ABSTRACT
Although efficacious and safe, current vaccines for FMD suffer from drawbacks. Among these are that the immune response to the vaccine interferes with the ability to detect vaccinated animals that have subsequently become infected and could carry and shed the virus, creating an obstacle to re-instating disease-free status to countries/regions that vaccinate to control outbreaks. Multiple diagnostic tests are available to identify animals that have been infected with FMDV by detection of antibodies to viral non-structural proteins (NSP) that are present in low concentration in traditional vaccines and are poorly immunogenic in vaccine preparations. However, these tests are not 100% reliable. To circumvent this problem, we have developed a new generation of vaccines that express the "empty capsid" subunit of the virus, in the absence of one of the most immunogenic NSPs, 3Dpol. Here we describe delivery of the empty capsid subunits by recombinant replication-defective human adenovirus type 5 (Ad5). These Ad5-vectored empty capsid vaccines can protect pigs from FMDV challenge as early as 7 days post-vaccination. A second problem with current FMD vaccines is that they do not induce protective immunity quickly, a drawback that is likely to be shared by our Ad5-vectored empty capsid vaccine. To overcome this problem, we have developed a prophylactic antiviral treatment consisting of an Ad5 encoding porcine interferon alpha (pIFNalpha). Administration of Ad5-pIFNalpha protects swine from FMD as early as one day post-administration. The combination of this antiviral treatment and the empty capsid subunit vaccine should induce rapid and complete protection from FMD, and could overcome current diagnostic problems.
Subject(s)
Foot-and-Mouth Disease Virus/immunology , Foot-and-Mouth Disease/immunology , Viral Vaccines/biosynthesis , Adenoviruses, Human/genetics , Animals , Capsid/immunology , Cell Line , Foot-and-Mouth Disease/prevention & control , Foot-and-Mouth Disease/transmission , Foot-and-Mouth Disease Virus/genetics , Genetic Engineering/methods , Genetic Vectors , Humans , Viral Vaccines/genetics , Viremia/immunology , Viremia/prevention & control , Virus ReplicationABSTRACT
We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-alpha/beta). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-alpha or -beta individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-dependent protein kinase (PKR). In support of these findings, treatment of porcine and bovine cells with 2-aminopurine, an inhibitor of PKR, increased the yield of virus 8.8- and 11.2-fold, respectively, compared to that in untreated infected cells. In addition, results of FMDV infection in mouse embryonic fibroblast cells derived from gene knockout mice lacking the gene for RNase L(-/-) or PKR(-/-) or both indicated an important role for PKR in the inhibition of FMDV replication.
Subject(s)
Aphthovirus/physiology , Endopeptidases/metabolism , Interferon-alpha/pharmacology , Interferon-beta/pharmacology , Virus Replication/drug effects , Animals , Aphthovirus/enzymology , Aphthovirus/genetics , Cattle , Cell Line , Endopeptidases/genetics , Endoribonucleases/metabolism , Foot-and-Mouth Disease/virology , Genetic Complementation Test , Interferon-alpha/genetics , Interferon-alpha/metabolism , Interferon-beta/genetics , Interferon-beta/metabolism , Protein Kinases/metabolism , RNA, Double-Stranded/metabolism , SwineABSTRACT
A replication-defective adenovirus 5 encoding foot-and-mouth disease virus (FMDV) capsid and 3C proteinase coding regions (Ad5-FMDV3CWT) was used to vaccinate swine. A single inoculation utilizing 1 x 10(8) plaque forming units (pfu) or an inoculation of 1 x 10(8) followed by a boost of 5 x 10(8) pfu Ad5-FMDV3CWT were tested, along with an inoculation and boost using an adenovirus encoding the FMDV capsid coding region and an inactive form of the 3C proteinase (Ad5-FMDV3CMUT). Sera collected from these animals were examined for the presence of FMDV-specific antibodies using immunoprecipitation, neutralization, and ELISA assays specific for IgM, IgG1 and IgG2. Efficacy studies were performed by placing the vaccinated swine in contact with an FMDV-infected swine and monitoring for signs of disease and changes in serum antibody levels. Ad5-FMDV3CMUT, which is unable to produce FMDV capsid structures, did not elicit FMDV-neutralizing antibodies or protect against FMD. Single inoculation with Ad5-FMDV3CWT generated FMDV-specific neutralizing antibodies, and reduced clinical signs in challenged swine, but failed to completely protect the majority of swine from FMD. Swine which received a primary vaccination with Ad5-FMDV3CWT followed by the boost at 4 weeks generated high levels of FMDV-neutralizing antibodies resulting in complete protection of five of the six swine and limited disease in the remaining animal. Increased efficacy of the two-dose regimen was associated with heightened levels of FMDV-specific IgG1 and IgG2 antibodies.
