Cytopathic effect induced by rabies virus in McCoy cells.

Both fixed and street rabies virus when cultivated in McCoy cells caused cytopathic changes 24 to 72 h after infection, depending on the multiplicity of infection. The cytopathic effect (CPE) was easily recognizable and resembles that induced by other members of the Rhabdovirus group, such as vesicular stomatitis virus, in several cell cultures. Higher titers of the Pasteur strain (PV) of fixed rabies virus were found in supernatants of McCoy cells when compared to those in VERO cells. The virus titer increased with the number of passages attaining a high titer after three passages. Rabies antigens were detected by direct immunofluorescence labeling in most McCoy cells of the infected culture, and specific antibodies neutralized the virus growth and CPE. There was also inhibition by treatment of the cells with human interferon (HuIFN) -alpha or -gamma, but not by murine interferon (MuIFN) -alpha, -beta or -gamma. Rabies-infected McCoy cell cultures may provide a useful assay system, based on the induction of CPE, the high virus production and the sensitivity to IFN.     

Bluetongue virus propagation and plaque assay: variation due to medium and serum supplement

Two base media, minimal essential medium (MEM) and RPMI 1640, were supplemented with a variety of serum extenders and/or substitutes for the purpose of defining a medium formulation capable of supporting good cell (VERO) growth and virologic assays. Bluetongue virus (BTV), the prototype Orbivirus in the Reoviridae, was used in all studies. In general, VERO cells grown in RPMI performed better than those grown in MEM relative to cell growth, virus production and plaque assay. RPMI was better for supporting cell growth when serum extenders (NuSerum or SerXtend) were employed as supplements. Relative to virologic techniques, cells grown in RPMI produced higher virus titers in both propagation studies and plaque assays. The interval from infection to greater than 90% cytopathic effect (CPE) was consistently shorter with RPMI as the base medium. Cell cultures supported with RPMI base medium, supplemented with 3.5% FBS with SerXtend, provided the best overall performance relative to: (a) amount of virus produced by infected cell monolayers, (b) sensitivity to productive infection under overlay conditions (revealed the highest titer of a standard virus stock) and (c) plaque assay quality including cell quality, plaque size and plaque clarity.     

Subtype-specific identification of influenza virus in cell cultures with FITC labelled egg yolk antibodies

We report on results obtained with a direct immunofluorescence test for subtype-specific identification of influenza virus in detached cells of MDCK cultures after inoculation of 281 clinical specimens from patients with influenzalike disease. Influenza virus antibodies were produced in eggs from immunized hens and labelled with FITC. In 157 cases CPE was found in MDCK cells. A total of 57 cases of influenza A (H3N2), 86 cases of influenza A (H1N1), and 14 cases of influenza B were identified. In 33 cases of influenza A (H1N1) infection with massive CPE guinea pig but not chicken erythrocytes were agglutinated by the cell culture supernatants. The single step immunofluorescence test described proved easy to perform and results were obtained within 1 h after CPE was observed in contrast to the conventional HIT which is very time-consuming.Dedicated to Professor Rudolf Rott on the occasion of his 60th birthday     

The growth of attenuated influenza vaccine donor strains in continuous cell lines

The growth of the Russian live attenuated influenza vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57 and B/USSR/60/69 was studied in cells of the VERO and Madin-Darby canine kidney (MDCK) lines as six-well cultures and cell factories infected at different multiplicities of infection. Yields for A/Leningrad/134/17/57 and A/Leningrad/134/47/57 were comparable in either cell line over a range of multiplicities but were about 10-fold lower than in the allantoic fluids of infected chicken embryos. For both A/Leningrad/134/47/57 and B/USSR/60/69, yields from the MDCK line were about 10-fold higher than for the VERO line. For B/USSR/60/69, yields in eggs were approximately 100-fold higher than those obtained in the MDCK line. A feature of the growth of B/USSR/60/69 was its reduced capacity to produce infectious progeny in either cell line at multiplicities of infection of 2.0 or 1.0 pfu/cell. Inhibition was due probably due to the presence of defective-interfering particles and was not detected with A/Leningrad/134/17/57 or A/Leningrad/134/47/57 in cultures of either line infected at the same multiplicities. Yields for both A/Leningrad/134/47/57 and B/USSR/60/69 in cells of the MDCK line were comparable when grown in six-well cultures or cell factories.     

