Factors affecting the assay of bovine type I interferon on bovine embryonic lung cells

One preparation of interferon (IF) on 7-day-old bovine embryonic lung cultures free of bovine viral diarrhea virus had titers ranging from 20 to 10,240 in plaque-reduction tests, using bovine vesicular stomatitis virus. Factors believed to contribute to this variation were investigated. Although titers differed on different cell strains, different passages, and on cultures of different ages, variations between the two assays definitely could not be established as a function of these factors. However, IF titers on low passage cultures were usually lower than were titers on subsequent passages of the same cell strain. Calf serum in the diluent for IF reduced the titer one or two twofold dilutions. Over the range of 7 to 24 hours contact of IF with bovine embryonic lung cultures, and titer decreased steadily and was one twofold dilution less at 24 hours than it was at 7 hours. Latent viruses were not found in cultures by electron microscopy. Treatment of cultures with a noncytopathogenic strain of bovine viral diarrhea virus almost eliminated the effect of IF. Naturally occurring production of IF was not a major problem. While IF was on the cells, the pH of IF had a pronounced effect on the titer and may have been responsible for the vatiation observed with different passages, different cell strains, use of cultures of different ages, and use of calf serum in the diluent. Interferon at a low pH had a titer three or four twofold dilutions less than did the IF at a high pH.     

Minimal interferon induction in dogs, using a modified polyriboinosinic-polyribocytidylic acid complex

Adult Beagles failed to respond to high concentrations of interferon (IF) when they were injected with a nuclease-resistant complex poly I:C with poly-L-lysine and carboxymethylcellulose (PICLC), by the IV or intrathecal route. An IV dose of 1 mg of PICLC/kg of body weight was lethal to 1 of 3 adult dogs, but induced IF in only 2 dogs. Smaller doses were less toxic, but also were less effective. The injection of a high dose of a known IF inducer (3 X 10(8) egg LD50 of Newcastle disease virus) also failed to induce IF in Beagles. Interferon could not be induced in vitro when primary cultures of neonatal dog lung or kidney were treated with cultures of neonatal dog lung or kidney were treated with PICLC. When these primary cell cultures were compared with the cell line Madin-Darby canine kidney in an IF assay, no difference in sensitivity to IF-induced protection from infection with vesicular stomatitis virus could be shown. This indicated that the sensitivity of the Madin-Darby cell line was not the only factor in determining the lack of IF response in dogs and indicates that the dogs are poor responders to IF induction.     

Cultivation and partial characterization of bovine astrovirus

Bovine astrovirus serotype 2 (US2) was adapted to primary neonatal kidney cell (NBK) cultures by the addition of 50 micrograms ml-1 of trypsin in the medium. Infectious virus was released from the cells within 7 days post-infection in early passages and within 3 days in later passages. In the absence of trypsin, neither passage of infected cells nor release of infectious virus occurred. The virus was shown to be similar to the fecal astrovirus by a neutralization test and by ultrastructural studies of infected cells. Primary embryo bovine kidney (EBK) and NBK cell cultures supported infection with both fecal and tissue culture adapted (TCA) astrovirus. The time-related development of infection, as studied by immunofluorescence, was similar for both fecal and TCA astrovirus and for both cell culture types. The first indication of viral infection and expression of viral antigens occurred at 7 h post-infection and was characterized by the appearance of a diffuse faint immunofluorescence (IF) of the cytoplasm. Soon after, two or three brilliant IF granules were observed in the nucleus, which appeared to involve the nucleoli. Subsequently, dense granular IF was seen in the perinuclear region of the cytoplasm, which later extended to involve all the cytoplasmic area. In both EBK and NBK cultures infected with either fecal or tissue culture adapted astrovirus, only a minority of cells became infected, even when the multiplicity of infection exceeded one. Occasionally 10-20% of cells were infected, but in most cultures the proportion did not exceed 2% and in NBK cultures, from 3/9 calves, no infected cells were observed. The virus did not infect bovine cell lines. Infectivity of the virus was not removed by treatment with chloroform, and iododeoxyuridine and actinomycin D when added to the medium, did not block replication. Masses of virions were observed by electron microscopy in discrete areas in the cytoplasm, with similar distributions as the viral antigen foci as seen by IF. The mean diameter of the virions was 34 nm. In conclusion, bovine astrovirus lacks both essential lipids and an envelope, probably has an RNA genome, may have a nuclear phase of replication involving the nucleoli which is not blocked by DNA inhibitors, and has a selective cell tropism.     

Sensitivity of seven different types of cell cultures to three serotypes of foot-and-mouth disease virus.

