• View in gallery
    Figure 1—

    Mean ± SEM rectal temperatures for calves inoculated on day 0 with Npro BVDV (squares) or Npro-deleted BVDV (triangles). *Significant (P < 0.05) difference between groups.

  • View in gallery
    Figure 2—

    Photomicrograph of ileal Peyer's patches of a calf inoculated with Npro BVDV and Npro-deleted BVDV. A—Notice almost complete lymphoid depletion of Peyer's patches in a calf inoculated with Npro BVDV. H&E stain; bar = 200 μm. B—Notice multifocal lymphoid depletion (arrow) of Peyer's patches in a calf inoculated with Npro-deleted BVDV. H&E stain; bar = 200 μm.

  • View in gallery
    Figure 3—

    Photomicrograph of ileal Peyer's patches in a calf 9 days after inoculation with Npro BVDV. A—Notice almost complete lymphoid depletion of center follicle (arrow). H&E stain; bar = 50 μm. B—Marked deposition of BVDV antigen (arrow) is evident. Immunohistochemical stain; bar = 50 μm.

  • View in gallery
    Figure 4—

    Photomicrograph of ileal Peyer's patches in a calf 9 days after inoculation with Npro-deleted BVDV. A—Notice mild lymphoid depletion of center follicle (arrow). H&E stain; bar = 50 μm. B—Mild deposition of BVDV antigen is evident (arrows). Immunohistochemical stain; bar = 25 μm.

  • View in gallery
    Figure 5—

    Mean ± SEM serum interferon concentrations of calves inoculated on day 0 with Npro BVDV (squares) or Npro-deleted BVDV (triangles). See Figure 1 for key.

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Effect of the viral protein Npro on virulence of bovine viral diarrhea virus and induction of interferon type I in calves

Jamie N. HenningsonDepartment of Veterinary and Biomedical Sciences, College of Agricultural Sciences and Natural Resources, University of Nebraska, Lincoln, NE 68583.

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Christina L. TopliffDepartment of Veterinary and Biomedical Sciences, College of Agricultural Sciences and Natural Resources, University of Nebraska, Lincoln, NE 68583.

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Laura H. V. GilFundação Osvaldo Cruz, Centro Aggeu Magalhães, Av. Professor Moraes Rego, s/n CEP-50670-420, Recife-PE, Brazil.

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Ruben O. DonisInfluenza Division, National Center for Immunization and Respiratory Diseases, Coordinating Center for Infectious Diseases, CDC, 1600 Clifton Rd, Mail Stop G-16, Atlanta, GA 30333.

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David J. SteffenDepartment of Veterinary and Biomedical Sciences, College of Agricultural Sciences and Natural Resources, University of Nebraska, Lincoln, NE 68583.

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Bryan CharlestonPirbright Laboratory, Institute for Animal Health, Woking, Surrey, GU24 0NF, England.

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Abstract

Objective—To characterize the influence of the viral protein Npro on virulence of bovine viral diarrhea virus (BVDV) and on type I interferon responses in calves.

Animals—10 calves, 4 to 6 months of age.

Procedures—BVDV virulence and type I interferon responses of calves (n = 5) infected with a noncytopathic BVDV with a deleted Npro were compared with those of calves (5) infected with a noncytopathic BVDV with a functional Npro. Rectal temperatures, clinical signs, platelet counts, and total and differential WBC counts were evaluted daily. Histologic examinations and immunohistochemical analyses of tissues were conducted to assess lesions and distribution of viral antigens, respectively. Serum type I interferon concentrations were determined.

Results—Calves infected with Npro-deleted BVDV developed leukopenia and lymphopenia, without developing increased rectal temperatures or lymphoid depletion of target lymphoid organs. There was minimal antigen deposition in lymphoid organs. Calves infected with Npro BVDV developed increased rectal temperatures, leukopenia, lymphopenia, and lymphoid depletion with marked BVDV antigen deposition in lymphatic tissues. Interferon type I responses were detected in both groups of calves.

Conclusions and Clinical Relevance—Deletion of Npro resulted in attenuation of BVDV as evidenced by reduced virulence in calves, compared with BVDV with a functional Npro. Deletion of Npro did not affect induction of type I interferon. The Npro-deleted BVDV mutant may represent a safe noncytopathic virus candidate for vaccine development.

Abstract

Objective—To characterize the influence of the viral protein Npro on virulence of bovine viral diarrhea virus (BVDV) and on type I interferon responses in calves.

