Effect of mosquito salivary gland treatment on vesicular stomatitis New Jersey virus replication and interferon alpha/beta expression in vitro.

The sensitivity of vesicular stomatitis (VS) viruses to interferon (IFN)-mediated antiviral effects has been well documented. Previous studies in our laboratory have shown the ability of mosquito saliva to enhance vesicular stomatitis New Jersey (VSNJ) virus infection in mice. To investigate the effect of mosquito saliva on virus replication and IFN alpha/beta expression, virus titers were analyzed at various time points after infection in cells that were treated with mosquito salivary gland homogenate (SGH). Salivary gland treatment of mouse fibroblast cells (L929) resulted in a significant increase in virus growth kinetics compared with untreated controls. In contrast, Vero cells, which are deficient in the IFN alpha/beta response, did not yield increased viral titers in the time points examined. Treatment of L929 cells with an IFN alpha/beta neutralizing antibody also slightly increased virus yield. Ribonuclease protection assays revealed that induction of IFN alpha2 expression was reduced in L929 cells treated with SGH. Modulation of IFN alpha/beta by mosquito saliva may be a critical determinant of the transmission and pathogenesis of VSNJ virus.

Potentiation of vesicular stomatitis New Jersey virus infection in mice by mosquito saliva.

Saliva of arthropod vectors can modulate vertebrate host immunological functions in many ways. To investigate if vesicular stomatitis New Jersey virus (VSNJ) infection could be potentiated by arthropod saliva, mice in three different age groups (3 days, 3 weeks, or > 8 months) were exposed to VSNJ-infected mosquitoes or were needle injected with an equivalent dose of VSNJ (titre 1.5-3 logs). Previous studies have demonstrated that VS viruses do not replicate in mice older than 3 weeks of age. Infection was monitored by examining serum for the presence of VSNJ at 2 days postinfection (PI) or for neutralizing antibody on days 7 and 14 PI. All 3-day-old mice succumbed to viral infection by mosquito transmission or delivery by injection. Ninety-four percent of the 3-week-old mice bitten by infected mosquitoes developed antibody, whereas antibody was detected in only 13% of inoculated mice. Adult mice developed neutralizing antibody (73%) when fed upon by infected mosquitoes, but only 11% developed antibody when virus was injected. Day 2 serum samples from 3-week and adult age groups were negative by virus isolation. These data indicate that mosquito mediated delivery of VSNJ exacerbates virus infection in mice older than 3 weeks.

The effects of pharmacological and lentivirus-induced immune suppression on orbivirus pathogenesis: assessment of virus burden in blood monocytes and tissues by reverse transcription-in situ PCR.

We investigated the effects of pharmacological and lentivirus-induced immunosuppression on bluetongue virus (BTV) pathogenesis as a mechanism for virus persistence and induction of clinical disease. Immunologically normal and immunosuppressed sheep were infected subcutaneously with BTV serotype 3 (BTV-3), a foreign isolate with unknown pathogenicity in North American livestock, and with North American serotype 11 (BTV-11). Erythrocyte-associated BTV RNA was detected earlier and at greater concentrations in sheep treated with immunosuppressive drugs. Similarly, viral RNA and infectious virus were detected in blood monocytes earlier and at higher frequency in immunosuppressed animals: as many as 1 in 970 monocytes revealed BTV RNA at peak viremia, compared to <1 in 10(5) monocytes from immunocompetent sheep. Animals infected with BTV-3 had a higher virus burden in monocytes and lesions of greater severity than those infected with BTV-11. BTV RNA was detected by in situ hybridization in vascular endothelial cells and cells of monocyte lineage, but only in tissues from immunocompromised animals, and was most abundant in animals infected with BTV-3. In contrast, reverse transcription-in situ PCR showed BTV RNA from both viral serotypes in high numbers of tissue leukocytes and vascular endothelial cells from both immunosuppressed and, to a lesser extent, immunocompetent animals. Collectively, these findings show that BTV infection is widely distributed during acute infection but replication is highly restricted in animals with normal immunity. These findings also suggest that in addition to virulence factors that define viral serotypes, immunosuppression could play a role in the natural history of orbivirus infection, allowing for higher virus burden, increased virus persistence, and greater potential for acquisition of virus by the arthropod vector.