Subject(s)
Adenoviruses, Human/immunology , Aphthovirus/immunology , Foot-and-Mouth Disease/prevention & control , Swine Diseases/prevention & control , Viral Vaccines/immunology , Viral Vaccines/pharmacology , Animals , Antibodies, Viral/blood , Foot-and-Mouth Disease/immunology , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Swine , Swine Diseases/immunology , Vaccines, Synthetic/immunology , Vaccines, Synthetic/pharmacologyABSTRACT
Four calves were exposed via aerosol to 1 of 2 strains of foot-and-mouth disease virus. Two animals received virus derived from an infectious clone virus (A12-IC) and 2 received virus derived from the same clone but which lacked the leader coding region (A12-LLV2) that codes for a protein responsible for turning off host protein synthesis. Animals were euthanized at 24 and 72 h post exposure. Cattle receiving A12-IC had a rapid course of disease with more virus in tissues while A12-LLV2-infected cattle did not develop clinical signs of disease. Formalin-fixed, paraffin-embedded tissue sections were probed with digoxigenin-labeled riboprobes corresponding to the coding sequence for bovine interferon (IFN) alpha and IFNbeta. Staining for IFNalpha mRNA was noted in mononuclear cells of the lungs of all animals and in respiratory lymph nodes of cattle receiving A12-IC. Staining for IFNbeta mRNA was confined to bronchiolar epithelium and present only in the animals infected with A12-IC. Inability of the A12-LLV2 virus to achieve levels of spread seen with A12-IC may be related to translation of IFNalpha in A12-LLV2-infected cells, which renders adjacent cells less susceptible to productive infection.
Subject(s)
Aphthovirus/pathogenicity , Foot-and-Mouth Disease/immunology , Interferon-alpha/biosynthesis , Interferon-beta/biosynthesis , Animals , Cattle , Cloning, Molecular , In Situ Hybridization/veterinary , Interferon-alpha/analysis , Interferon-beta/analysis , RNA, Messenger/analysisABSTRACT
A recombinant replication-defective human adenovirus serotype 5 vector containing FMDV capsid, P1-2A, and viral 3C protease coding regions was constructed. Two viral clones were isolated, Ad5-P12X3CWT, containing the wild-type (WT) 3C protease that processes capsid polyprotein precursor into mature capsid proteins, and Ad5-P12X3CMUT, containing a point mutation in the protease coding region that inhibits processing. In 293 cells infected with either virus, synthesis of the FMDV capsid polyprotein precursor occurred, but processing of the polyprotein into structural proteins VP0, VP3, and VP1 occurred only in 3CWT virus-infected cells. Immunoprecipitation with monospecific and monoclonal antibodies indicates possible higher order structure formation in Ad5-P12X3CWT virus-infected cells. The viruses were used to elicit immune responses in mice inoculated intramuscularly (im). Only virus containing the 3CWT elicited a neutralizing antibody response. After boosting, this neutralizing antibody response increased. Swine inoculated im with Ad5-P12X3CWT virus developed a neutralizing antibody response and were either completely or partially protected from contact challenge with an animal directly inoculated with virulent FMDV. This adenovirus vector may be an efficient system for the delivery of FMDV cDNA into animals, leading to a high level of neutralizing antibody production and protection from FMDV challenge.