Animals—10 calves, 4 to 6 months of age.

Procedures—BVDV virulence and type I interferon responses of calves (n = 5) infected with a noncytopathic BVDV with a deleted Npro were compared with those of calves (5) infected with a noncytopathic BVDV with a functional Npro. Rectal temperatures, clinical signs, platelet counts, and total and differential WBC counts were evaluted daily. Histologic examinations and immunohistochemical analyses of tissues were conducted to assess lesions and distribution of viral antigens, respectively. Serum type I interferon concentrations were determined.

Results—Calves infected with Npro-deleted BVDV developed leukopenia and lymphopenia, without developing increased rectal temperatures or lymphoid depletion of target lymphoid organs. There was minimal antigen deposition in lymphoid organs. Calves infected with Npro BVDV developed increased rectal temperatures, leukopenia, lymphopenia, and lymphoid depletion with marked BVDV antigen deposition in lymphatic tissues. Interferon type I responses were detected in both groups of calves.

Conclusions and Clinical Relevance—Deletion of Npro resulted in attenuation of BVDV as evidenced by reduced virulence in calves, compared with BVDV with a functional Npro. Deletion of Npro did not affect induction of type I interferon. The Npro-deleted BVDV mutant may represent a safe noncytopathic virus candidate for vaccine development.

Bovine viral diarrhea virus, a member of the genus Pestivirus in the family Flaviviridae, causes economically important disease costing $10 to $40 million/million calvings.1,2 Bovine viral diarrhea virus is an enveloped, single-stranded, positive-sense RNA virus that is approximately 12.5-kilobases long with 1 large open reading frame flanked by 5′ and 3′ untranslated regions.3 The genome encodes a polyprotein that is co- and posttranslationally cleaved to generate 11 or 12 viral proteins, including Npro, C, Erns, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B.4,5

Bovine viral diarrhea virus consists of 2 biotypes, noncytopathic and cytopathic. In vitro cytopathic BVDV infection results in host cell death via apoptosis, whereas in vitro noncytopathic BVDV infection does not.6–8 Bovine viral diarrhea virus causes a variety of diseases in cattle, including acute subclinical and clinical infections, congenital defects, abortion, immunosuppression with secondary infections, mucosal disease, and persistent infection of calves infected in utero.9 Maintenance and transmission of the virus in cattle populations are attributed to cattle persistently infected with noncytopathic virus. Control of BVDV infections in herds is based on biosecurity measures to eliminate persistently infected cattle and the use of safe, effective BVDV vaccines.

A key mediator of innate immunity is the interferon system, comprising types I, II, and III interferons.10,11 Type I interferons, specifically A and B subtypes, induce transcription of a number of genes important for establishing an antiviral state.12–14 Proteins induced by type I interferon that are important in the antiviral response include dsRNA-dependent protein kinase R; 2′, 5′-oligoadenylate synthetase; and Mx proteins.15 The Mx proteins are large guanine triphosphatases that are induced by type I interferon and play a role in establishment of the antiviral state by interfering with viral replication.16

The Npro is a nonstructural protein unique to pestiviruses with autoprotease activity17 that interferes with host type I interferon responses.18,19 Classical swine fever virus mutants with deletion of the Npro segment are viable viruses with defective growth properties resulting in attenuated virulence.20 Classical swine fever virus infection of macrophages inhibits type I interferon induction by use of polyinosinic-polycytidylic acid in macrophages and porcine kidney cells (ie, PK-15 cells),21 whereas CSFV Npro gene deletion mutants induce type I interferon in the absence of polyinosinic-polycytidylic acid in those cells.22 The role of Npro in inhibiting type I interferon secretion is associated with the amino terminal region of the protein.18 Bovine viral diarrhea virus Npro inhibits type I interferon by targeting interferon regulatory factor-3, which is involved in interferon regulation, for proteosomal degradation.23,24

Increased rectal temperatures, increased respiratory rates, viremia, leukopenia, lymphopenia, and infection of the thymus are clinical manifestations of acute BVDV infection that may be prevented by vaccination.25,26 However, live attenuated noncytopathic BVDV vaccines have some residual degree of virulence, commonly resulting in transient leukopenia, lymphopenia, and lymphodepletion of lymphoid organs of vaccinated cattle.25,26 The leukopenia and lymphopenia induced by use of live noncytopathic BVDV vaccination contribute to immunosuppression, which may render the vaccinated animal susceptible to secondary infections. However, if Npro plays a role in virulence and in suppressing the immune system, it is possible that deletion of the Npro of a live BVDV vaccine strain could result in a safer, yet efficacious, vaccine because of viral attenuation.

