Evaluation of techniques to demonstrate foot-and-mouth disease virus in bovine tongue epithelium: comparison of the sensitivity of cattle, mice, primary cell cultures, cryopreserved cell cultures and established cell lines

Tongue epithelia infected with each of the 7 serotypes of foot-and-mouth disease virus (FMDV) were used to evaluate in vivo and in vitro systems for the detection of FMDV. Cattle inoculated by the intradermal route in the tongue (IDL) and suckling mice inoculated intraperitoneally were compared for susceptibility to FMDV with freshly prepared bovine thyroid cell cultures; cultures from cryopreserved bovine thyroid, bone marrow, mammary gland, myocardium, tongue, ovary and kidney cells; cultures from cryopreserved embryonic ovine kidney, newborn ovine kidney, ovine testicle, bone marrow, and chloroid plexus cells; and the continuous porcine kidney cell lines MVPK-1 and S6. The mean titers determined for each serotype in each system were statistically compared. The FMDV titers obtained in freshly prepared bovine thyroid cell cultures and by cattle IDL inoculation were the highest and were statistically indistinguishable. The titers obtained by suckling mouse inoculation were significantly lower than the titers obtained in thyroid cultures for serotypes A, C, Asia 1, and SAT 3. The cattle IDL assay was significantly more sensitive than the mouse assay for serotype A. The cell cultures from the cryopreserved newborn ovine kidney and embryonic ovine kidney were significantly less susceptible to serotype Asia 1 when compared with the fresh bovine thyroid cultures, but not significantly different when compared with the cattle assay for all serotypes. Cryopreservation of bovine thyroid cells directly after trypsinization resulted in the loss of susceptibility to FMDV serotype SAT 2. The other cryopreserved cell culture systems exhibited no or minimal susceptibility to all 7 serotypes, or exhibited considerable inconsistency. The established cell lines MVPK-1 and S6 were not susceptible to serotype A, and were less sensitive to serotype C than other culture systems. Quality control of cell cultures used to evaluate field specimens for FMDV was critical. The cell cultures of cryopreserved ovine kidney cells provided the most practical diagnostic system.     

Susceptibility of a new fetal pig kidney cell line (MVPK-1) to foot-and-mouth disease virus.

The Mengeling-Vaughn Porcine Kidney (MVPK-1) cell line, derived in October, 1970, from fetal pig kidneys, is susceptible to all 7 types of foot-and-mouth disease (FMD) virus. A plaque assay was developed for FMD virus that depended on washing MVPK-1 cells in serum-free medium before infection and excluding serum from 0.6% gum tragacanth overlay during plaque formation. The numbers of plaques that formed on MVPK-1 cells by a representative strain of each FMD type were comparable with the numbers of those on primary bovine calf kidney (BK) cells. Virus passaged in BK cell cultures did not have to be adapted to the cell line to obtain these results. The cell line lost susceptibility rapidly at 37 C after confluency was reached but retained susceptibility if maintained at room temperature. The cell line has the potential of replacing BK cells for many diverse purposes.     

A continuous bovine kidney cell line for routine assays of foot-and-mouth disease virus

A continuous bovine kidney cell line, LF-BK, arose from primary bovine calf kidney cells that survived infection with a temperature-sensitive mutant of foot-and-mouth disease virus. No virus was recovered after the first passage. Cells of Passage 48 were inoculated into two steers which remained healthy and did not develop neutralizing antibodies to the virus. The karyotype of cells of the 53rd and 87th passages was similar and revealed that the cells were markedly transformed. The modal number of diploid chromosomes was 52 at both passage levels. LF-BK cells and primary bovine kidney cells were equally susceptible in plaque assays to each of the 7 types of foot-and-mouth disease virus. The cell line and primary bovine kidney cells were less susceptible than primary bovine thyroid cells to several subtypes of the virus in suspensions of tongue epithelium. The LF-BK continuous cell line is recommended for routine plaque assays or plaque neutralization tests as a substitute for primary bovine kidney cells.     