Scanning electron microscopy of CV-1 and HeLa cells infected with herpes simplex viruses types 1 and 2

Production of virus particles in CV-1 or HeLa BU-25 cells was investigated after their infection with several strains of herpes simplex virus (HSV) types 1 and 2, especially in relation to the association of virus particles with cells, the ratio of plaque forming units (PFU) to the whole number of virus particles and the morphological characteristics of cytopathic effect (CPE). Growth curves of the viruses differed according to the combination of cells and infecting virus strains. At 20 hr p.i., the number of cell-associated or cell-free viruses ranged from 5 X 10(8) to 5 X 10(9) PFU/35 mm dish or from 5 X 10(2) to 1.5 X 10(4) PFU/cell irrelevant of virus serotype or the morphology of CPE. In the case of CV-1 cells, the ratios of the number of the virus particles to PFU ranged from 100 to 640 and/or 18 to 110, respectively, depending on the CPE of rounding type or of fusion type. In case of CPE of fusion type, a higher rate of infectious particles was observed.     

Studies on the replication of African horse-sickness virus in two different cell line cultures

Summary Single-passage growth patterns of African horse-sickness (AHS) virus in two different cell lines, MS and VERO, of monkey origin were compared by titrating extracellular virus in cultures infected at high multiplicity.Both by fluorescent-antibody and acridine orange staining techniques, large antigenic bodies containing RNA were found in the cytoplasm of infected VERO cells. This was not so evident in infected MS cells.Metabolic analogs, bromodeoxyuridine (BUDR) and iododeoxyuridine (IUDR) did not inhibit the yield of AHS virus either in MS or VERO cell cultures indicating that synthesis of new DNA is not required for replication of this virus. The yield of AHS virus was not inhibited by actinomycin D and mitomycin C, suggesting that synthesis or function of DNA is not directly required for the replication of the virus. MS cells were more sensitive to the toxic effect of these chemicals than VERO cells.This work was undertaken at the Near East Animal Health Institute, a project established by the United Nations Development Program Special Fund through the Food and Agriculture Organization in cooperation with the Ministry of Agriculture of Iran.     

Enhanced isolation of respiratory syncytial virus in cell culture.

During two winter seasons, we found that the combination of WI-38 or MRC-5 human lung fibroblasts plus primary rhesus monkey kidney (RhMK) and HEp-2 cell cultures yielded maximal isolation of respiratory syncytial virus. Cytopathic effects (CPE) developed earliest in RhMK cells and slowest in the human fibroblast lines. In RhMK cells, 50% of ultimately positive cultures showed CPE in 5 days, and 90% of positive cultures showed CPE within 7 days during both respiratory syncytial virus seasons.     

Abortive infection of human diploid cells by murine cytomegalovirus

Inoculation of human diploid cells (WI-38) with murine cytomegalovirus (MCMV) did not result in the synthesis of any infectious virions. However, morphological changes typical of the cytopathic effects (CPE) of MCMV were detectable within 12 hr of infection. The CPE included rounding, swelling, and detachment of cells. The nuclei of infected cells were enlarged, and intranuclear inclusions were visible by May Grunwald-Giemsa staining and by the indirect fluorescent-antibody test. All cells infected at a multiplicity of infection of 3 showed CPE, and these cells could not be passaged successfully. Cell lysates and exhausted media from infected WI-38 cultures did not produce any CPE in WI-38 cells. The virus absorbed to WI-38 cells with the same efficiency as to mouse embryo fibroblast cells (MEF). Samples of MCMV in which virus infectivity for MEF cells had been inactivated by ultraviolet irradiation or by exposure to 56 C failed to produce any of the above signs. MCMV-specific CPE did not occur in the presence of actinomycin D (1 mug/ml) or puromycin (20 mug/ml), but 5'-fluoro-2'-deoxyuridine at 1 x 10(-4)m did not prevent CPE or the development of intranuclear inclusions.     