Variation in immune responsiveness of sheep to the antigens of intestinal nematodes and blowfly larvae.

The total and IgG1 antibody responses to the intestinal nematode parasites Haemonchus contortus and Trichostrongylus colubriformis were measured in the serum of 160 lambs, 4 months of age. These antibodies had developed as the result of natural exposure to the parasites on pasture. Three sires were examined and strong sire effects on half-sib progeny were found. Plotting of ELISA antibody results in two dimensions revealed clustering of responses within sire groups. Bimodal antibody distributions were also observed within sire groups and the whole population for T. colubriformis. A bimodal distribution of antibodies to H. contortus was found for one sire group but not for the whole population. The injection of blowfly larvae (Lucilia cuprina) extract into 42/160 lambs at a later age (12 months) was followed by increased antibodies to L. cuprina and an apparent increase in antibodies to T. colubriformis. A bimodal distribution for antibodies to L. cuprina was found in one sire group and in the whole population. These bimodal distributions of antibodies to L. cuprina did not coincide with the distribution of antibodies to T. colubriformis or H. contortus, measured on the same serum samples. It was concluded that high and low responder sire groups could be differentiated in lamb populations for all three parasites. These effects persisted during lamb maturation and appeared to be genetic effects. Finally, cross-reacting antibodies between L. cuprina and T. colubriformis appear to be stimulated by injection of L. cuprina antigens.

Ovine lentivirus expression and disease. Virus replication, but not entry, is restricted to macrophages of specific tissues.

To better define the relationship between lentivirus infection and lymphoproliferative or inflammatory disease, we studied postmortem specimens of 38 sheep naturally infected with ovine lentivirus (OvLV) and with different clinical manifestations of OvLV-associated disease. Immunohistochemistry, in situ hybridization, and virus isolation were used to localize viral protein, viral RNA, and infectious virus to specific cells and tissues. Viral protein or infectious virus was found in cells morphologically and histochemically compatible with macrophages (M phi s), but only in lung, bone marrow, mammary gland, lymph node, spleen, synovium, brain, and spinal cord, frequently in association with lymphocyte infiltrates. In contrast, viral RNA was found in a variety of cell types, including epithelium, M phi s, and M phi-like cells, and in a wider range of tissues, with or without OvLV-associated lesions. In summary, these findings suggest that in vivo: 1), OvLV can enter a variety of cell types, 2), productive infection is restricted to cells of M phi lineage, and 3), cells expressing viral proteins are limited to specific tissues, those associated with OvLV-induced diseases.

Monoclonal antibodies that distinguish between encephalitogenic bovine herpesvirus type 1.3 and respiratory bovine herpesvirus type 1.1.

Seven mouse hybridoma cell lines producing monoclonal antibodies (MAb) against an encephalitogenic strain of bovine herpesvirus type 1.3 (BHV-1.3) were established. The clones producing MAb were selected to be specific for BHV-1.3 by enzyme-linked immunosorbent assay. Only L1B neutralized virus without complement. With the addition of complement, five of the MAb neutralized BHV-1.3 but not the respiratory strain BHV-1.1. The anti-BHV-1.3-specific MAb Q10B, L6G, and L1B precipitated glycoproteins from BHV-1.3 that were analogous to the gI, GIII, and gIV glycoproteins of BHV-1.1, respectively. The other four MAb precipitated unknown proteins. None of the anti-BHV-1.3 MAb precipitated BHV-1.1 glycoproteins. The majority of the anti-BHV-1.3 MAb did not react with BHV-1.1 by immunoblotting, but O7E (unknown protein pattern by radioimmunoprecipitation) was reactive with five proteins (M(r)s of 33,000, 43,000, 70,000, 141,000, and 190,000) of BHV-1.3 and with a different pattern of proteins of BHV-1.1 (M(r)s of 30,000, 38,000, 83,000 and 144,000). Two of the MAb, L6G and O7E, conjugated with peroxidase were found to be useful for detecting BHV-1.3 antigen by immunochemistry in Formalin-fixed brain tissue from experimentally infected calves.