We hypothesized that deletion of the Npro coding region of noncytopathic BVDV would result in attenuation of virulence in vivo and that absence of Npro function would result in an adequate type I interferon response in experimentally infected calves. The objective of the study reported here was to characterize the influence of Npro on BVDV virulence and on the type I interferon responses in calves. Evidence of attenuation was based on evaluation of clinical signs, histopathologic lesions of target organs, BVDV antigen deposition, and type I interferon responses in calves infected with the noncytopathic Npro-deleted BVDV.

Materials and Methods

Calves—Ten Holstein calves that were 4 to 6 months old and weighed approximately 240 kg were used in the study. All calves were BVDV-free and seronegative, as confirmed via virus isolation and assays for serum neutralizing antibodies. Calves were housed in biosecurity level 2 isolation rooms and fed a pelleted complete feeda at a rate of 2% to 2.5% of their body weight daily. The project was reviewed and approved by the University of Nebraska Institutional Animal Care and Use Committee.

Cell cultures, viruses, and vaccine—Bovine turbinate cellsb were used for virus isolation and titration assays. All cells were tested for extraneous BVDV (via immunohistochemical evaluation), bacteria (including mycoplasmal organisms), and fungi, as described.27,28 Cells were grown as monolayers in DMEM supplemented with equine serum (10%) in a humidified incubator with 5% CO2. Each group of calves (n = 5/group) was inoculated with noncytopathic BVDV with Npro or noncytopathic BVDV with Npro deleted. The viruses were cloned from wild-type virus from the National Animal Disease Laboratory (a cytopathic BVDV virus), and then cINS in the NS2 region was deleted to produce a noncytopathic variant.29,30 Most of the Npro region was deleted from Npro-deleted BVDV, with the first 14 amino acids remaining. Polymerase chain reaction amplification and DNA sequencing confirmed the presence of the Npro region in Npro BVDV and the deletion of the Npro region in Npro-deleted BVDV stock virus before inoculation of the calves.

Inoculation—Five calves each were randomly allotted to 2 groups. Calves in groups 1 and 2 were inoculated on day 0 via intranasal aerosolizationc of 10 mL of DMEM containing 108 TCID50 of Npro BVDV or Npro-deleted BVDV, respectively. Individually assigned identification numbers for group 1 were 1 through 5 and for group 2 were 6 through 10. Calves were housed individually or in pairs. Calves were observed for clinical signs for 9 days and necropsied on day 9.

Clinical observations—Beginning 2 days before calves were inoculated, rectal temperature and clinical signs of infection were recorded daily for each calf by individuals who were not aware of the treatment groups. Nasal swab specimens were collected from each calf for use in BVDV isolation.

Clinical signs of disease were assigned numeric values on the basis of a scoring system.28 Respiratory rate and dyspnea were scored by use of a 3-point scale (0 = normal; 1 = rapid shallow breathing; and 2 = dyspnea). Nasal secretions were scored by use of a 3-point scale (0 = normal; 1 = excessive serous secretion; and 2 = excessive mucopurulent secretion). Coughing was recorded as a binary variable (0 = not detected and 1 = detected). Lethargy was determined by observing whether calves were responsive each time the observer entered an isolation room and scored by use of a 4-point scale (0 = responsive; 1 = mild signs of depression and inactivity; 2 = severe signs of depression and inactivity; and 3 = recumbent and unresponsive). Fecal characteristics were scored by use of a 3-point scale (0 = normal; 1 = unformed feces; and 2 = watery feces).

Hematologic evaluation—Blood samples were collected daily on days −2 to 9. Blood was collected from the external jugular vein into sterile tubes containing EDTA and was used for determination of total and differential WBC and platelet counts and for preparation of peripheral blood mononuclear cells. Blood samples were also collected daily into sterile tubes without coagulant for separation of serum.