Isolation and propagation of the virus of transmissible

Summary Of four strains of TGE virus tested, the Purdue strain could be propagated serially and produced a cytopathic effect (CPE) in primary monolayer cultures of adult and fetal pig kidney cells, pig thyroid cells, and pig salivary gland cells. In kidney and salivary gland cell cultures, a distinct CPE was observed only after several passages. In thyroid cell cultures, a marked CPE was evident in the first passage. High cell passage virus caused the rapid and complete destruction of and produced plaques regularly in pig thyroid cell cultures, while plaque production in pig kidney cell cultures was irregular. The CPE was accompanied by acid production. New infectious virus appeared in fetal pig kidney cells less than 5 hours after infection. TGE virus was found to be ether, acid, and heat labile. Serial passage in fetal pig kidney cells led to a considerable attenuation of TGE virus.This investigation was supported in part by funds provided by the Research Committee of the Graduate School and Fromm Laboratories, Grafton, Wisconsin.Published with the approval of the Director of the Wisconsin Agricultural Experiment Station as paper N. S. 517.     

Isolation, characterization, and serial propagation of a bovine group C rotavirus in a monkey kidney cell line (MA104).

A virus (designated the Shintoku strain) which was morphologically indistinguishable from group A rotaviruses was detected in the feces of adult cows with diarrhea in Japan. The virus contained 11 segments of double-stranded RNA and had an electrophoretic migration pattern in polyacrylamide gels similar to that of other group C rotaviruses (4-3-2-2). Feces containing the bovine virus reacted with antiserum to porcine group C rotavirus (Cowden strain) but not group A or B rotaviruses in immunoelectron microscopy. The virus was adapted to serial propagation in roller tube cultures of a rhesus monkey kidney cell line (MA104) by using high concentrations of trypsin. Evidence for viral replication in MA104 cell cultures was demonstrated by immunoelectron microscopy and indirect immunofluorescence by using antiserum to porcine group C rotavirus and by electrophoretic analysis of extracted viral double-stranded RNA. A significant antibody response against the isolate was detected in convalescent-phase sera of cows which excreted the virus: no increased antibody response to bovine group A rotavirus was observed. To our knowledge, this is the first isolation of a group C rotavirus from cattle.     

Susceptibility of various cell lines to virulent and attenuated strains of pseudorabies virus.

Various cell lines were infected with virulent and attenuated strains of pseudorabies virus (PRV). According to the type of the cytopathic effect (CPE), the cells could be divided into 3 groups: cells in which all strains formed syncytia; cells in which all strains caused rounding of cells; and cells in which virulent strains caused syncytium formation while attenuated ones rounding of cells. Neither the form (fibroblastoid or epithelial) nor the origin of cells influenced the type of the CPE. L cells and some cell lines derived from human tissue proved to be less sensitive to PRV than other cells. In certain human cells (e.g. HeLa and D6) virulent PRV strains propagated better than attenuated ones.     

Human rotavirus infection enhances invasiveness of enterobacteria in MA-104 cells

To study the interaction between common pathogens causing infectious diarrhea in humans, MA 104 cell cultures were infected with human rotavirus and Salmonella typhimurium or Shigella flexneri or enteroinvasive Escherichia coli (EIEC) concomitantly. When MA-104 cells were preinfected with human rotavirus, invasiveness of S. typhimurium was significantly enhanced. The enhancement was evident after 48 h of virus preincubation. At this time virus specific antigens were demonstrated in the cell cultures. Also, the invasiveness of S. flexneri and EIEC was enhanced after virus preincubation, though not significantly before 72 h. When the virus preincubation time was prolonged to 96 h a further increase in invasive ability was observed. No cytopathogenic effect of the virus on the cells was demonstrated. Two control strains of non-enteropathogenic E. coli did not show any invasiveness in MA-104 cells pretreated with virus. The results indicate a specific interaction between rotavirus infected cells and facultatively intracellular enteropathogenic bacteria.     