Genetically engineered multi-component virus-like particles as veterinary vaccines.

Multiprotein structures can be constructed to mimic virus particles. These engineered particles lack genetic material and are not infectious but they can elicit protective immune responses in animals against challenges with infectious viruses. As a prototype, insect cells were co-infected with two recombinant baculoviruses. One recombinant baculovirus contained an insert of L2 and M5 genes, which encode for outer capsid proteins VP2 and VP5, respectively, from bluetongue virus (BTV) downstream of duplicated copies of the baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) polyhedrin promoter. Another recombinant baculovirus expressed two major core proteins (VP3 and VP7) from BTV virions. The co-infected cells synthesized non-infectious, double-shelled, virus-like particles (VLP). The VLP resembled the authentic BTV in size, appearance, and biochemical constitution but they lacked the double-stranded-RNA genome and three minor polypeptides normally contained in the icosahedral inner capsid. The VLP consisted of an outer shell of VP2 and VP5 from US BTV-10 attached to an icosahedral framework formed by the two major core proteins VP3 and BTV-17 and VP7 from US BTV-10. Sheep immunized with VLP expressing BTV capsid proteins produced antibodies that neutralized homologous serotypes of BTV. The assembly of VLP from different proteins simultaneously expressed indicates the potential of this novel approach for producing safe and effective vaccines against several viral agents.

Host-virus interaction as defined by amplification of viral DNA and serology in lentivirus-infected sheep.

To correlate the presence of ovine lentivirus (OvLV) as detected by polymerase chain reaction (PCR) with detection of antibody, 42 sheep from a flock with enzootic OvLV infection were studied. The results of agar gel immunodiffusion (AGID), ELISA, and immunoblotting assays were compared, and leukocytes (blood, bone marrow, lymph node, and lung cells) were assessed for viral DNA by PCR using pol and LTR primers; amplified products were detected by specific DNA and RNA probes. Based on the number of animals that had detectable viral DNA, the specificities of AGID, ELISA, and immunoblotting were 77%, 92%, and 95 or 100% (depending on which criterion was used to interpret immunoblot results), respectively. Only in animals with OvLV-associated disease was OvLV DNA detected in leukocyte DNA prior to the amplification of virus in culture and only in this group was high titer antibody detected to the OvLV major surface (gp 105) and transmembrane (gp 55) antigens. Animals that were both antibody and PCR-negative lacked histopathologic evidence of disease. From this study there was no indication that OvLV infection without the development of antibody occurs, and detection of OvLV DNA in animals with weak or partial serological reactions likely indicates early OvLV infection rather than false-positive PCR results.

Analysis of ovine lentivirus infectivity and replication by using a focal immunoassay and an antigen-capture enzyme-linked immunosorbent assay.

A focal immunoassay and an antigen-capture enzyme-linked immunosorbent assay (antigen-capture ELISA) were developed to quantify infectious ovine lentivirus (OvLV) and OvLV capsid protein (CA) (p27), respectively. The in vitro kinetics of replication and cytopathogenicity of distinct biological clones of OvLV (rapid/high and slow/low phenotypic variants) were assessed. Both viruses were detected by focal immunoassay within 48 h postinfection, 2 days before syncytia were observed in goat synovial membrane cells infected with rapid/high OvLV and 4 days before they appeared in cultures infected with slow/low OvLV. CA was first detected by antigen-capture ELISA in supernatants of cells infected with rapid/high OvLV 4 days postinfection, and it reached a plateau by 10 days, 4 days after peak syncytium formation. In contrast, in cultures infected at the same multiplicity of infection with slow/low OvLV, CA was detected 8 days postinfection, and the titer gradually increased over the following 12 days while the number of syncytia gradually decreased. Peripheral blood mononuclear cells (PBMC) from seropositive sheep treated with phorbol 12-myristate 13-acetate (PMA) generally expressed CA earlier and at higher levels than PBMC treated with either phytohemagglutinin or concanavalin A. Serum CA levels above 3 ng/ml were found in 58% (18 of 31) of seropositive sheep. However, there was no correlation between PMA-induced CA expression and levels of antigenemia. Viral heterogeneity may account for variations both in CA expression in cultures of PBMC and in antigenemia, humoral immune response, and viral pathogenicity in infected animals.