Sensitivity of primary cells immortalised by oncogene transfection for the detection and isolation of foot-and-mouth disease and swine vesicular disease viruses.

Primary cells derived from calf thyroid (CTY), calf kidney (CK) and piglet kidney (PK) were immortalised by oncogene transfection and their susceptibility to infection by foot-and-mouth disease (FMD) virus and swine vesicular disease (SVD) virus examined. Eighty-five immortalised cell lines (47 CTY, 20 CK and 18 PK) proved stable upon repeated cell culture passage and many supported the growth of FMD virus and several of the PK cell lines supported SVD virus. However, none of the immortalised lines exhibited either the degree of sensitivity or the specificity for all virus serotypes and strains as shown by primary CTY and IB-RS-2 cell cultures which are routinely employed for vesicular virus diagnosis.     

Selection of foot-and-mouth disease viruses by passage in bovine and swine kidney cell cultures

Two uncloned populations of foot-and-mouth disease virus, one pathogenic for adult mice and the other nonpathogenic, were passaged in cultures of primary bovine kidney (BK) cells and a line of pig kidney (MVPK) cells. Within 10 passages in MVPK cells, the nonpathogenic virus became pathogenic for adult mice, but similar passages of this virus in BK cells did not affect its pathogenicity. In contrast, passage of the pathogenic virus in MVPK cells resulted in a decrease in pathogenicity, but again passage in BK cells had no effect on this characteristics. Neither of the viruses changed in pathogenicity for infant mice during the four passages that were tested. The nonpathogenic virus passaged in BK cells was more infectious for BK than for MVPK cells, but after passage in MVPK cells, this virus was about equally infectious for the two types of cells. Infectivity of the pathogenic virus was relatively unchanged by passage in either type of cell. The parent nonpathogenic virus and the 10th BK cell passage of this virus were much more resistant to adsorption with homogenized mouse kidney than were the MVPK cell passages of nonpathogenic virus. The parent and passaged pathogenic viruses were readily adsorbed. The results demonstrated that passage of the two viruses in MVPK cells had a pronounced selective effect.     

Effect of Incubation Temperature and Serum Content in Agar Overlay on Plaque Production by Foot-and-Mouth Disease Virus

The numbers of plaques produced by foot-and-mouth disease virus in primary cultures of calf kidney cells conditioned to 24, 30, and 37 C were essentially the same if virus was absorbed at 37 C. Adsorption was as effective at 30 as at 37 C in cultures conditioned to the respective temperature, but 24 C was less effective under this condition. However, when the adsorption temperature for cultures conditioned to 37 C was decreased to 30 or 24 C, fewer plaques were produced than in cultures conditioned to and maintained at the lower temperatures during absorption. Comparison of the number of plaques produced in cultures overlaid with nutrient agar containing from 0.5 to 10% bovine serum revealed no difference in relation to serum concentration. However, plaque size was related directly to the concentration of serum in the overlay.     

A parapoxvirus isolated from nasal secretion of a calf with respiratory disease

A virus was isolated in cultures of calf kidney and calf testicle cells from the nasal secretion of a 4-week-old calf with acute respiratory disease. This virus, designated I-74, was identified by electron microscopy as a parapoxvirus.     

Detection of African malignant catarrhal fever virus antigens in cell cultures by immunofluorescence

A fluorescent antibody (FA) test for antigens of African bovine wildebeest-derived malignant catarrhal fever virus was developed. Serum from one of the few survivors of the experimental disease in steers was used to prepare the conjugate. Both a virulent and an attenuated strain of malignant catarrhal fever virus were used to infect bovine thyroid cell cultures. Cells infected with both strains were readily detected by FA staining as early as 24 h post infection, whereas cytopathic effect could be observed by bright-field microscopy only after days 5 or 6 post infection. Controls consisting of normal bovine thyroid cells or infected cells treated with conjugated normal globulins did not show autofluorescence. The reaction was blocked by treatment of infected cells with homologous positive antisera but not by treatment with normal bovine serum or antisera to foot-and-mouth disease, rinderpest, bovine virus diarrhea, Ibaraki, infectious bovine rhinotracheitis, or bovine herpes mammilitis viruses. Treated with African malgnant catarrhal fever virus conjugate did not react.     