Criteria of primary screening of attenuated strains for live vaccine against Influenza A

Reassortant strains for live influenza vaccine (LIV) were selected using two additional markers: intensity of cytopathic effect (CPE) at 40 degrees C in MDCK cells and toxicity for mice (induction of acute hemorrhagic pulmonary edema after intranasal challenge with undiluted virus). All wild-type viruses induced a high CPE in MDCK cells, while the reassortants differed by this sign. Only vaccine strains and attenuation donors were characterized by a low CPE. Modern epidemic viruses are highly toxic for mice, causing the death of 60-100% animals from hemorrhagic pulmonary edema on days 3-4 after intranasal infection. Attenuation donors and vaccine strains were not toxic for mice, the level of toxic effect correlating with CPE in MDCK culture. Evaluation of CPE in MDCK culture and toxicity for mice can be used for primary screening of candidates for LIV.     

Growth of Junin virus,the etiological agent of argentinian hemorrhagic fever,in cell cultures

Summary Propagation of Junin virus, the etiological agent of Argentinian hemorrhagic fever, has been studied in a variety of cell cultures. One line of African green monkey kidney cells (Vero) at the 107th to 165th passage levels supported the multiplication of the virus and developed a marked cytopathic effect (CPE), while no CPE appeared in another line (BS-C-1) at the 34th passage level. CPE was markedly enhanced by rolling the cultures, and the cytocidal nature of virus replication was markedly more pronounced under these conditions. Junin virus also produced well-defined plaques in monolayer cultures of Vero cells under agar overlay. Virus assays obtained by titration endpoint of CPE or by plaque counts in Vero cells were in almost all instances higher than those obtained by titration in suckling mice. A linear relationship was observed between plaque counts and virus concentration. Maximum adsorption was obtained by 120 minutes incubation at 37°C. When the cultures were infected with <0.001 PFU per cell, the maximum virus titer was obtained on the 5th day after inoculation.Neutralization tests employing the 80% plaque reduction endpoint give reproducible results and indicate the absence of antibody for Junin virus in the Tacaribe and Machupo antisera that were tested.     

Isolation and characterization of a canine rotavirus

Summary Canine rotavirus particles were visualized by direct electron microscopy in the feces from a clinically normal dog. The virus was subsequently propagated in cell cultures; it was chracterized and compared with rotaviruses from other species. Replication of the virus in cell culture was found to be less dependent upon trypsin than that of human, bovine and porcine rotaviruses. Reproducible, sharp-edged plaques of various sizes were produced by the canine rotavirus in an established cell line of fetal rhesus monkey kidney, MA104, under overlays of carboxymethyl cellulose or agarose. Intracytoplasmic inclusion bodies of different sizes and shapes were produced in infected MA104 cells. By plaque reduction neutralization assay, a two-way antigenic relationship was found between the canine (CU-1) and simian (rhesus MMU 18006 and SA-11) rotaviruses. The canine rotavirus had a one-way antigenic relationship with feline (Taka), bovine (NCDV), and porcine (OSU) rotaviruses.With 4 Figures     

Sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus.

A sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus was developed in Vero and MA 104 cell cultures. The tests were performed in semimicrotoiter trays containing 24 wells, 16 mm in diameter. Parainfluenza virus type 1 formed plaques in Vero and MA 104 cells only when trypsin was added to the overlay medium. Plaquing of parainfluenza virus type 1 was more sensitive and technically reproducible in MA 104 cells than in Vero cells. Parainfluenza virus types 2 and 3 and respiratory syncytial virus readily formed plaques in Vero cells. Plaques with all viruses were necrotic in character, except for plaques produced by parainfluenza virus type 3, which appeared red due to an increased uptake of neutral red by infected cells. Different conditions for plaquing of the four viruses had to be used to obtain plaques of suitable size. Antibody titers of commercially prepared guinea pig typing sera were 5- to 50-fold higher by the plaque neutralization test than by complement fixation. The addition of guinea pig immunoglobulin G antiglobulin to the serum-virus mixtures enhanced the conventional neutralization test 5- to 10-fold. The sensitivity and specificity of the plaque neutralization test was also determined with sera of marmosets experimentally infected with parainfluenza virus types 1 and 3. The generally low postinfection titers could be enhanced, on the average, 40-fold by using human immunoglobulin G antiglobulin in the neutralization test. A low degree of cross-reactivity was shown between parainfluenza virus types 1 and 3 both in the conventional neutralization test and in the anti-immunoglobulin enhanced neutralization test.     