Subject(s)
Adenoviruses, Human/genetics , Aphthovirus/immunology , Capsid/immunology , Cysteine Endopeptidases/immunology , Defective Viruses/genetics , Viral Proteins , Viral Vaccines/immunology , 3C Viral Proteases , Adenoviruses, Human/classification , Adenoviruses, Human/physiology , Animals , Antibodies, Viral/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , Antigens, Viral/metabolism , Aphthovirus/enzymology , Aphthovirus/genetics , Aphthovirus/growth & development , Capsid/genetics , Capsid/metabolism , Cell Line , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Defective Viruses/physiology , Foot-and-Mouth Disease/immunology , Foot-and-Mouth Disease/pathology , Foot-and-Mouth Disease/prevention & control , Foot-and-Mouth Disease/virology , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , Point Mutation , Protein Processing, Post-Translational , Swine/blood , Swine/immunology , Swine/virology , Time Factors , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Vaccines/genetics , Virus ReplicationABSTRACT
A genetic variant of foot-and-mouth disease virus lacking the leader proteinase coding region (A12-LLV2) is attenuated in both cattle and swine and, in contrast to wild-type virus (A12-IC), does not spread from the initial site of infection after aerosol exposure of bovines. We have identified secondary cells from susceptible animals, i.e., bovine, ovine, and porcine animals, in which infection with A12-LLV2, in contrast to A12-IC infection, does not produce plaques; this result indicates that this virus cannot spread from the site of initial infection to neighboring cells. Nevertheless, A12-LLV2 can infect these cells, but cytopathic effects and virus yields are significantly reduced compared to those seen with A12-IC infection. Reverse transcription-PCR analysis demonstrates that both A12-LLV2 and A12-IC induce the production of alpha/beta interferon (IFN-alpha/beta) mRNA in host cells. However, only supernatants from A12-LLV2-infected cells have significant antiviral activity. The antiviral activity in supernatants from A12-LLV2-infected embryonic bovine kidney cells is IFN-alpha/beta specific, as assayed with mouse embryonic fibroblast cells with or without IFN-alpha/beta receptors. The results obtained with cell cultures demonstrate that the ability of A12-IC to form plaques is associated with the suppression of IFN-alpha/beta expression and suggest a role for this host factor in the inability of A12-LLV2 to spread and cause disease in susceptible animals.
Subject(s)
Aphthovirus/physiology , Interferon-alpha/immunology , Interferon-beta/immunology , Virus Replication , Animals , Aphthovirus/immunology , Cattle , Cell Line , Cricetinae , Cytopathogenic Effect, Viral , Mice , Sheep , Swine , Viral Plaque AssayABSTRACT
We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.
Subject(s)
Aphthovirus/genetics , Aphthovirus/immunology , Foot-and-Mouth Disease/prevention & control , Vaccines, DNA/pharmacology , Animals , Aphthovirus/physiology , Biotechnology , Foot-and-Mouth Disease/immunology , Foot-and-Mouth Disease/virology , Humans , Mice , Swine , Swine Diseases/immunology , Swine Diseases/prevention & control , Swine Diseases/virology , Vaccines, DNA/genetics , Viral Vaccines/genetics , Viral Vaccines/pharmacology , Virus Replication/geneticsABSTRACT
Over the last few years we have utilized a system to genetically engineer foot-and-mouth disease virus (FMDV) to produce live-attenuated vaccine candidates. These candidates have been generated in the genetic background of a tissue culture-adapted strain of serotype A12 virus. Based on this A12 system, we created a virus lacking the sequence encoding the leader (L) proteinase (Piccone et al., 1995), and demonstrated that this leaderless virus, A12-LLV2 was avirulent in bovine and swine, and could be used as an attenuated vaccine (Mason et al., 1997; Chinsangaram et al., 1998). The current study shows that a similar leader-deleted chimeric virus containing the genome of the type A12 virus with a substituted type O1 capsid coding region from a bovine-virulent virus can be constructed, and that the virus has low, but detectable virulence in swine. A second chimera specifying a tissue culture-adapted type O1 capsid lacking the RGD cell binding site, was avirulent in swine, but was not sufficiently immunogenic to provide protection from challenge. These results are described with respect to mechanisms of attenuation and antigen formation in live-attenuated virus-inoculated animals.
