FJ13. Other pathogenic agents were excluded as possible pathogenic agents. Phylogenetic analyses of the whole genome, ORF (open reading frame) and CCR (capsid coding region) using the neighbour-joining method revealed that EMCV isolates cluster into two groups (group 1 and 2) with two sub-clusters within group 1 (group 1a and 1b), and FJ13 belongs to group 1a. Animal experiment showed that the isolated strain FJ13 could cause clinical symptoms and pathological changes. The results of this study indicated that FJ13 caused myocarditis of tigers and provided new epidemiologic data on EMCV in China.

Detection of Hepatitis A Virus by Using a Combined Cell Culture-Molecular Beacon Assay

Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5' noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.

An Authentic 3′ Noncoding Region Is Necessary for Efficient Poliovirus Replication

Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).

Development and Analysis of Recombinant Adenoviruses for Gene Therapy of Cystic Fibrosis

A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.

Antiviral properties of Ro 31-8959, an inhibitor of human immunodeficiency virus (HIV) proteinase

Ro 31-8959 inhibits the spread of HIV infection and the production of cytopathic effects in cultures of acutely infected cells. IC 50 values for these effects are in the range 0.5–6.0 nM and IC 90 values are in the range 6.0–30.0 nM. This inhibitor is effective even when added to cultures at a late stage of infection, after syncytia have started to form. Virus antigen, virus particles and virus cytopathic effects can largely be cleared from cultures treated with compound from 3 days until 6 days post infection. In chronically-infected cells, inhibition of virus maturation can be detected after 24 hours' treatment with 10 nM Ro 31-8959. In addition, a significant reduction of the proteolytic processing of p56 to p24 can be demonstrated in these cells with compound at picomolar concentrations. These properties indicate that Ro 31-8959 is highly effective against HIV with the potential to inhibit acute, established acute and chronic infections.

Intracellular distribution of the envelope glycoprotein of human immunodeficiency virus and its role in the production of cytopathic effect in CD4+ and CD4- human cell lines

Human CD4+ and CD4- monocytoid cell lines were transfected with a constructed plasmid that has the envelope gene of human immunodeficiency virus under the transcriptional control of human metallothionein IIA promoter; the transfected cells were then cloned. These CD4+ and CD4- transfectant cell clones, both of which expressed almost the same amount of gp160 after induction with metal ions, were used for ultrastructural analysis of the distribution of the envelope glycoprotein in the cytoplasm. Immunofluorescence microscopy with an anti-envelope glycoprotein monoclonal antibody showed localized distribution of gp160 in the CD4+ cell clone and diffuse distribution of gp160 in the CD4- cell clone. These observations were substantiated by immunoelectron microscopy, in which the aggregated form of gp160 was observed in the cytoplasm of CD4+ cells but was scarce in that of CD4- cells. A notable finding was that the sites corresponding to the nuclear pores were occupied with aggregates of gp160 in CD4+ cells, exhibiting cytopathic effects. Both freeze-fracture and transmission electron microscopy also showed abnormal morphology around the nuclear pores and perinuclear space. These results support the possibility that such gp160 complexes accumulated around the nuclear pores primarily disturb the transportation of many molecules between the nucleus and the cytoplasm, resulting in a cytopathic effect in the CD4+ cell clone.

Isolation of a rickettsiales-like organism from diseased coho salmon (Oncorhynchus kisutch) in Chile.

A rickettsiales-like organism was isolated in cell culture from coho salmon (Oncorhynchus kisutch) reared in seawater net pens in the vicinity of Puerto Montt, Chile S. A. and experiencing an epizootic with accompanying mortality. The organism, observed in fish and isolated in cell culture, was GRAM-negative, pleomorphic, predominantly coccoid in shape, and approximately 1μm in diameter. It replicated, producing cytopathic effect (CPE) in cell lines from four species of salmonids, but failed to grow in any of eleven standard bacteriological media. The optimal temperature for replication was 15-18°C based on the production of CPE. Streptomycin, gentamicin, and tetracycline were inhibitory for the organism, but it was not sensitive to penicillin. The isolated organism stained with GIEMSA and PINKERTON'S stains and with a modified GIMENEZ stain. It did not react with a monoclonal antibody against the group-specific LPS chlamydial antigen. On the basis of available information, we suggest this organism is a member of the order Rickettsiales and represents the first isolation of a member of this group from an aquatic poikilotherm.