Comparison of shell vials and conventional tubes seeded with rhabdomyosarcoma and MRC-5 cells for the rapid detection of herpes simplex virus.

Shell vials (SV) and conventional tubes (CT) were seeded with rhabdomyosarcoma (RD) and MRC-5 cells and inoculated with clinical specimens, and the systems were evaluated for the rapid diagnosis of herpes simplex virus (HSV) infections by detection of cytopathic effects (CPE) (for CT, for 7 days) and by using fluoresceinated monoclonal antibodies (for SV, 16 h postinoculation). Of 245 genital specimens (16 from males and 229 from females) 56 (23%) seeded with MRC-5 cells (14 type 1 and 42 type 2) and 55 (22%) seeded with RD cells were detected in CT; however, CPE were recognized in only 26 (46%) of the total HSV-positive cultures 1 day postinoculation. Forty-eight (86% sensitivity, MRC-5) and 46 (84% sensitivity, RD) HSV strains were detected immunologically in SV 16 h postinoculation. Early CPE in CT or fluorescent foci in SV were easier to detect in MRC-5 than in RD cell cultures. MRC-5 and RD cells were equally sensitive to infection with HSV. CT cell cultures were more sensitive than SV but less rapid for the detection of HSV infection (P less than 0.01). We recommend using SV for the rapid diagnosis of HSV infections, but in addition, CT must be inoculated with MRC-5 or RD to ensure maximum detection of this virus.     

Replication of white spot syndrome virus in ovarian primary cultures from the kuruma shrimp, Marsupenaeus japonicus

Propagation of white spot syndrome virus (WSSV) was investigated in primary ovarian cultures from the kuruma shrimp Marsupenaeus japonicus. A WSSV strain, purified by sucrose density gradient centrifugation, was inoculated into 10-day-old primary ovarian cultures. WSSV infection induced marked cytopathic effect (CPE) on primary ovarian cells. Initially, virus-infected cells began to shrink 72 h post-inoculation, followed by the rounding of most cells which detached finally from flask surface. Electron microscopic observations clearly showed that the replication of WSSV occurred in nuclei of ovarian cells. Immunoblot analysis with antibodies against the WSSV envelope protein VP28 provided the evidence that the levels of WSSV antigens in culture supernatant gradually increased during the period between 24 and 120 h after virus inoculation. The results suggest that the use of primary ovarian cultures of the kuruma shrimp will facilitate characterization of the WSSV infection.     

Loss of surface fibronectin after infection of cultured cells by HSV-1 and 2

Summary Fibronection is lost from the surface of HSV infected cells during cell rounding. In order to investigate also the fate of fibronection during the process of HSV-induced cell-fusion, BHK, Vero as well as primary or secondary rabbit kidney cells were infected with HSV-1 strains producing cell-fusion. By immunofluorescence and immunoelectron microscopy a considerable loss of fibronectin after HSV infection could be demonstrated leaving only irregular clumps of fibronectin containing virus particles on the cell surface. Decrease and disarrangement of fibronectin was similar during cell rounding and cell fusion. Loss of Fibronectin was closely connected with the two types of the cytopathic effect (CPE) and could not be prevented by protease inhibitors. The immediate-early protein 175K is essential for induction of CPE and loss of fibronectin. The damage to the cell membrane during HSV infection shows certain analogous mechanisms with events induced by Cytochalasin B and might be explained by the loss of hypothetical fibronectin receptors.With 7 Figures     

Isolation of human rotavirus subgroups 1 and 2 in cell culture.

One strain of human rotavirus subgroup 1 (KUN) and one strain of subgroup 2 (MO) were isolated with the MA104 cell line, a fetal rhesus monkey kidney cell line. Their subgroup specificities and RNA patterns were identical to those of rotaviruses present in stools before cultivation. Distinct cytopathic effects consisting of obscure cell boundaries, cell fusion, cell rounding, cell detachment, and lytic foci were recognized at passage 3 of MO and passage 6 of KUN. No differences in cytopathic changes were found between the two isolates.