Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach.

Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV), and border disease virus (BDV) are closely related pestiviruses. BVDV and BDV are found worldwide but seldom cause disease in swine. In contrast, HCV has been successfully eradicated from swine in several nations but poses a potentially devastating threat to them because of its great virulence. Rapid differential diagnosis of HCV from BVDV and BDV infections in swine is vital for detection of the possible reintroduction of HCV into national herds from which it has been eradicated. Nested polymerase chain reactions (PCRs) for each of two pestiviral genomic segments are described. Amplification of the relatively conserved 5' genomic terminus identified 59 of 61 HCV, BVDV, and BDV isolates generically as pestiviruses. Nested amplification of the second region was designed to differentiate HCV from BVDV and BDV by exploiting relatively conserved differences in the nucleotide sequences that encode the major envelope glycoprotein. This second PCR correctly identified 36 of 36 diverse HCV isolates while failing to recognize any of 25 BVDV and BDV isolates. Multiple restriction fragment length analyses confirmed the identities of both external and nested PCR products. The two sets of PCRs may help confirm the generic identity of most pestiviruses and may permit presumptive differential diagnosis of HCV from BVDV and BDV.     

Characterization of bovine viral diarrhea viruses

Summary Embryonic bovine kidney cell cultures infected with the NADL strain of bovine viral diarrhea (BVD) virus, and PK-15 cells infected with an adapted strain of BVD virus were stained with fluorescein-isothiocyanate-conjugated anti-BVD serum globulins at sequential time periods. The development of virus-induced fluorescing antigens within infected cells was then studied. From this time-study, it appears that BVD virus-induced fluorescing antigens originate in the nucleus of the host cell, and are seen next in a paranuclear position (probably in the Golgi Apparatus); the antigen later appears diffusely throughout the cytoplasm of the cell, forms granular cytoplasmic bodies, then fades and eventually disappears unless the host cell is destroyed by the virus. The time-sequential development of fluorescing antigen in BVD virus-infected cells correlates approximately with the appearance of soluble antigen. The disappearance of fluorescence from the cytoplasm corresponds approximately with the synthesis of mature infective viral particles in the virus-cell system.This investigation was conducted, in part, under the U.S. Government Training Act, Public Law 85-507.     

Competitive and blocking enzyme-linked immunoassay for detection of fetal bovine serum antibodies to bovine viral diarrhea virus

A competitive blocking enzyme-linked immunoassay (CELIA) was developed to detect bovine viral diarrhea virus (BVDV) antibodies in undiluted fetal bovine serum (FBS). The CELIA was based on competition of serum BVDV antibodies with biotin-labelled anti-BVDV immunoglobulins (Ig) for a limited quantity of solid-phase BVDV antigen. Antigen preparation was simple, FBS could be tested undiluted, and detergent-containing washes were unnecessary. A series of dilutions of postnatal bovine BVDV antiserum prepared in FBS and a set of 147 undiluted abbatoir FBS samples were tested by both CELIA and serum neutralization tests (SNT). CELIA results on both sets of specimens correlated positively with SNT titers (r = 0.99 and r = 0.85). Relative to the SNT, CELIA sensitivity was 100%; specificity was 76%. CELIA detected a level of BVDV antibody below the 1:2-titer threshold detectable with the SNT. Advantages, limitations, and theoretical differences between the CELIA and SNT are discussed. A similar comparison of CELIA with non-competitive enzyme-linked immunoassay approaches to BVDV serodiagnosis is made. It is concluded that the CELIA is valuable in selecting only BVDV-seronegative FBS for use in virologic cell culture media.     

Antigenic variations in bovine viral diarrhea viruses detected by monoclonal antibodies.