Proteinase activities of Entamoeba histolytica cytotoxin

The proteinase activity of the low molecular weight cytotoxin of Entamoeba histolytica was correlated with its cytotoxicity. Gel-filtered amebal toxin (mol wt 10–30,000) proteinase activities could be assayed on azocasein at pH 6 or on hemoglobin at pH 4.5. Proteinase activity was inhibited by serum fractions, thiol reagents, heavy metals, leupeptin, and antipain. The cytotoxic activity of gel-filtered amebal toxin was inhibited by serum fractions, leupeptin, and antipain. Increased proteinase and cytotoxic activity was produced by treatment with cysteine. These data support the action of a thiol proteinase in the production of cytopathic effects by gel-filtered amebal toxin in vitro. The cytotoxic and proteinase activities were further purified using a combination of column chromatography and preparative isoelectric focusing. Two low molecular weight cytotoxins with proteinase activity on both substrates were isolated. The major cytotoxin had an isoelectric point of 4.5 and a molecular weight of 22,000; the other cytotoxin had a basic isoelectric point. These substances may be cathepsin B-like proteinase and elastase or cathepsin G-like proteinases of E. histolytica. The major proteinase activity in the high molecular weight fraction was not cytotoxic. The isoelectric points of the high molecular weight proteinase activities corresponded to that of mammalian cathepsin D. The major cell rounding cytotoxic activity of E. histolytica extracts in vitro is probably due to the activity of a thiol-containing cathepsin B-like proteinase.

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.

Tickborne viruses in western North America. III. Viruses from man-biting ticks (Acari: Ixodidae) in Oregon.

Numerous isolations of a new, serologically unique, filterable agent, Cascade virus, were made from adult Dermacentor occidentalis ticks in southwestern Oregon during 1979 and 1980. The virus was not found in Ixodes pacificus collected the same general areas and at the same times. Dermacentor andersoni ticks collected in eastern Oregon were occasionally infected with Colorado tick fever virus, but not with the new agent. Cascade virus could readily be isolated in Xenopus laevis (XTC-2) or Dermacentor variabilis (RML-15) cells incubated temperatures of 34 or 27 °C, respectively, but could not be isolated when tick material was inoculated into laboratory animals or homeothermic cell lines. The virion contains RNA and essential lipids and is sensitive to exposure to heat and acid; its diameter is between 100 and 220 nm. Virus growth in XTC-2 cells is accompanied by the production of cytopathic effects and plaques, but growth in other cell lines (RML-15, Vero, PS, BHK-21) is demonstrable only upon secondary assay. The virus caused cytopathic effects and plaques in XTC-2 cells incubated at 27 or 34 °C, but failed to grow in or visibly affect these cells when incubated at 37 °C. Vero, PS and BHK-21 cell lines permitted the virus to grow at an incubation temperature of 34 °C, but growth was inhibited at 37 °C in at least 2 of these lines (Vero, PS). Cascade virus failed to replicate in mouse fibroblasts (L cells) or Aedes albopictus (ATC-15) mosquito cells. Intracerebral (ic) inoculation of virus in suckling Microtus pennsylvanicus caused illness and death only sporadically. Suckling white mice, hamsters and wet chicks showed no signs of infection after ic or peripheral inoculation, and the virus failed to grow in the yolk sac of embryonated chicken eggs. Although no evidence of human infection with Cascade virus was obtained from a small sample of sera selected from Californians who became sick after tick exposure, recent evidence in Oregon and California of tick-associated disorders of undetermined cause (erythema chronicum migrans, Lyme disease) suggests further study.

Persistent infection of cells in culture by respiratory syncytial virus.

The virus-cell relationship of RS virus and the HEp-2 cell line has been examined. The production of cytopathic effect (c.p.e.) on HEp-2 cells has been found to be dependent upon the passage level of the cell line. Cells at lower passage levels exhibit c.p.e. in the form of syncytium formation, while those at higher passage levels no longer exhibit this effect. Cells infected at higher passage levels are covertly infected and continue to produce large amounts of infectious virus which remains cell-associated. On continued passage, these cells remain infected with virus but show no c.p.e. and release little if any infectious virus into the medium. Examination of the RNA species present in infected cells revealed that similar species are present in both the overtly and covertly infected cells.

Studies on viral arthritis (teno‐synovitis) in Italy

Three of several virus isolates made from different groups of chickens affected with arthritis were serially passaged in chicken embryos, and then cultured on chicken kidney cells with the production of cytopathic effects and development of syncytia. Resistance to inactivating agents and electron microscopic features showed their relationship to reoviruses. By the agar gel diffusion test the isolates were shown to be serologically related to each other and to 2 American strains. A tissue suspension from infected embryos when inoculated into the footpad of one to 15 day-old chicks reproduced arthritis.

