Antibody and complement-mediated cytotoxicity for bovine parainfluenza-3 virus-infected cells

An antibody-complement-mediated cytotoxicity assay was employed to determine whether susceptible host cells infected with bovine parainfluenza-3 virus could be destroyed by the humoral immune mechanism. The cytotoxicity assay yielded 21.5% to 76.9% specific release of 51Cr with sera collected from calves 28 days after inoculation with the virus. A multiplicity of infection of 5, a postinfection period of 2 days, an antibody dilution of 1:10, and an incubation period of 12 hours with antibody and complement were found to be optimal for this assay.     

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen.     

Replication of swine and equine influenza viruses in canine kidney cells.

Both swine and equine influenza viruses were replicated in Madin-Darby canine kidney cells. Hemagglutinin and complement-fixing antigens were synthesized at 4 to 8 hours postinfection and reached maximal synthesis at 16 to 20 hours. Neuraminidase activity increased 9- to 11-fold; 50% of the total hemagglutinin antigen was made before the infective virus progeny was detected.     

Observations on recovery mechanisms from feline viral rhinotracheitis.

Experiments were designed to determine immunological mechanisms responsible for controlling dissemination of feline rhinotracheitis virus in feline cell cultures. Virus infected cells could be destroyed by three mechanisms--antibody and complement mediated lysis, direct lymphocyte cytotoxicity and antibody dependent cell-mediated cytotoxicity. This latter immune parameter was mediated by both lymphocytes and macrophages and varied in extent in different cats. To ascertain the potential importance of the immunological parameters in curtailing viral spread, the time when virus infected cells could be destroyed by each component was related to the chronological events of viral replication and dissemination. Intracellular infectious virus and intracellular spread occurred at six to seven hours postinfection and extracellular spread at nine to ten hours postinfection. Antibody complement lysis and antibody dependent cell-mediated cytotoxicity occurred at six hours postinfection and direct cytotoxicity at eight hours postinfection. The relevance that these findings might have in relation to the occurrence and frequency of recrudescent disease is discussed.     

Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus

Unsuckled specific pathogen free calves were inoculated at 3-4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responses, in most calves, were detected in fecal and nasal speciments at 7-10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10-14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14-21 DPE.     

Duration of protection of calves against rhinovirus challenge exposure by infectious bovine rhinotracheitis virus-induced interferon in nasal secretions

Six calves inoculated intranasally with a vaccinal strain of infectious bovine rhinotracheitis (IBR) virus and 6 control calves were given a placebo. All calves were subsequently challenge exposed (by aerosol) with rhinovirus--3 of the calves from each group at 2 days after they were inoculated with IBR virus or with placebo and the remaining calves at 6 days. Nasal excretion of viruses, interferon (IFN) concentrations in nasal secretions (NS), and neutralizing antibody in sera and NS were determined. All calves given the vaccinal IBR virus subsequently had IFN in their NS. Interferon was detected as early as 1 day, reached maximal titers at 2 to 4 days, and persisted in individual calves for 5 to 10 days after inoculation. Rhinovirus shedding was not detected from IBR virus-inoculated calves whose NS contained both rhinovirus antibody and IFN at the time of challenge exposure; such calves were protected at either 2 or 6 days after IBR virus inoculation. The outcome of rhinovirus challenge exposure of calves whose NS contained IFN, but not rhinovirus antibody, varied with the day of challenge exposure. Rhinovirus excretion was detected from 2 of these calves challenge exposed 2 days after IBR virus inoculation, but was not detected from a calf challenge exposed 6 days after inoculation. However, while IFN was present in NS from the former 2 calves, rhinovirus shedding was markedly reduced as compared with that from control calves without IFN or NS antibody at the time of challenge exposure. Consistent relationship was not observed between the rhinovirus neutralizing antibody titer of calves' sera and NS. The antibody titer of NS more closely correlated with protective immunity to rhinovirus infection than did the serum antibody titer.     