Necropsy and histologic findings—Calves were euthanatized on day 9. The investigator (DJS) who examined the tissues grossly was unaware of calf identification and treatment group. Thymic atrophy was assessed by reduction in size; calves at the age of 6 months typically have a prominent thymus extending well beyond the thoracic inlet into the caudal cervical region. Specimens were obtained from the ileum for virus isolation from Peyer's patches and from the lungs, liver, kidneys, and ileum for aerobic bacterial culture. Specimens obtained from the tonsils, thymus, trachea, esophagus, lungs, liver, kidneys, spleen, rumen, abomasum, duodenum, jejunum (approx 2 m proximal to the ileocecal valve), mesenteric lymph nodes, ileum (approx 15 cm proximal to the ileocecal valve), cecum, and colon were fixed in neutral-buffered 10% formalin. The following day, tissues were processed, embedded in paraffin, sectioned at a thickness of 5 μm, stained with H&E, and examined by use of light microscopy. The investigator (JNH) who examined the stained tissue sections and scored the lesions was unaware of calf identification and treatment group. Histologic changes in tissues (reactive follicles and lesions) were evaluated and scored by use of a 4-point scale (0 = none; 1 = mild; 2 = moderate; and 3 = severe). Lymphocytolysis was scored by use of a 5-point scale (0 = none or rare; 1 = few cells in follicle of nodes or Peyer's patches [normal rate of lymphocytolysis]; 2 = 10% to 25% of follicles in node affected; 3 = 26% to 75% of follicles in node affected; and 4 = > 75% of follicles in node affected). Lymphoid depletion was scored by use of a 5-point scale representing the percentage of follicles depleted in lymphoid tissues (0 = none or rare; 1 = 1% to 10%; 2 = 10% to 25%; 3 = 25% to 50%; and 4 = > 50%).25

Immunohistochemical analyses—Paraffin-embedded tissues were sectioned at 5 μm and stained for detection of BVDV antigen by use of an avidin-biotin-alkaline phosphatase method. Sections were deparaffinized in xylene, rehydrated in a series of graded alcohol solutions, and treated with protease XIV in 0.5M TBSS (pH, 7.6) for 15 minutes at 37oC. Sections were blocked for 30 minutes at 18° to 24°C in TBSS with 4% equine serum. After blocking, primary antibody (anti-BVDV monoclonal antibody 15C531) directed against Erns/glycoprotein 48 (diluted 1:1,000 in TBSS) was added, and sections were incubated for 1 hour at 18° to 24°C. Sections were washed twice in TBSS with 1mM EDTA and 0.05% Tween 20 (4 min/wash). Biotinylated horse antimouse IgGd diluted 1:200 in TBSS with 2% normal bovine serum and 4% horse serum was applied, and slides were incubated for 30 minutes at 18o to 24°C. Slides were washed as described, and a conjugated avidin-alkaline phosphatase complexe was added and allowed to incubate for 15 minutes. After washing, substratef was applied to the tissue sections and slides were allowed to incubate for 10 minutes at 20o to 22oC in darkness. Slides were washed in tap water for 2 minutes, counterstained in Mayer hematoxylin, and dehydrated. A coverslip was applied, and each slide was examined for staining as described.28 Slides for immunohistochemical evaluation were scored by use of a 5-point scale (0 = negative; 1 = rare or scattered cells; 2 = clearly positive, moderate, regional to scattered staining; 3 = widespread multifocal staining; and 4 = intense widespread staining in multiple regions or diffuse staining).

Virus isolation—Virus isolation was performed on ileum. Three grams of ileum containing Peyer's patches was homogenized in a stomacher with 6 mL of DMEM, 100 μg of gentamicin/mL, and 0.25 μg of amphotericin B/mL. The tissue homogenate was frozen at −80°C until tested for BVDV. Ninety-six–well plates were set up with 50 μL of bovine turbinate cells (2 × 105 cells/mL). Dulbecco modified Eagle medium with 2% horse serum, 100 μg of gentamicin/mL, and 0.25 μg of amphotericin B/mL was used. Samples were filtered through a 0.8and 0.45-μm filter, respectively. Nine 5-fold serial dilutions were performed with DMEM, 2% horse serum, 100 μg of gentamicin/mL, and 0.25 μg of amphotericin B/mL, and 100 μL of sample or diluent was added to the plates. Plates were incubated for 4 days at 37oC with 5% CO2. On day 4, cells were fixed with 20% acetone-PBS buffer (vol/vol). Cells were stained the following day by use of an indirect immunoperoxidase test with monoclonal antibody 348g directed against glycoprotein E2, as described.26