The ability of bovine tongue origin foot-and-mouth disease virus serotypes A, O and C to replicate in seven different types of cell cultures was studied. Primary and secondary calf thyroid cells were equivalent in susceptibility to bovine kidney cell cultures passaged up to five times. Calf thyroid cells lost their susceptibility after two passages. Cryopreserved bovine kidney cell cultures passaged three and four times were equivalent in susceptibility to sensitive calf thyroid and bovine kidney cells. Susceptibility to foot-and-mouth disease virus serotype C was most variable among the cells tested. Lamb testicle and porcine kidney cells were susceptible to foot-and-mouth disease virus while goat and calf testicle and calf lung cells were refractory.     

Interferon induction in bovine and feline monolayer cultures by four bluetongue virus serotypes.

The interferon inducing ability of bluetongue viruses was studied in bovine and feline monolayer cultures inoculated with each of four bluetongue virus serotypes. Interferon was assayed by a plaque reduction method in monolayer cultures with vesicular stomatitis virus as challenge virus. Interferon was produced by bovine turbinate, Georgia bovine kidney, and Crandell feline kidney monolayer cultures in response to bluetongue virus serotypes 10, 11, 13 and 17. The antiviral substances produced by the bluetongue virus infected cultures had properties of interferon.     

Comparative susceptibility of Marek's disease cell lines to chicken infectious anemia virus

Chicken infectious anemia virus (CIAV) is known to infect and replicate in various Marek's disease chicken cell lines (MDCCs) derived from Marek's disease (MD) tumors. One line, MDCC-MSB1, has been the substrate used in most studies. We compared a total of 26 MDCCs, including two sublines of MDCC-MSB1, MSB1 (L) and MSB1 (S), four other MD tumor-derived lines, and 20 lines derived from MD virus-induced local lesions, for susceptibility to the Cux-1 and CIA-1 strains of CIAV. The cell lines represented six phenotypic groups of T cells based on the expression of CD4, CD8, and TCR-2 and -3 surface markers. Susceptibility was measured by the number of cells positive for viral antigen in immunofluorescence (IF) tests at 3-10 days postinfection. No clear-cut differences were found in susceptibility related to phenotype, although CD4-/8+ lines and CD4-/8- lines might be more susceptible than CD4+/8- lines. However, several individual lines were more susceptible to Cux-1 than the two MSB1 sublines tested. Contrary to an earlier report, cells of MDCC-CU147, a CD8+, TCR3+, local-lesion derived line, were found to be susceptible to CIA-1. In fact, CU147 was distinguished by very high susceptibility to both CIAV strains. In direct comparisons with MSB1, CU147 detected approximately 10-fold lower doses of virus. Also, virus spread was faster (P < 0.05) in CU147 than in MSB1 and other lines. Results from polymerase chain reaction (PCR) tests to detect infection in titrations were in general agreement with IF test results although PCR detected infection in a few terminal dilution cultures that were negative by IF.     

Factors affecting the production of bovine type I interferon on bovine embryonic lung cells by polyriboinosinic-polyribocytidylic acid

Variables believed to affect the amount of interferon (IF) produced in bovine embryonic lung cell cultures by polyriboinosinic-polyribocytidylic acid (poly (rI:rC)) were investigated. A concentration of 100 micron of poly (rI:rC)/ml consistently induced substantial amounts of IF. Bovine viral diarrhea virus infection of cultures did not interfere with induction of IF by poly (rI:rC). Neither the age of cells nor the passage level of the cultures seemed to affect the amount of IF produced. Priming of cultures with large concentrations of IF before treatment with poly (rI:rC) was more effective than priming with small concentrations of IF. Furthermore, priming for 24 hours was more effective than was priming for 12 hours. The use of 2% bovine fetal serum in the medium, subsequent to treatment with poly (rI:rC) markely enhanced IF production. Practically all IF was produced within a few hours after treatment with poly (rI:rC).     

In vitro interferon production by bovine tissues: induction with infectious bovine rhinotracheitis virus.

The interferon-inducing ability of infectious bovine rhinotracheitis (IBR) virus was determined in tissue cultures of bovine origin inoculated with untreated and ultraviolet (UV) irradiated IBR viruses. Interferon was assayed by the plaque-reduction method in bovine fetal kidney (BFK) cell cultures, using vesicular stomatitis virus as challenge virus. Highest interferon concentrations were produced by cultures of bovine fetal (BF) spleen cells and aveolar macrophage cultures derived from adult cattle. Moderate interferon concentrations were produced by peripheral blood leukocyte (PBL) suspension cultures from adult cattle with serum-neutralizing antibodies against IBR virus. Cultures of PBL from 1 cow without detectable serum-neutralizing antibodies against IBR virus did not produce detectable interferon in response to IBR virus. Cultures of PBL from cattle with or without detectable serum-neutralizing antibodies against IBR virus produced interferon when stimulated with phytohemagglutinin (PHA). Low levles of viral inhibitors were detected infrequently in monolayer cultures of BFK and BF nasal mucosa inoculated with UV-irradiated IBR virus and in BF tracheal organ cultures inoculated with untreated IBR virus. Interferon was not detected in fluids collected from IBR virus-exposed monolayer cultures of primary and secondary BF lung, secondary BF tracheal mucosa, secondary BF liver, secondary BF adrenal, and PBL in the 4th and 7th passages. The antiviral inhibitors from BF spleen, bovine alveolar macrophage, and PBL cultures induced with IBR virus, as well as inhibitors from PBL cultures induced with PHA, had the usual properties of interferon.     