Effects of virus load in the pathogenesis of lentivirus-induced lymphoid interstitial pneumonia.

To better define the relationship between ovine lentivirus (OvLV) infection and respiratory disease, pulmonary leukocytes and postmortem lung specimens from 42 sheep seropositive or at risk for OvLV infection were obtained. The lungs were examined for lesions of lymphoid interstitial pneumonia (LIP), and animals were categorized into five groups by severity of LIP and OvLV serologic status. The presence of OvLV in alveolar macrophages was established by proviral DNA amplification using the polymerase chain reaction (PCR), and the proportion of infected cells was determined by a quantitative focal immunoassay (FIA) and by immunohistochemistry. The concentration of OvLV p25 in serum was measured by capture ELISA. In contrast to animals with mild or no pulmonary lesions, sheep with moderate or severe LIP (17/42) were all seropositive, 71% had antigenemia (greater than 2 ng/mL), and 82% had proviral DNA in 1.5 x 10(5) alveolar macrophages. Of sheep positive by PCR, those with moderate or severe LIP (79%) had an average of 3 infected cells/10(3) alveolar macrophages by FIA. These results implicate alveolar macrophages as important target cells in the pathogenesis of OvLV-induced respiratory diseases.

Differentiation of parapoxviruses by application of orf virus-specific monoclonal antibodies against cell surface proteins.

Monoclonal antibodies were produced against orf virus-specified cell surface proteins in an attempt to develop reagents capable of differentiating between members of the Parapoxviridae. Two immunization protocols were used to induce an anti-orf response in BALB/c mice, one of which resulted in virus replication in the recipient. The monoclonal antibodies produced were tested for crossreactivity with bovine papular stomatitis virus (BPS) and milker's node virus (MNV) by indirect immunofluorescence assay (IFA) and immunoblotting. The results indicate that significant antigenic overlap exists between isolates of orf, MNV and BPS, even at the level of specificity provided by monoclonal antibodies. One monoclonal antibody reacted strongly in IFA with orf virus isolates, very weakly with MNV, and not at all with BPS. On immunoblots this same antibody recognized a 40-43 kDa protein in orf virus-infected cells, and also a 45-48 kDa protein in cells infected with MNV or BPS virus. The data suggest that it may be possible to define parapoxvirus strains on the basis of small variations in specific virus-directed cell surface proteins.

Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates.

Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats.

Production and assay of ovine T cell growth factor by concanavalin A stimulated peripheral blood leukocytes.

Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. Supernatants were harvested after 30 hr and concentrated for further characterization. A 28 hr proliferation assay with 2.5 X 10(4) 24 hr Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30-37 KD molecular weight fraction. Production and assay of TCGF were performed under autologous conditions to reduce background stimulation which occurred when fetal bovine serum was present. This methodology required no cell lines or inbred animals and should be adaptable to the study of immunostimulatory molecules of other outbred species.

Intestinal absorption of maternal leucocytes by newborn lambs.

Fluorescein isothiocyanate (FITC)-labelled maternal colostral or peripheral blood leucocytes appeared in the blood of newborn lambs following oral administration. A small population of autofluorescent cells was found in the blood of control lambs, but lambs receiving labelled leucocytes had significantly higher numbers of fluorescent cells in their blood. The peak appearance of labelled cells in the blood of newborn lambs occurred between 6 and 12 h after ingestion. Labelled cells were also absorbed by 3-day-old lambs. Cells from unrelated ewes were absorbed as well as cells from natural mothers.

Fluorescein isothiocyanate as a cell marker for tracing lymphocyte circulation in sheep.

The use of fluorescein isothiocyanate (FITC) as a cell marker in ovine lymphocyte circulation studies was investigated. The effects of the label on lymphocyte function were assessed by lymphocyte blastogenesis, cell-mediated cytotoxicity and normal lymphocyte transfer reactions. Labelling lymphocytes in 0.05 mg FITC/ml for 10 min at 37 degrees C was found to be optimum. Circulation kinetics of FITC-labelled efferent lymph lymphocytes were monitored as they disappeared from peripheral blood following intravenous infusion and reappeared in the efferent lymphatic duct of the popliteal lymph node. No functional changes were detected in lymphocytes labelled with the optimal regimen.