Type I interferon analysis—An Mx protein chloramphenicol acetyltransferase reporter gene assay was used to measure interferon in serum samples as described by Fray et al.32 Madin-Darby bovine kidney-t2 cells,h stably transfected with a plasmid containing a human MxA promoter driving a chloramphenicol acetyltransferase cDNA, were used for the assay. The Madin-Darby bovine kidney-t2 cells at a concentration of 1 × 106 cells/well were seeded into 6-well plates. Cell culture medium was adjusted to 2 mL of DMEM/well with 10% FBS and 10 μg of blasticidin/mL.i Plates were incubated for 24 hours at 37°C in 5% CO2. The following day, duplicate serum samples from calves were heat inactivated at 56°C for 30 minutes. Samples were diluted 1:5 in DMEM with 2% FBS. Medium was discarded from wells, and heat-inactivated serum samples diluted with DMEM were added to the wells and incubated for 24 hours at 37°C in 5.0% CO2. The next day, chloramphenicol acetyltransferase expression was determined by use of a commercial ELISA kit.j The assay was performed following the manufacturer's instructions. Cells were lysed for 20 minutes with Triton X-100 lysis buffer, and 200-μL aliquots were added in duplicate to wells of the chloramphenicol acetyltransferase-ELISA 96-well plate. A standard curve was developed by use of the same procedure with known amounts of recombinant human interferon-A A/Dk by use of a 1:2 serial dilution (0.132 to 270 U/aliquot). Interferon activity (U/mL) in test samples was calculated from the standard curve.

Statistical analysis—An ANOVA for repeated measures in a completely randomized design with an autoregressive error covariance structure and comparison of least squares means was used to detect significant differences between treatment groups for mean values of rectal temperature, WBC counts, lymphocyte counts, neutrophil counts, monocyte counts, and platelet counts. A Fisher exact test, extended for > 2 outcomes when necessary, was used to detect differences in histologic characteristics between treatments. A value of P < 0.05 was considered significant.

Results

Clinical observations—In group 1, rectal temperatures in calves inoculated with Npro BVDV were increased from days 6 through 8. Three calves developed rapid, shallow respiratory patterns (score, 1) on days 7 through 9. Calf 2 occasionally had a cough (binary score, 1) on days 8 and 9. The animals were scored as lethargic with diminished appetite during periods of fever. Calf 5 had a serous nasal discharge (score, 1) on days 6 and 7. Mean WBC counts for calves inoculated with Npro BVDV decreased from days 3 through 9, with the lowest count on day 4 at 8.0 × 103/μL. Mean lymphocyte counts for calves inoculated with Npro BVDV decreased from days 3 through 7, with the lowest count on day 7 at 5.0 × 103/μL.

In group 2, increased temperatures were not observed throughout the 9-day period. Mean temperatures for the 2 groups were significantly different on day 7 only (Figure 1). No abnormalities were detected in appetite, condition, respiratory character, and other clinical observations in group 2. Mean WBC counts decreased from days 3 through 7, with the lowest count on day 6 at 6.7 × 103 cells/μL. Mean lymphocyte counts decreased from days 3 through 7, with the lowest count on day 6 at 4.1 × 103 cells/μL.

Figure 1—
Figure 1—

Mean ± SEM rectal temperatures for calves inoculated on day 0 with Npro BVDV (squares) or Npro-deleted BVDV (triangles). *Significant (P < 0.05) difference between groups.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1117

Pathologic findings—On postmortem examination, some calves in both groups had soft to watery colonic contents, mild thymic atrophy, atrophied Peyer's patches, and mildly enlarged mesenteric lymph nodes.

In group 1, watery colonic contents were observed in 3 calves and semisolid to firm colonic contents were observed in 2 calves. Mild thymic atrophy characterized by reduced size with limited extension into the cervical region was evident in 3 calves. Peyer's patches appeared atrophied in 1 calf.

In group 2, Peyer's patches appeared atrophied in 1 calf. Enlarged mesenteric lymph nodes were detected in 1 calf. One calf had a slightly mucoid nasal discharge. Mild thymic atrophy characterized by reduced size with limited extension into the cervical region was evident in 2 calves. Semisolid to firm colonic contents were observed in all 5 calves. Additional gross lesions were not observed, and no bacterial pathogens were cultured from the ileum, kidneys, lungs, or liver of calves from either group.