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.     

Induction of chicken interferon by avian infectious bronchitis virus.

The ability of nine strains of avian infectious bronchitis virus (IBV) to induce chicken interferon has been investigated using Semliki Forest virus for the tests. The Beaudette, H120 and Connecticut 46 strains induced interferon in the allantoic fluids of embryonated hens' eggs, the highest titre (1 : 30) being associated with Beaudette; but these as well as the Massachusetts M-41 and H52 strains failed to yield interferon in primary monolayer cultures of chick kidney cells as did all nine strains in organ cultures of chick embryo trachea. None of six strains of IBV investigated was susceptible to the inhibitory effects of chicken interferon.     

马流感病毒血凝素基因甲病毒复制子重组表达质粒的构建及其免疫效力评价

为评价马流感病毒(EIV)HA基因核酸免疫效果,本研究以甲病毒复制子载体pSFV1CS分别构建了表达EIV H3N8亚型的美洲型和欧洲型HA基因的重组真核表达质粒。并将其转染293T细胞,经间接免疫荧光鉴定表明HA基因获得表达;以重组质粒免疫的BALB/c鼠能够检测到特异性抗体产生,而且HI抗体水平持续升高,同时小鼠体内IFN-γ、IL-4分泌水平也有所升高。攻毒后小鼠表现轻度临床症状,但病毒分离和RT-PCR均未检测到病毒。上述结果表明,该重组质粒pSFV1CS-EIV-HA具有良好的免疫原性并且可以诱导免疫动物产生较高免疫应答的能力。     

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.

     

Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus.

Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.     

Sheep erythrocyte and bluetongue virus antibody responses of spleen cell cultures from mice.

The optimum conditions for the culture of cells from dissociated spleens were determined. Routinely, 10(7) cells were seeded per ml of RPMI 1640 medium supplemented with 20% pre-tested foetal calf serum. For the assay of the immune response, cultures were supplemented with 30 muMolar mercaptoethanol. The immune responses to sheep erythrocyte and bluetongue virus antigens were determined by the haemolytic plaque-forming cell assays described by Oellermann (1974) and Oellermann, Carter & Marx (1976a). The optimum sheep erythrocyte antigen concentration was 2 X 10(6) erythrocytes per 10(7) spleen cells and maximum IgM plaque-forming cells were detected after 4 days in culture. Successful stimulation of the immune response to bluetongue virus was achieved in spleen cell cultures from mice previously primed with bluetongue virus. The optimum antigen concentration was 30-40 ng bluetongue virus per 10(7) spleen cells and the maximum plaque-forming cell response was observed after 4 days in culture.     

Temporal relationships of viremia, interferon activity, and antibody responses of sheep infected with several bluetongue virus strains.

Sheep had viremias that were first detected on day 3 (+/- 1) after infection with several strains of bluetongue virus (BTV) representing United States serotypes 10, 11, 13, and 17. Diphasic peaks of infectivity were attained on days 6 and 10 (+/- 2). Interferon (IFN) was first detected in serum samples on day 5 (+/- 1), and reached greatest concentrations on day 6 (+/- 2), which coincided with the first viremic peak; IFN concentrations then decreased toward zero by day 10 (+/- 2). Interferon peak concentrations induced approximately a 90% decrease in virus titer. The decrease in IFN concentrations by day 9 (+/- 2) corresponded with the second viremic peak on day 10 (+/- 2). Onset of the decrease in detectable concentrations of virus after the second peak of viremia corresponded to the initial detection of serum antibody to BTV by day 10 (+/- 2). Virus titer decreased and antibody production increased until approximately days 21 to 28, when the titers plateaued and virus was not detected. Febrile responses peaked on day 7 (+/- 1) during the peak viremic period. The WBC count was depressed at the time the virus titer increased, but returned to normal values while the sheep were still viremic. Diphasic viremias in BTV-infected sheep were attributed to induction of high concentrations of IFN concurrent with the first virus titer peak, followed by production of antibody to specific BTV strains and a subsequent reduction in viremia at the second virus titer peak.     