Propagation of bovine coronavirus in clones of the Caco-2 cell line showing different levels of alkaline phosphatase activity

The aim of the present study was to determine whether bovine coronavirus (BCV) has the ability to initiate infection in a human colon carcinoma cell line, Caco‐2, that has been established to spontaneously differentiate after confluence. When Caco‐2 cells were infected with BCV, a titer of 5.5 × 10⁶ plaque‐forming units (p.f.u.)/mL was found in the culture supernatant at 5 days postinfection. Two clones, Caco‐2/CA1 and Caco‐2/CA2, were then isolated by monitoring alkaline phosphatase (ALP) and cell proliferation activities. The ALP activity level of CA1 cells was significantly higher than that of CA2 cells, while the level of cell proliferation activity of CA1 was significantly lower than that of CA2. When CA1 and CA2 cells were infected with BCV at confluence, virus hemagglutination (HA) was detected in the culture supernatant at 5 days postinfection for CA1 cells and at 8 days postinfection for CA2 cells. Thus, BCV propagation was substantially delayed in CA2 cells, suggesting that a cellular factor(s) that appears at the differentiation stage may control BCV propagation. BCV‐susceptible CA1 and CA2 cells showing different levels of ALP activity would be useful for further experiments to elucidate the mechanism of BCV propagation.     

Plaque assay and propagation in rat cell line LBC cells of rat coronavirus and 5 strains of sialodacryoadenitis virus

Various factors influencing the plaque formation of rat coronavirus (RCV) and sialodacryoadenitis virus (SDAV) in LBC cell monolayers were studied to develop the practical method for plaque assay. By this method, 4 Japanese isolates of SDAV also produced clear plaques. In one-step growth experiments of these viruses, newly formed virus was first recognized within 7.5 h postinfection and showed subsequently a rapid exponential increase. The virus was released rapidly from the infected cells. By indirect immunofluorescence virus specific antigen was detected as perinuclear granules in the cytoplasm of the cells at 5-6 h postinfection, and all the cells revealed fluorescence at 12 h postinfection.     

Detection of bovine herpesvirus-1 in peripheral blood mononuclear cells eight months postinfection.

Peripheral blood mononuclear cells (PBMCs) from 5 calves (3 controls and 2 vaccinates) used in a bovine herpesvirus 1 (BHV-1) vaccine study with a BHV-1 Cooper strain challenge were collected 6 months after challenge. The PBMCs from the control animals were positive by immunofluorescence for the BHV-1 glycoprotein D (gD) while the vaccinates were negative. The PBMC samples from 4 of the 5 animals were examined for BHV-1 DNA by polymerase chain reaction (PCR) and for gD immunofluorescence at 8 months after challenge. The BHV-1 DNA and viral antigen were detected in PBMC samples at 8 months postinfection, but no virus was isolated.     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.     

Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.     

Propagation of infectious bovine rhinotracheitis (bovine herpes-1) virus in murine primary cell cultures

Virus synthesis in BALB/C mouse lung and kidney primary cultures infected with infectious bovine rhinotracheitis (IBR) virus started between 6 and 8 hours after virus inoculation and reached a maximum titer of 5.5 log₁₀ plaque forming units (PFU) at 48 hours postinfection (PI). Cytopathic effect (CPE) in cell cultures occurred at 8–10 hours and over 90% of the cells had CPE by 48 to 72 hours PI. The bulk of the newly replicated virus (60–80%) was cell-associated as determined by plaque assay of extracellular and intracellular virus. Pulse-chase experiments demonstrated incorporation of radioactive precursors into viral DNA and protein macromolecules. Viral DNA synthesis was initiated between 2 and 4 hours PI and was maximum at 4 to 6 hours. Viral proteins were detected at 4 hours and peaked between 6 and 8 hours PI. Enzyme-linked immunosorbant assay (ELISA) confirmed synthesis of specific viral proteins, which gradually increased during the virus growth cycle. Abstract in French is given at the end of the article.