Cell culture propagation of porcine rotavirus (reovirus-like agent).

Two isolates of porcine rotavirus (reovirus-like agent) were isolated and passaged in primary procine kidney cell cultures. Viral infectivity for cells was monitored by immunofluorescence because viral cytopathic effect was moderate. Successful passage of virus in cell culture required that viral suspensions obtained from infected cell cultures be treated with pancreatin prior to inoculation onto cell monolayers. Porcine rotavirus passage in cell culture also was accomplished, using trypsin treatments in lieu of pancreatin treatments. Porcine rotavirus passaged 10 times in cell culture infected gnotobiotic pigs and caused diarrhea. Gnotobiotic pigs that recovered from this infection were resistant to challenge exposure with porcine rotavirus but were susceptible to challenge exposure with transmissible gastroenteritis virus. As determined by immunofluorescent cross reactions, porcine rotavirus was found to be antigenically related to the human and bovine rotaviruses but not to reovirus type 3 or to transmissible gastroenteritis virus.     

A heteroploid permanent cell line originating from embryonic calf thyroid supporting the replication of all known bovine adenovirus serotypes

An embryonic calf thyroid cell culture was established as a permanent heteroploid cell line, which is now in its 150th subculture. It allowed replication of all nine bovine adenovirus serotypes at its 15th as well as its 60-150th passages. All viruses induced typical cytopathic effects. Yields obtained on the permanent calf thyroid line were, on average, 0.8 log10 lower than those obtained on primary calf testicle cells.     

Production of large quantities of interferon with calf testicular cells and leucocytes.

Interferon (IF) was induced in calf testicle monolayer cultures and leucocycte suspension cultures with Semliki forest virus and Newcastle disease virus to evaluate the potency of those cells for producing IF in large quantities for clinical experiments. Interferon activity was measured by an RNA-inhibition test using Semliki forest virus as challenge. To produce 10(6) units of IF, leucocytes from 67-5 litres of blood or 7-2 X 10(9) testicle cells were needed.     

A sensitive plaque assay for bovine rotavirus in cultures of the bovine cell line GBK

The Lincoln strain of bovine rotavirus was found to replicate with cytopathic effects in cultures of GBK cells, a stable cell line derived from bovine kidney, when the cultures were maintained in the presence of trypsin. The virus was readily passaged and the infected cells were shown to contain specific viral antigen by indirect immunofluorescent staining. The virus formed plaques in GBK cell monolayers, when trypsin was incorporated in the agar overlay medium. The plaque count increased about twofold when diethylaminoethyl dextran was further included in the overlay medium. Plaque assay in GBK cells was more sensitive than that in MA-104 cells previously reported by Matsuno et al. The specificity of plaques was confirmed by specific inhibition with antiserum against the Lincoln strain.     

Studies with Bovine Parainfluenza — 3 Virus in U.A.R. (Egypt)

A bovine strain of myxovirus parainfluenza-3 (MP3) virus, designated S virus, was isolated from lung tissue collected from cattle with respiratory illness in 1963. The virus agglutinates mammalian and avian erythrocytes, and is sensitive to ether, sodium desoxycholate and trypsin. It grows in primary calf kidney, buffalo kidney, dog kidney, camel kidney and MS cell cultures. The S virus forms well-defined plaques in buffalo and calf kidney cells on the 5th or 6th day after inoculation. Examination of cell cultures following inoculation with S virus revealed giant cell formation, and introcytoplasmic and intranuclear inclusions. At 37°C the virus titer dropped from 10^10.4 to 10^2.6 in 3 days. Virus was completely inactivated at 56°C within 15 minutes. Growth-curve studies in tissue culture monolayer cells revealed a latent period of 10 hours. The intracellular virus titer was slightly lower than that of extracellular virus. The isolate was identified as MP3 virus by serum neutralization and hemagglutination-inhibition tests. Antibodies (HI) to S virus were shown to be present in a significant proportion of Egyptian cattle. The epidemiological significance of MP3 (bovine strain) virus in U.A.R. is discussed.     