Microscopic findings—In both groups, lymphoid depletion was irregular in all tissues other than the ileal Peyer's patches. Lymphoid depletion was mild in other lymphoid organs of infected calves. Lymphocytolysis was also variable but was detected with greater regularity in group 1. Increased thymic lymphocytolysis was detected in calves from both groups; however, it was more common in group 1 calves.

In group 1, calves had lymphoid depletion scores of 3 to 4 in ileal GALT or Peyer's patches (Figures 2 and 3). In calf 4, 25% to 50% of Peyer's patches (score, 3) were depleted. Calves had thymic lymphoid depletion with scores of 0 to 2. Lymphoid depletion was again variable and evident in other lymphoid tissues in all calves inoculated with Npro BVDV. Lymphocytolysis in Peyer's patches was dectected in group 1 with scores of 2 to 3. Four calves inoculated with Npro BVDV had a score of 2 for thymic lymphocytolysis. In other organs, such as the prescapular lymph nodes, tonsils, and GALT, lymphocytolysis was common in calves inoculated with Npro BVDV, with scores of 2 to 3.

Figure 2—
Figure 2—

Photomicrograph of ileal Peyer's patches of a calf inoculated with Npro BVDV and Npro-deleted BVDV. A—Notice almost complete lymphoid depletion of Peyer's patches in a calf inoculated with Npro BVDV. H&E stain; bar = 200 μm. B—Notice multifocal lymphoid depletion (arrow) of Peyer's patches in a calf inoculated with Npro-deleted BVDV. H&E stain; bar = 200 μm.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1117

Figure 3—
Figure 3—

Photomicrograph of ileal Peyer's patches in a calf 9 days after inoculation with Npro BVDV. A—Notice almost complete lymphoid depletion of center follicle (arrow). H&E stain; bar = 50 μm. B—Marked deposition of BVDV antigen (arrow) is evident. Immunohistochemical stain; bar = 50 μm.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1117

In group 2, lymphoid depletion of Peyer's patches received a score of 1 to 2 (Figures 2 and 4). Calves had thymic lymphoid depletion with scores of 0 to 2. One calf had lymphoid depletion in GALT of the jejunum with a score of 1. Four calves had lymphocytolysis scores of 2 in Peyer's patches without complete lymphoid depletion, and 2 calves had a score of 2 for thymic lymphocytolysis. Increased lymphocytolysis with a score of 2 was observed in individual calves in the tonsils, mediastinal lymph nodes, and GALT of the jejunum. Lymphoid depletion of the ileal Peyer's patches was significantly different between the 2 groups. The mean ileal Peyer's patches lymphoid depletion score in calves inoculated with the Npro BVDV was 3.8, whereas the score in calves inoculated with Npro-deleted BVDV was 1.6.

Figure 4—
Figure 4—

Photomicrograph of ileal Peyer's patches in a calf 9 days after inoculation with Npro-deleted BVDV. A—Notice mild lymphoid depletion of center follicle (arrow). H&E stain; bar = 50 μm. B—Mild deposition of BVDV antigen is evident (arrows). Immunohistochemical stain; bar = 25 μm.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1117

Immunohistochemical staining—Bovine viral diarrhea virus antigen deposition was most abundant in Peyer's patches. Antigen deposition in Peyer's patches followed a characteristic pattern for each group and was significantly different between groups. The mean antigen deposition score for the Npro BVDV calves was 3.8, whereas the score for the Npro-deleted BVDV calves was 1.4. Staining was variable in other organs and among calves; however, staining was detected more commonly in organs other than the Peyer's patches in group 1 calves, compared with group 2 calves.

In group 1, all calves received an immunohistochemical score of 4 in Peyer's patches, except for 1 calf that received a score of 3 (Figure 3). Thymus immunohistochemical staining received scores of 0 to 3. Other sites with antigen deposition receiving scores of 1 to 4 observed in group 1 included the following: mesenteric, mediastinal, and retropharyngeal lymph nodes; ileocecal valve; tonsils; and jejunal GALT.

In group 2, 3 calves received a score of 1 (rare single cell or group) for immunohistochemical staining in Peyer's patches, whereas 2 calves received a score of 2 (clearly positive scattered staining; Figure 4). Thymic BVDV antigen deposition was observed in only 1 calf with a score of 1.

Peyer's patch virus titers—There was no significant difference between groups for mean virus titers of the Peyer's patches. Group 1 titers were < 1.07 × 104 TCID50/mL, and group 2 titers were < 6.30 × 102 TCID50/mL.