Macrophages in efferent lymph of sheep and their role in lectin-induced lymphocyte blastogenesis.

Very low numbers (3.9 +/- 3.0/10(5) cells) of macrophages (cells with intense cytoplasmic staining for nonspecific esterase) were found in seven popliteal lymph samples taken at surgery from 6 sheep. 67% of these cells were phagocytic for latex beads. Dose response and limiting dilution experiments indicated that efferent lymph lymphocytes responded to concanavalin A and pokeweed mitogen when at least 10 macrophages/10(5) cells were present, but macrophages appeared not to be necessary for phytohemagglutinin responses. Further depletion of macrophages from efferent lymph lymphocytes by nylon wool columns almost abolished concanavalin A and pokeweed mitogen responses and reduced phytohemagglutinin responses by 67%. The responses to all mitogens were restored to near normal efferent lymph lymphocyte levels by addition of 1% adherent cells and were elevated to near normal cell levels by 5% adherent cells. These results indicate that macrophages are present in efferent lymph and that, despite their low numbers, they may be of functional significance in regulation of immune responses.

Immunologic responses of the bovine fetus to parvovirus infection.

Effects of bovine parvovirus (PBV) infection on the bovine fetal immune system were studied. Five fetuses obtained by cesarean section at 5, 10, 20, 40, and 60 days after they were inoculated and 2 control fetuses at 130 and 153 days of gestation were studied. Virus was recovered from the BPV-inoculated fetuses. between 5 and 10 days postinoculation (PI) fetal blood lymphocyte counts tripled, due primarily to an increase in E-rosetting lymphocytes. There were transient increases in fetal serum immunoglobulin (Ig) M (peaking at PI day 20), marked and gradual increase in IgG1, and minimal increase of IgG2. Serum-neutralizing antibodies were detected at PI day 10 and increased to a titer of 1:2056 by day 60. Peripheral blood lymphocytes of all fetuses responded well to the nonspecific mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. Histopathologically, there was lymphoid hyperplasia, most evident in lymph nodes draining the site of PBV inoculation. There was no indication of lymphoid depletion or necrosis in lymph nodes, spleen, or thymus of PBV-inoculated fetuses. These data indicate that under the conditions of this study, inoculation of the isolate of PBV may stimulate an immune response in the fetus.

Variation in mitogen-induced ovine lymphocyte blastogenesis: adherent cells or 2-mercaptoethanol restore randomly depressed responses.

Peripheral blood leucocytes (PBL) isolated 23 times over a 6-week period from four normal sheep showed considerable variation in serially tested responses to predetermined optimal concentrations of concanavalin A (Con A), phytohaemagglutinin (PHA), and pokeweed mitogen (PWM). Con A responses, in particular, varied widely and were often randomly depressed (21 of 91 times compared to 15 of 91 times for PHA or PWM). The addition of as few as 1% adherent cells (AC) to depressed cultures fully restored the PBL proliferative response to normal levels. Addition of greater numbers of AC (5 or 10%) had little further enhancing effect on depressed cultures. The addition of 1, 5, or 10% AC to cultures that were responding at normal levels increased responses only slightly. Autologous or allogeneic AC were equally effective. Addition of 2-mercaptoethanol (2-ME) to depressed cultures only partially restored the blastogenic response to Con A and had little effect on normal cultures.

Mitogen-induced blastogenesis of peripheral blood and efferent lymph lymphocytes from sheep.

Peripheral blood lymphocytes and efferent lymph lymphocytes (ELL) from 20 healthy adult sheep were studied for their blastogenic responses to the phytolectins concanavalin A, phytohemagglutinin, and pokeweed mitogen. Peripheral blood lymphocytes generally incorporated significantly (P less than 0.00001) more [3H]thymidine than did ELL. The lower response in ELL, especially in concanavalin A- and pokeweed mitogen-stimulated cultures, may be due to an insufficiency of accessory cells that may be required for optimal proliferation.