Expression of the mouse Fc receptor B2 in bovine cells rescues the infectivity of conditionally neutralized bovine viral diarrhea virus

We examined the infectivity of bovine viral diarrhea virus (BVDV) particles opsonized with monoclonal antibodies on bovine cells expressing the murine Fcgamma receptor B2 (FcgammaRB2). Incubation of BVDV with each of five monoclonal antibodies (Mabs) to the envelope glycoprotein E2 led to efficient virus-neutralization, as evidenced by the failure to infect standard bovine testicle cells. In contrast, inoculation of four of these Mab-virus complexes onto transfectant bovine testicle cells expressing FcgammaRB2 resulted in a significant rescue of virus infectivity. Mab-virus complexes were 13.1, 7.37, 5.56 and 4.49 times more infectious for FcgammaR-expressing cells than for cells lacking FcgammaR. Because Mab-opsonized BVDV virion complexes uninfectious for standard cells may initiate productive infection in cells expressing the FcgammaR, the virion-Mab interaction should be described as a conditional neutralization. Interestingly, the infectivity of BVDV complexed with a specific virus neutralizing Mab (10f9) could not be rescued in FcgammaRB2-expressing cells. We postulate that attachment of antibody-virus complexes to FcR may only result in productive infection if the binding of antibody to virions does not interfere with post-attachment entry functions. Conditionally neutralized virions may play a role in the pathogenesis of any of the multiple diseases resulting from BVDV infections in cattle.     

Inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon

Replication of feline infectious peritonitis virus (FIPV) in feline cell cultures was inhibited after incubation of cells with either human recombinant leukocyte (alpha) interferon (IFN) or feline fibroblastic (beta) IFN for 18 to 24 hours before viral challenge exposure. Compared with virus control cultures, FIPV yields were reduced by ranges of 0.1 to 2.7 log10 or 2 to 5.2 log10 TCID50 in cultures treated with human alpha- or feline beta-IFN, respectively; yield reductions were IFN dose dependent. Sensitivity to the antiviral activities of IFN varied with cell type; feline embryo cells had greater FIPV yield reductions than did similarly treated feline kidney or feline lung cells. Comparison of the virus growth curves in IFN-treated and virus control cultures indicated marked reduction in intracellular and extracellular FIPV in IFN-treated cultures. Compared with virus control cultures, intracellular and extracellular infectivity in IFN-treated cultures was delayed in onset by 12 and 30 hours, respectively, and FIPV titers subsequently were reduced by 3 to 3.5 and 5 log10 TCID50, respectively. Frequently, immunofluorescent and electron microscopy of IFN-treated cells or cell culture fluids did not reveal virus; however, even in cultures without viral cytopathic changes, small amounts of virus occasionally persisted in cells.     

In vitro interferon production by bovine tissues: effects of hydrocortisone.

The effect of hydrocortisone on bovine interferon production in vitro was studied. Infectious bovine rhinotracheitis virus was used as an inducer. Interferon was assayed by the plaque-reduction method in bovine fetal kidney cultures, using vesicular stomatitis virus as challenge virus. Hydrocortisone decreased interferon production in bovine fetal spleen and peripheral blood leukocyte cultures. Hydrocortisone did not decrease interferon production by bovine alveolar macrophages, in 1 experiment. Properties of viral inhibitors were those of interferon.     

Susceptibility of various ceil culture systems to pseudorabies virus

A comparative study was carried out to determine the susceptibility of five different cell lines to pseudorabies virus (PRV), a herpes virus of pigs. The cell systems tested were swine testicle (ST), mink lung (ML), equine dermal (ED), porcine kidney (PK₁₅), and bovine turbinate (BT) cells. Virus titers obtained were 10^4.88, 10^4.38, 10^3.75, 10^2.63, and 10^0.25 for ML, ST, PK₁₅, BT and ED cells, respectively indicating that ML, ST, and PK₁₅ are optimal cell lines for the growth of PRV whereas BT and ED are not very sensitive.     

Serological diagnosis of infection with the putative bovine leukemia virus.

We report on an accurate, rapid and inexpensive test for the identification of animals infected with the Bovine C-type virus (BLV). The test involves the detection of serum antibodies to BLV using the immunofluorescent (IF) technique on acetone-fixed, infected cells. The specificity of the test was demonstrated by the fact that virus was found by electron microscopy in 90% of cattle showing positive reactions. In contrast, virus was not found despite extensive examination in antibody negative animals. Thus, the presence of IF antibody is an accurate indicator of current rather than past BLV infection. In order for the IF test to be specific it is of critical importance that the target cells used are infected only with BLV. BLV antibodies can also be detected by the immunoprecipitation (Ouchterlony) technique. However, a significant proportion of BLV infected animals showing positive reactions in the IF test failed to show precipitin antibodies to the virus. Likewise, BLV infection was demonstrated by both the IF test and electron microscopy in many animals with persistently normal levels of blood lymphocytes. Thus, neither the precipitin test nor the blood lymphocyte count (Bendixen's key) can be used to rule out BLV infection.