Type I interferon response—For all calves, serum interferon type I concentrations were increased from basal concentrations from days 3 through 6 (Figure 5). A significant difference between groups was detected on days 3 and 4, with group 1 calves having higher values. Serum interferon type I returned to basal concentrations for both groups of calves at day 7. In group 1, concentrations peaked at day 4 with a value of 11.1 U/mL. In group 2, concentrations peaked at day 5 with a value of 10.3 U/mL.

Figure 5—
Figure 5—

Mean ± SEM serum interferon concentrations of calves inoculated on day 0 with Npro BVDV (squares) or Npro-deleted BVDV (triangles). See Figure 1 for key.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1117

Discussion

The present study confirmed that Npro played a crucial role in virulence of noncytopathic BVDV and that deletion of Npro from noncytopathic BVDV resulted in attenuation of virulence. Calves infected with Npro-deleted BVDV developed subclinical infection with less lymphoid depletion of lymphoid organs and less antigen deposition in target tissues, compared with calves infected with the parent noncytopathic BVDV. In addition, Npro-deleted BVDV and Npro BVDV both induced serum interferon type I production that was significantly different on days 3 and 4. The attenuated phenotype BVDV is a promising candidate vaccine virus with desirable attenuation features.

In our study, clinical signs of disease were evident in calves infected with Npro BVDV, which had a functional Npro. The clinical signs were consistent with those of previous reports25,26,28 of experimental acute noncytopathic BVDV infections of calves. Conversely, calves infected with Npro-deleted BVDV did not have clinical disease, and the infection remained subclinical throughout the 9-day observation period.

Shifts in leukocyte populations were evident in both groups of calves. Leukopenia is a salient feature of calves with acute BVDV infection,25,26,28 and in the present study, leukopenia and lymphopenia developed in both groups on days 3 to 7, with no significant difference between groups. The clinical signs, leukopenia, and lymphopenia in group 1 calves were comparable to those of previous studies25,26 of acute BVDV infection in calves with NY-1 and 890 BVDV performed in the authors' laboratory. Lymphopenia, in addition to lymphoid depletion and antigen deposition, provided clear evidence that the infection resulted in systemic viral replication in both groups of calves.

Limited, mild gross lesions were evident at necropsy in the present study and included watery colonic contents in several calves infected with Npro BVDV, which was not observed in calves infected with Npro-deleted BVDV. These findings were similar to gross lesions reported in calves acutely infected with cytopathic and noncytopathic BVDV that typically developed subtle and nonspecific gross lesions such as flaccid, edematous intestines (sometimes with ecchymotic and petechial hemorrhages) and mildly reactive mesenteric lymph nodes.33,34

The most severe histologic lesions were evident in Peyer's patches. Severe lymphoid depletion developed in all calves inoculated with Npro BVDV, whereas in calves inoculated with Npro-deleted BVDV, Peyer's patch lymphoid depletion was substantially reduced, compared with that of calves infected with Npro BVDV. Lymphocytolysis was also most common in Peyer's patches of all calves and was increased in other lymphoid tissues of calves inoculated with Npro BVDV. Taken together, these findings indicated that Npro-deleted BVDV had reduced virulence, compared with Npro BVDV. Lymphoid depletion observed in the present study was consistent with those of published reports25,26,35,36 in which Peyer's patches were often the most prominent site for development of histologic lesions and for deposition of BVDV antigen in calves acutely infected with BVDV. Calves infected with noncytopathic BVDV strains BVDV 7937 or BVDV 126 had variable degrees of lymphoid depletion in Peyer's patches,36 whereas calves infected with noncytopathic type I BVDV strain, NY-1 BVDV, or type II BVDV (strain 890) had lymphopenia, lymphoid depletion, and substantial lymphocytolysis in Peyer's patches and other lymphoid organs.25,26 Because lesions in calves in our study were almost exclusively limited to Peyer's patches, findings were consistent with the lymphoid depletion described for Peyer's patches; however, viruses in this study, Npro-deleted BVDV and Npro BVDV, did not cause consistent lymphoid tissue depletion in other lymphoid tissues and therefore were less virulent than BVDV strains WRL, NY-1, and 890.25,26,36