Detection of Border disease antigen in tissues of affected sheep and in cell cultures by immunofluorescence.

An account is presented of the distribution of fluorescence in cryostat sections of tissues from eight lambs with Border disease (BD). In young lambs fluorescence was observed in almost every organ, indicating a generalised infection with BD virus. Fluorescence was most prominent in the secretory glands of the alimentary and respiratory tracts, the basal cell layers of the epidermis and mucous membranes, and in the medullary rays of the kidneys. Abomasum, pancreas, kidneys, testicles and thyroid were most consistently affected. Although the number of fluorescing tissues decreased with age, viral antigen could still be detected in two sheep of 22 and 52 weeks old. Border disease viral antigen was demonstrated in cell cultures derived from the brain, kidney and testicle of six out of seven lambs despite the presence of neutralising antibody against bovine virus diarrhoea virus in three of them. The presence of the virus in the skin, the vascular walls and the endocrine system is discussed in relation to the aberrant development of fetal hair follicles, periarteritis and growth retardation respectively.     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange.

Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes.

Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.

     

Growth of Foot-and-Mouth Disease Virus in Dispersed Tissue Cells: I. Methods of Production

Methods are described for rapid and economical production of large quantities of foot-and-mouth disease virus in stationary cultures of trypsin-dispersed bovine kidney cells in a simple medium. Yields of between 10⁷ and 10⁸ plaque-forming units per milliliter were obtained from serum-free cultures containing approximately a million and a half viable trypsin-dispersed cells per milliliter.

Some of the advantages and disadvantages of these methods of virus production are discussed.

     

Epizootic diarrhoea of adult cattle associated with a coronavirus-like agent

An epizootic of acute diarrhoea of adult cattle occured in Japan during the winter of 1976 to 1977. A majority of adult cattle clinically diagnosed as having the disease, showed a significant rises in antibody titres to bovine coronavirus, whereas only a smal; minority showed serological evidence of recent infection with calf rotavirus, bovine adenovirus type 7, parainfluenza virus type 3, or bovine viral diarrhoea-mucosal disease virus. A coronavirus-like agent was detected by electron-microscopy in faecal material from a cow with diarrhoea, and was subsequently isolated in primary bovine kidney cell cultures.     

Use of in situ hybridization for the detection of foot-and-mouth disease virus in cell culture

Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.     

Susceptibility of bovine macrophage and tracheal-ring cultures to bovine viruses.

Cultures of macrophages initiated from peripheral blood monocytes and organ cultures of tracheal rings were tested for their susceptibility to bovine viruses. With several notable exceptions, viruses cytopathogenic for bovine embryonic lung cultures were cytopathogenic for macrophages. Although cowpox virus replicated in macrophages, pseudocowpox did not, and although pseudorabies virus replicated within macrophages, infectious bovine rhinotracheitis and DN-599 herpesviruses did not. Bluetongue virus established an interesting relationship with macrophages. Whereas bluetongue virus was initially cytopathogenic for macrophages, it lost its cytopathogenicity on repeated passage, although it was capable of continued replication in macrophages. When subsequently passaged onto bovine embryonic lung cultures, it regained its cytopathogenicity. Parainfluenza-3, bovine viral diarrhea, and infectious bovine rhinotracheitis viruses readily destroyed ciliary activity in tracheal-ring cultures, as contrasted with the inability of bovine respiratory syncytial virus to destroy ciliary activity, even though bovine respiratory syncytial virus was able to replicate within ciliated epithelial cells of tracheal rings.