Five murine monoclonal antibodies (MAbs) against the NADL strain of bovine viral diarrhea (BVD) virus were developed, identified, and characterized. Four of the MAbs were directed against a 53-kilodalton (kDa) viral protein, and one was specific to a 47-kDa polypeptide. Competitive radioimmunoassay showed that two MAbs were specific to related epitopes of the 53-kDa protein, and the other three MAbs were each specific to a different epitope. The MAbs were used to study heterogeneity among BVD virus strains. Various degrees of reactivity of cytopathic and noncytopathic virus isolates were detected by virus neutralization and immunofluorescence assays. The virus isolates were divided into six groups based on the neutralization test. The results indicated that the 53-kDa glycoprotein of BVD virus is the major protein involved in virus neutralization and that only a few epitopes of the protein contribute to the neutralization. None of the MAbs neutralized all the BVD virus isolates tested in this study, suggesting antigenic variations among BVD virus isolates.     

Neutralizing antibodies to type 1 and 2 bovine viral diarrhea viruses: detection by inhibition of viral cytopathology and infectivity by immunoperoxidase assay.

Neutralizing antibodies to type 1 and 2 bovine viral diarrhea virus (BVDV) strains were measured by a microtiter virus neutralization test (MVNT) in cell culture. Antibodies (neutralizing) were detected by inhibition of viral infectivity, by the absence of viral cytopathology for cytopathic strains, or by immunoperoxidase staining for noncytopathic strains. The immunoperoxidase-stained monolayers could be detected without the aid of light microscopy. Twenty BVDV strains were used as challenge viruses in the in vitro MVNT, including 14 type 1 and 6 type 2 strains. Representative noncytopathic and cytopathic strains of both types were used. Positive control serum samples available for diagnostic testing contained both type 1 and type 2 BVDV antibodies. There did not appear to be major differences in antibody titers among the respective type strains, regardless of biotype (cytopathic or noncytopathic). In a study with sera from calves receiving a modified live virus or inactivated BVDV vaccine, the calves receiving type 1 strains responded with higher antibody titers to type 1 strains than to type 2 strains.     

A rapid serum neutralization test in microplates for the detection of antibodies to hog cholera virus

The fluorescent antibody serum neutralization (FASN) test for the detection of antibodies to hog cholera virus was developed utilizing 96-well and Terasaki microplates. This microtechnique, especially when performed in Terasaki plates, offers some advantage if compared with conventional FASN in coverslip cell cultures, being easier and more rapid, saving of reagents and allowing simple microscopic observation.     

Immune response to bovine viral diarrhea virus induced by anti-idiotypic antibodies.

We have previously prepared rabbit anti-idiotypic antibodies (anti-Ids) against the neutralizing monoclonal antibodies (MAbs) specific for the gp53 of bovine viral diarrhea virus (BVDV). The anti-Ids, purified by sequential immunoaffinity chromatography, inhibited the immunizing MAb from binding to the original antigens and blocked BVDV infection of cell cultures. This study evaluated immune responses in mice to the purified anti-Id reagents. BVDV-specific neutralizing antibodies were induced by the anti-Ids. The antisera (Ab3) induced by the anti-Ids immunoprecipitated gp53 from BVDV-infected Madin-Darby bovine kidney (MDBK) cell lysates. However, lymphocyte-proliferative responses were specific only for the respective immunizing antigens. These results suggest that the anti-Ids may bear an internal image of the gp53 to stimulate production of antibody but not to stimulate a virus-specific cellular immune response in mice.     

Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins

The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione S-transferase-tagged recombinant proteins in an Escherichia coli system. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.     

Phylogenetic analysis of bovine viral diarrhea viruses using five different genetic regions

Phylogenetic analysis of the five different regions (5' non-coding region (5'NCR), N(pro), E2, NS3 and NS5B-3'NCR) of 48 Japanese and reported bovine viral diarrhea virus (BVDV) genomes was performed. Japanese BVDVs were segregated into BVDV1 subdivided into six subgroups and BVDV2. One isolate, So CP/75, isolated in 1975 and previously proposed as subgroup 1e according to its 5'NCR sequence, was quite unique and formed an independent lineage in the tree of any region. Another isolate, 190CP, obtained from an experimental mucosal disease case was classified as subgroup 1e, defined by Becher et al. in the 5'NCR, N(pro) and E2 regions, whereas it was classified as subgroup 1a in the NS5B-3'NCR region. The genomic sequences of the American isolates ILLC and ILLNC obtained from the GenBank database were assigned into subgroup 1b in the 5'NCR, N(pro), E2 and NS5B-3'NCR regions, whereas they were assigned into subgroup 1a in the NS3 region, suggesting that recombination between the virus strains classified into different subgroups had occurred in an animal. These findings suggest that phylogenetic analysis of several genetic regions is useful for the further characterization of field BVDV isolates.     