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Resume La synthèse du virus de la rhinotrachéite infectieuse bovine (virus IBR) sur des cultures primaires de poumon et de reins de souris (souche Balb/C) commençait entre 6 et 8 heures après inoculation du virus et atteignait un titre maximal de 5.5 log PFU (Plaque Forming Unit) 48 heures après infection. L'effet cytopathogenic (CPE) sur ces cultures cellulaires apparaissaient en 8 à 10 heures et plus de 90% des cellules montraient un CPE en 48 à 72 heures après infection. La détermination par la méthode des plages sous agarose, du virus extracellulaire et du virus intracellulaire, indiquait que 60–80% du virus synthétisé était associé avec les cellules. Les expériences de pulsechase demontraient l'incorporation des précurseurs radioactifs dans les protèines et ADN viraux. La synthèse virale d'ADN était initiée entre 2 et 4 heures après infection et était maximale en 4 à 6 heures. Les protéines virales étaient détectées en 4 heures avec un maximum de synthèse entre 6 et 8 heures après infection. Enzyme-linked immunosorbant assay (ELISA) confirmait la synthèse de protéines virales spécifiques qui montrait une augmentation graduelle pendant le cycle de replication du virus.

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Supported in part by grants 0831 from the Cooperative State Research Service of the US Department of Agriculture and published as contribution 85-160-J, Kansas Agricultural Experiment Station.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of an immunofluorescent antibody test to detect bovine herpesvirus 1-specific IgM.

An indirect immunofluorescent antibody test (IIFAT) was developed to detect bovine herpesvirus 1 (BHV-1)-specific IgM. All sera were treated with protein-G agarose prior to testing to eliminate the possibility of false-positive results due to IgM-isotype rheumatoid factor (IgM-RF). Specific IgM was first detected 8 days after experimental infection of 3 calves free of maternally derived antibody, with peak responses occurring 2-7 days later. Seroconversion was detected in all 3 calves using a single-dilution enzyme-linked immunosorbent assay. Following reinfection at 30 days postinfection, a low-level IgM response was detected in only 1 calf. Seroconversion was detected in 2 calves. There was no evidence of activation of IgM-RF by infection or reinfection with BHV-1. When 87 acute and convalescent serum pairs collected from 21 outbreaks of respiratory disease were tested, specific IgM was detected in 58 animals (66.6%) from 19 (90.5%) outbreaks. Seroconversion was detected in 44 of these animals (50.6%) from 17 outbreaks (81.0%). The correlations between these 2 assays on a calf and outbreak basis were 79.3% and 90.5%, respectively. Specific IgM was detected in 17/20 sera (85.0%) collected from an additional outbreak. No virus was detected by virus isolation or immunofluorescent staining in nasal mucus samples collected at the same time. Detection of specific IgM by IIFAT is a useful technique for the serodiagnosis of BHV-1 infection.     

Cell-mediated cytotoxicity of peripheral blood mononuclear cells stimulated in vitro for infectious bovine rhinotracheitis virus-infected cells

Peripheral blood mononuclear cells (PBMC) from calves infected with and hyperimmunized to infectious bovine rhinotracheitis virus (IBRV) were stimulated in vitro with viral antigens to evaluate their cytotoxicity for a variety of cells. The 51-Cr release assay was used to measure cytotoxicity. Cytotoxicity was not present in fresh nonstimulated cells, but was detected in cultured, IBRV-stimulated cells at day 3, was maximal at day 7, and declined thereafter. PBMC stimulated in vitro with IBRV expressed a preference for killing IBRV-infected cells compared to pseudorabies virus (PRV)-infected cells. IBRV-infected, but not PRV-infected, cold target cells inhibited lysis of IBRV-labeled target cells. High concentrations of IBRV hyperimmune serum partially blocked cytotoxicity. Cells expressing a viral preference for cytotoxicity showed no preference for lysis of autologous compared to heterologous bovine cells. PBMC from calves that were either IBRV-immune or not immune were cultured without IBRV stimulation and had similar levels of cytotoxicity for IBRV-infected cells as cells from IBRV-infected cattle.     

Diagnosis of bovine viral diarrhea virus infection using monoclonal antibodies

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.     