Calves infected with Npro BVDV had marked antigen deposition in Peyer's patches and more widespread antigen distribution than calves infected with Npro-deleted BVDV. Thymic BVDV antigen staining was most extensive in the group of calves infected with Npro BVDV. Distribution of BVDV antigen with various noncytopathic strains, such as BVDV 7939, BVDV 126, NY-1, and 890, is most prominent in Peyer's patches, but is also evident in various lymph nodes such as the mesenteric lymph nodes, GALT, and thymus.25,26,36 Antigen deposition in calves infected with Npro-deleted BVDV, compared with BVDV antigen deposition in tissues of calves infected with Npro BVDV and other BVDV strains in the previous studies, was mild and indicated that virus replication was either reduced or more transient because of the attenuation associated with deletion of Npro in the Npro-deleted BVDV mutant.

In the present study, the influence of Npro on type I interferon induction was evaluated by use of experimental infection of calves with Npro-deleted BVDV and Npro BVDV. Calves infected with Npro BVDV had greater type I interferon production than calves inoculated with Npro-deleted BVDV on days 3 to 6, with a significant difference detected on days 3 and 4. The reduced concentration of interferon produced by calves infected with Npro-deleted BVDV may have been caused by reduced growth of virus as an expression of reduced virulence in vivo, despite less efficient inhibition of type I interferon expected with Npro-deleted BVDV based on in vitro work. A study37 examining serum type I interferon responses in CSFV infection reported that at the time of lymphoid depletion in lymphoid organs and lymphopenia, high serum concentrations of type I interferon were observed. The type I interferon responses were detected as early as 2 days after infection, with maximum concentrations detected between days 3 and 5 after infection, and the type I interferon response depended on the virulence of the infecting virus.37 Our findings were similar in that in both groups of calves, serum type I interferon concentrations increased on day 3, reaching maximum concentration from days 4 to 6, which was the same time period that lymphopenia was evident.

In a previous study,38 findings suggest that the Npro-deleted BVDV mutant virus, with most of the Npro gene deleted and retaining only 5 of the Npro codons next to the 5′ untranslated region, has acceptable growth characteristics and induces low-level type I interferon responses in inoculated fetuses, resulting in development of persistent infection. This finding was similar to that of the present study, in which deletion of a major portion of the Npro gene resulted in a viable virus able to infect and replicate in vivo and induce an adequate type I interferon response.

The Npro played an integral role in virulence, and deletion of the Npro gene resulted in attenuation of the noncytopathic BVDV as evidenced by reduced severity of infection in calves, compared with calves infected with the parent BVDV with a functional Npro. The Npro-deleted BVDV did not cause widespread lymphodepletion in lymphoid organs. In light of its low virulence and apparent in vivo safety, assessment of the ability of this recombinant noncytopathic BVDV I mutant to protect vaccinated cattle against systemic infection and fetal infection from virulent BVDV is warranted.

ABBREVIATIONS

BVDV

Bovine viral diarrhea virus

CSFV

Classical swine fever virus

DMEM

Dulbecco modified Eagle medium

FBS

Fetal bovine serum

GALT

Gut-associated lymphoid tissue

TBSS

Tris-buffered saline solution

a.

Plain Ration, No. 51786BHB24, ADM Alliance Nutrition Inc, Quincy, Ill.

b.

National Veterinary Services Laboratory, USDA, Ames, Iowa.

c.

Atomizer No. 163, DeVilbiss Health Care Inc, Somerset, Pa.

d.

Biotinylated horse anti-mouse IgG (H&L), Vector Laboratories, Burlingame, Calif.

e.

Alkaline phosphatase substrate kit 1, Vector Laboratories, Burlingame, Calif.

f.

Vector Red, Vector Laboratories, Burlingame, Calif.

g.

VMRD Inc, Pullman, Wash.

h.

Provided by Dr. Bryan Charleston, Institute of Animal Health, Compton, Berkshire, England.

i.

Blasticidin S HCl, Invitrogen, Carlsbad, Calif.

j.

Roche Applied Science, Indianapolis, Ind.

k.

Biosource PHC 4045, Invitrogen, Carlsbad, Calif.

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Contributor Notes

Supported in part by Animal Health funds, USDA NRI grant 2002-35-204-11619, and the Nebraska Veterinary Diagnostic Center, Lincoln, Neb.

Presented in part at the Conference of Research Workers in Animal Diseases, St Louis, December 2005.

This study is a contribution of the University of Nebraska Research Division.

Address correspondence to Dr. Kelling.