Direct enzyme immunoassay for detection of specific IgG antibody to rubella virus by use of labelled antigen

A new solid phase enzyme immunoassay (EIA) for detection of rubella-specific immunoglobulin G (IgG) antibody was developed. The test uses polystyrene microtiter strips coated with rabbit anti-human IgG immunoglobulins as the solid phase and an enzyme-labelled semipurified rubella antigen as indicator. The direct EIA was compared with hemagglutination inhibition (HI), single radial hemolysis (SRH), radioimmunoassay (RIA) and time-resolved fluoroimmunoassay (TR-FIA) using 52 serum specimens from patients with remote rubella infection. The overall agreement of direct EIA with HI was 96.1%, with SRH and RIA 98.1% and with TR-FIA 100%. The linear regression coefficient varied from 0.77 to 0.91, the best being obtained with direct EIA and SRH. The direct EIA was also suitable for diagnosis of acute infections, as a significant increase in antibody levels was detected in all paired specimens tested from patients with acute rubella infection. The sensitivity and were comparable to those of the assays employed. An advantage of the present assay is that the same method and same labelled antigen can be used to test for different classes of antibody using simply a solid phase with capture antibodies of different chain specificity.     

Characterization and application of monoclonal antibodies to bovine viral diarrhea virus nonstructural protein 5A

Bovine viral diarrhea virus (BVDV) nonstructural protein 5A (NS5A) is one the least studied of the BVDV proteins. Therefore, to develop a tool for unraveling the functions performed by BVDV NS5A, monoclonal antibodies (MAbs) were generated by fusion of myeloma cells with spleen cells from mice immunized with recombinant E. coli-expressed GST-NS5A protein. Two MAbs (1H12 and 2F9) were established on the basis of immunofluorescence and Western blot analysis. Both the MAbs were of IgG1 subclass and recognized an epitope clustered within the N-terminal region of NS5A. Furthermore, the MAb 1H12 was used successfully to detect NS5A protein in BVDV field isolates belonging to genotypes 1 and 2. Temporal expression pattern studies during an infectious cycle revealed that BVDV NS5A could be detected 12–60 h postinfection. Confocal microscopy studies showed a cytoplasmic staining pattern and revealed that NS5A is localized on the endoplasmic reticulum membrane in BVDV infected cells.     

Studies on genetic diversity of bovine viral diarrhea viruses in Danish cattle herds

Scandinavian countries have successfully pursued bovine viral diarrhea virus (BVDV) eradication without the use of vaccines. In Denmark, control and eradication of BVDV were achieved during the last two decades, but occasionally new BVDV infections are detected in some Danish cattle herds. The aim of this study was to determine recent BVDV subtypes isolated from 4 Danish herds (A, B, C, and D) isolated in 2009–2012 and to analyze the genetic variation of these isolates within the same herd and its relation with those of other herds. The results showed that three herds (B, C, D) were BVDV 1-b and only one herd (herd A) was BVDV 1-d, no other subtypes were detected. The deduced E2 amino acids result showed a high identity percent (99–100 %) between isolates originating from the same herd, but with higher variation compared to isolates of the other herds. Some of these new Danish strains have closer relationship to BVDVs from outside Denmark than to older Danish strains indicating that these are new introductions to Denmark. In conclusion, BVDV-1 subtypes recently detected in Denmark were only subtypes 1b and 1d, and BVDV infections established in a herd is genetically stable over a long time period.     