Different effects of temperaturs on Type 1 and Type 2 Herpesvirus hominis in cell culture.

SummaryEffects of various incubation temperatures (30, 37 and 40°) on type 1 and type 2 strains of Herpesvirus hominis (HVH) were studied in primary cultures of rabbit kidney cells. Eight strains of type 1 and 7 strains of type 2 HVH were used. These strains included both recent isolates and laboratory strains with long histories of passage in various tissues.Both type I and type 2 HVH strains, regardless of passage history, grew slower at 30 and 40° than at 37°, but this retardation of growth was more pronounced in cultures infected with type 2 strains of HVH at 40°. The production of cytopathic effects (CPE) and the growth of type 2 HVH in rabbit kidney cells were almost completely suppressed as long as the infected cells were maintained at 40°. Upon transferring the cells to 37°, however, CPE along with rising infectivity titers of the virus were noted. Further spread of CPE produced by type 2 HVH following incubation at 37° was successfully blocked by incubating the cells at 40°.Fig. 2. Minimum infect...

Role of interferon in the propagation of MM virus in L cells.

MM virus propagated in mouse brain replicates to low titers in L cells without production of cytopathic effect (CPE). After growing the virus in BHK-21 cells, however, the virus replicates to high titers in L cells with complete CPE. It was found that suspensions of MM virus propagated in L cells directly from the mouse brain contained much more interferon than did suspensions of virus which had first been grown in BHK-21 cells. Mouse brain suspensions of the virus were also found to contain high interferon titers. Treatment of L cells with actinomycin D before infection with mouse brain-grown virus resulted in full virus replication with CPE. BHK-21 cell-grown virus diluted in L cell interferon behaved like mouse brain-grown virus in L cells. It is concluded that the presence of interferon in the inoculum is largely responsible for the suppression of MM virus replication in L cells.

Characterization of viruses isolated from bats.

Antigenic relations among viruses isolated from bats were studied by complement-fixation, hemagglutination-inhibition, and neutralization testing. This information would help to determine which of these viruses could be identified as strains and which could be recognized as antigenically distinct agents. The viruses were also characterized with respect to their comparative infectiousness for several host systems. Most of the viruses isolated from bats have been previously placed in serogroup B; two were considered ungrouped. The group B viruses included Rio Bravo, Burns, Dakar, BP 180, Ib An 8646, Bukalasa, and Entebbe virus. Antigenic studies indicated that BP 180 and Ib An 8646 were strains of Dakar virus. Bukalasa virus was shown to be closely related to the Dakar virus strains. Rio Bravo and Burns viruses were indistinguishable and were distantly related to MML virus, which, by certain tests, was also distantly related to the Dakar virus strains. The two ungrouped viruses, Kern Canyon and Lagos, were not related to one another nor to any of the other viruses. Plaque formation on LLC-MK2 or Vero cell cultures, or both, and the production of cytopathic effects (CPE) in BHK-21 cell cultures were also studied. Lagos bat and Bukalasa viruses did not produce plaques and Lagos bat virus did not produce CPE in BHK-21 cell cultures. A comparison of the in vitro assays with assays performed in suckling mice indicated that the intracerebral inoculation of suckling mice provided a slightly more sensitive assay system than any of the in vitro systems. Neutralization in LLC-MK2 cell cultures was significantly enhanced by the presence of “fresh” normal serum in the neutralization mixtures, although in certain instances, the enhancement of neutralization was accompanied by a lessening in the specificity of the neutralization reaction.

Role of Interferon in the Propagation of MM Virus in L Cells

MM virus propagated in mouse brain replicates to low titers in L cells without production of cytopathic effect (CPE). After growing the virus in BHK-21 cells, however, the virus replicates to high titers in L cells with complete CPE. It was found that suspensions of MM virus propagated in L cells directly from the mouse brain contained much more interferon than did suspensions of virus which had first been grown in BHK-21 cells. Mouse brain suspensions of the virus were also found to contain high interferon titers. Treatment of L cells with actinomycin D before infection with mouse brain-grown virus resulted in full virus replication with CPE. BHK-21 cell-grown virus diluted in L cell interferon behaved like mouse brain-grown virus in L cells. It is concluded that the presence of interferon in the inoculum is largely responsible for the suppression of MM virus replication in L cells.