Standardization and kinetics of in vitro bovine blood lymphocyte stimulation with bovine rotavirus

Two groups of 3-month old calves were immunized intramuscularly with attenuated bovine rotavirus and boosted 21 and 42 days later. The first group of three calves were vaccinated with live virus emulsified with incomplete Freund's adjuvant (IFA) and the second group was immunized with live virus suspended in phosphate buffered saline (PBS). Three other calves, serving as controls, were inoculated with PBS emulsified with IFA. The specific cell-mediated and antibody responses of the animals were studied. Preliminary analysis of in vitro peripheral blood lymphocyte transformation to bovine rotavirus determined optimal conditions as: 96 h culture period, 5 X 10(5) cells per culture in RPMI 1640 medium containing 10% heat-inactivated bovine fetal serum and the use of inactivated virus in the cell culture at a concentration of 5 X 10(6) median tissue culture infective dose before inactivation. Specific blastic stimulation was observed on calves immunized with the rotavirus emulsified with IFA after the second and third vaccine inoculation with stimulation index values varying from 2.00 to 5.73. Serum neutralizing antibody titers of 1/25,600 were also induced in the same calves. Calves immunized with rotavirus-PBS suspension developed a mean antibody titer of 1/1,600, but showed no specific lymphocyte stimulation. No increase in specific immune responses was detected in the control animals.     

Bovine respiratory syncytial virus infection of bovine embryonic lung cultures: a kinetic study by the fluorescent antibody technique.

Development of fluorescence in bovine embryonic lung cells infected with bovine respiratory syncytial virus (BRSV) was studied by the fluorescent antibody (FA) test. Similar patterns of fluorescence were seen with the direct FA test, in which the immunoglobulin G fraction of antiserum to BRSV was conjugated with fluorescein isothiocyanate and used; and the indirect test, in which antiserum to the Long strain of respiratory syncytial virus and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G were used. In different trials, fluorescence was first detected between 16 and 18 hours after inoculation with BRSV. Fluorescence always was confined to the cytoplasm. Before 24 hours, fluorescence consisted of fine fibrils, usually parallel to the long axis of the cell, and cytoplasmic granules. After 24 hours, coincident with rounding of the cells, fluorescence slowly moved to the periphery of the cytoplasm. Under the growth conditions used, syncytia did not develop. By the FA test and as determined by the release of BRSV into the supernatant fluid, the minimal time for a single cycle of infection was between 24 and 26 hours.     

Detection of Bovine herpesvirus 4 DNA in aborted bovine fetuses

The presence of Bovine herpesvirus 4 (BoHV-4) was investigated by several methods in 24 aborted bovine fetuses. Polymerase chain reaction (PCR) and in situ DNA hybridization proved the presence of BoHV-4 DNA in 7 (29%) of the fetuses. The BoHV-4 genome was detected in the cytoplasm of splenic lymphocytes and monocytes, and sometimes in renal tubular epithelial cells or hepatic Kupffer cells, in all 7 PCR-positive fetuses. However, BoHV-4-specific monoclonal antibody failed to detect viral antigen in the formalin-fixed, paraffin-embedded tissue samples. No bacterial pathogens were found in the tissues of the BoHV-4-positive fetuses. Fungi were detected in 1 sample, and antibody to bovine viral diarrhea virus was detected in another. These results indicate that BoHV-4 could play a role in reproductive disorders of cattle, including abortion.     

Characterization of the bovine secondary in vitro antibody response

In the present report, the bovine secondary in vitro antibody response to keyhole limpet hemocyanin is described. The induction of anti-KLH antibody was not dependent upon the presence of mitogen but was antigen specific (KLH vs ovalbumin). Furthermore, this response was dependent upon cell density (10(6) per well), antigen dose (1 to 10(-5) ug/culture) and time in culture (5 days). The antibody produced was specific for KLH as measured in several binding assays. An unresponsive state was detected with high concentrations of KLH (more than 10 ug per culture) which was not due to the formation of immune complexes but to the inactivation of B and/or T cells. The induction of the antibody response was dependent on the presence of macrophages (syngeneic or allogeneic) and their presence could not be replaced with 2-mercaptoethanol.