Use of monoclonal antibodies for studying the classical hog cholera virus]

Numerous monoclonal antibodies (MAb) to hog cholera virus are a highly specific and effective instrument for studies of this agent. Panels of MAb for differential diagnosis of Pestiviruses are characterized. International reference panel of 30 MAbs is a result of cooperation of European scientists; it was approved as the official reference for assessing all available and new diagnostic agents. MAb permit intraspecies differentiation between hog cholera virus strains and, which is particularly important, between vaccine and field strains. Study of antigenic structure and functional characteristics of surface proteins of the virus with the use of MAb panels helped single out and map the functions of four antigenic sites of surface glycoprotein E2 and detect a relationship between RNAse activity and structural component of another surface glycoprotein E0.     

Antigen-binding radioimmunoassays for human IgG antibodies to bovine beta-lactoglobulin

Colloidal gold particles coated with rabbit anti-HCG (Au-(anti-HCG)) were used in a spectrophotometric ‘sandwich’ SPIA for HCG. The effects of several reagent properties and assay variables were investigated in order to optimize the assay. Best results were obtained with 55 nm particles. Optimum assay conditions involved a sample incubation period of 2 h followed by aspiration of the sample and two washings. A suitable Au-(anti-HCG) concentration corresponded to a value of $\text{A}{}_{\mathrm{<mtext>540</mtext><mtext>nm</mtext>}}{}^{\mathrm{<mtext>1</mtext><mtext>cm</mtext>}}\text{= 2.0}$. The steepest dose-response curves were obtained with an Au-(anti-HCG) incubation period of 16 h at 37°C. The test procedure was suitable for preparing dose-response curves for HCG in buffer, urine and serum. The practical measuring range for HCG dissolved in these media was 30–250 IU/l. The within-run coefficient of variation varied between approx. 4 and 12%. The detection limit for HCG (in buffer) was about 1 IU/l.     

A capture antibody ELISA for detection of antibodies against bovine enterovirus

A capture antibody ELISA was developed for detecting antibodies against bovine enterovirus. Chicken IgG F(ab')2 fragments against strain J2129 captured virus effectively. The captured virus was used to detect specific antibodies against the virus in bovine sera. The specificity of the ELISA is high. The factors affecting the specificity are discussed. The assay is more sensitive and economic than traditional serum neutralisation.     

Immunological findings in sensory carcinomatous neuropathy. Application of peroxidase labelled antibody

An antibody against neurones, which has previously been demonstrated by immunofluorescence, is present in the sera of patients with sensory carcinomatous neuropathy. This antibody was demonstrated by a new technique, namely the use of purified peroxidase-labelled sheep anti-human γ-globulin. In cryostat sections of brain treated with a first layer of serum antibody followed by a second layer of peroxidase labelled anti-human γ-globulin, heavy coarse brown deposits in nerve cells only from different parts of the central nervous system indicated a positive reaction. The special technical precautions necessary to obtain specific staining of neurones are outlined.     

Genotyping of bovine viral diarrhea viruses (BVDV) isolated from cattle in Sicily

Bovine Viral Diarrhea–Mucosal Disease (BVD–MD) is a widely spread infectious disease that causes important economic losses in farms. Several epidemiological studies indicate a high genetic heterogeneity among Bovine Viral Diarrhea Virus (BVDV) strains circulating in Italy. The aim of this study was to investigate the genotypes of BVDV in Sicily, a region in the South of Italy. For this purpose, 17 BVDV strains collected from cattle breed in Sicily between 2005 and 2008 were genetically typed by sequencing of the 5′-untraslated region (5′-UTR) of the viral genome. In this study, phylogenetic analysis showed that all 17 examined strains were clustered within the BVDV genotype 1. Particularly, 14 of them were clustered with the BVDV-1b subgroup, while the remaining three strains were clustered with the BVDV-1e. Moreover, the restriction analysis indicated a bovine origin for all of the 17 strains typed in this study. These results could be useful to carry out an epidemiological survey and to create vaccines that protect cattle against BVDV different subgroups.