Subject(s)
Aphthovirus/immunology , Capsid/genetics , Endopeptidases/genetics , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Amino Acid Substitution , Animals , Aphthovirus/genetics , Cattle , Cell Line , Cricetinae , Foot-and-Mouth Disease/prevention & control , Gene Deletion , Genetic Engineering , Immunity, Active , Integrins/genetics , Serotyping , Swine , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Attenuated/isolation & purification , Vaccines, Synthetic/genetics , Vaccines, Synthetic/isolation & purification , Viral Vaccines/genetics , Viral Vaccines/isolation & purification , VirulenceABSTRACT
Previously, we demonstrated that a genetically engineered variant of foot-and-mouth disease virus (FMDV) serotype A12 lacking the leader proteinase-coding region (A12-LLV2) was attenuated and induced an immune response that partially protected cattle from FMD. In this study, A12-LLV2 was tested in swine as a live or chemically inactivated vaccine. Animals vaccinated with chemically inactivated A12-LLV2 or wild-type (WT) virus in oil adjuvant developed high levels of neutralizing antibodies and were protected from FMD upon challenge. Animals vaccinated with live A12-LLV2 did not exhibit signs of FMD, did not spread virus to other animals, developed a neutralizing antibody response and antibodies to nonstructural protein 3D, and were partially protected from FMD. Animals given a similar dose of chemically inactivated A12-LLV2 in the absence of adjuvant developed a poor immune response and were not protected from FMD, indicating that limited replication was responsible for the improved immune response found in animals vaccinated with live A12-LLV2. The results demonstrate the potential of A12-LLV2 as a live-attenuated vaccine as well as a safe source of antigen for chemically inactivated vaccines.
Subject(s)
Aphthovirus/immunology , Foot-and-Mouth Disease/prevention & control , Swine Diseases/prevention & control , Viral Vaccines/therapeutic use , Animals , Antibodies, Viral/biosynthesis , Aphthovirus/classification , Aphthovirus/genetics , Cricetinae , Endopeptidases/deficiency , Endopeptidases/genetics , Female , Swine , Vaccines, Attenuated/immunology , Vaccines, Attenuated/therapeutic use , Vaccines, Inactivated/immunology , Vaccines, Inactivated/therapeutic use , Viral Vaccines/immunology , Virus Replication/physiologyABSTRACT
Candidate foot-and-mouth disease (FMD) DNA vaccines designed to produce viral capsids lacking infectious viral nucleic acid were evaluated. Plasmid DNAs containing a portion of the FMDV genome coding for the capsid precursor protein (P1-2A) and wild-type or mutant viral proteinase 3C (plasmids P12X3C or P12X3C-mut, respectively) were constructed. Cell-free translation reactions programmed with pP12X3C (wild-type 3C) and pP12X3C-mut produced a capsid precursor, but only the reactions programmed with the plasmid encoding the functional proteinase resulted in P1-2A processing and capsid formation. Baby hamster kidney (BHK) cells also produced viral capsid proteins when transfected with these plasmids. Plasmid P12X3C was administered to mice by intramuscular, intradermal, and epithelial (gene gun) inoculations. Anti-FMD virus (FMDV) antibodies were detected by radioimmunoprecipitation (RIP) and plaque reduction neutralization assays only in sera of mice inoculated by using a gene gun. When pP12X3C and pP12X3C-mut were inoculated into mice by using a gene gun, both plasmids elicited an antibody response detectable by RIP but only pP12X3C elicited a neutralizing antibody response. These results suggest that capsid formation in situ is required for effective immunization. Expression and stimulation of an immune response was enhanced by addition of an intron sequence upstream of the coding region, while addition of the FMDV internal ribosome entry site or leader proteinase (L) coding region either had no effect or reduced the immune response.
Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Aphthovirus/immunology , DNA, Viral/immunology , Vaccines, DNA/immunology , Viral Vaccines/immunology , 3C Viral Proteases , Animals , Antigens, Viral/genetics , Aphthovirus/genetics , Capsid/genetics , Capsid/immunology , Cell Line , Cricetinae , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/immunology , Mice , Protein Precursors/genetics , Protein Precursors/immunology , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
A variant of foot-and-mouth disease virus (FMDV) lacking the leader (L) coding region (A12-LLV2) was previously constructed and shown to be less virulent in cattle than its wild-type parent (A12-IC). In this study, cattle were tested for their clinical and immunological responses to subcutaneous inoculation with A12-LLV2 or A12-IC or to intramuscular vaccination with chemically inactivated A12-IC. Five weeks postinoculation animals were challenged by intradermal inoculation in the tongue with a virulent cattle-passaged virus. A12-LLV2-inoculated animals showed no clinical signs of disease and developed a neutralizing antibody response by 4 days postinoculation, whereas a companion control bovine did not seroconvert. After challenge, two of three inoculated animals did not develop lesions, but showed mild signs of infection. The third inoculated animal developed some lesions, but these were less severe than in the uninoculated control animal, which showed classical FMD. All animals inoculated with A12-IC developed a fever, two showed typical FMD lesions, and the companion control seroconverted, indicating that it had acquired infection by contact. The A12-IC-inoculated animals and the control were protected from challenge. Animals vaccinated with inactivated virus showed no clinical signs of disease and developed a neutralizing antibody response, and the control did not seroconvert. Upon challenge none of the vaccinated animals developed lesions, one developed a fever, and the control developed FMD. These experiments demonstrate the potential of a rationally designed live-attenuated FMDV vaccine.
Subject(s)
Cattle Diseases/prevention & control , Foot-and-Mouth Disease/prevention & control , Viral Vaccines/administration & dosage , Animals , Antibodies, Viral/biosynthesis , Aphthovirus/drug effects , Aphthovirus/genetics , Aphthovirus/immunology , Aphthovirus/pathogenicity , Aziridines/pharmacology , Cattle , Cattle Diseases/virology , Cell Line , Endopeptidases/genetics , Evaluation Studies as Topic , Injections, Intradermal , Injections, Subcutaneous , Male , Mutation , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunology , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/adverse effects , Vaccines, Inactivated/immunology , Viral Vaccines/adverse effects , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/geneticsABSTRACT
A polymerase chain reaction (PCR)-based detection assay that specifically detected Ehrlichia canis in dogs with acute infections was developed. A region of the 16S ribosomal RNA gene of E. canis was targeted for PCR amplification and chemiluminescent hybridization (CH) with a complementary internal 287-base pair (bp) oligonucleotide probe. The CH improved the PCR assay sensitivity 1,000-fold as compared with visualization on ethidium bromide-stained agarose gels. The PCR assay with CH (PCR/CH) detected as little as 30 fg of E. canis genomic DNA, the equivalent of approximately 150 E. canis organisms. The 495-bp product defined by the specific primers was not detected when genomic DNA from E. platys, E. chaffeensis, E. risticii, and E. equi were used in the PCR/CH assay. The PCR/CH assay was tested with unfractionated blood samples collected from 9 dogs experimentally infected with E. canis. The PCR/CH assay had greater detection sensitivity than did cell culture isolation (CCI) from infected blood. PCR/CH detected E. canis 7 days prior to CCI in 4 of 6 experimentally infected dogs. The results obtained with the PCR/CH assay otherwise consistently matched the results obtained by CCI. This PCR/CH assay is a rapid, sensitive, and specific method for E. canis detection with sensitivity comparable to or exceeding that of CCI. A diagnosis of E. canis using this PCR/CH assay can be made in 2 days as compared with 1-4 weeks for CCI. The PCR/CH assay appears to be an acceptable alternative or complement to current diagnostic techniques.
Subject(s)
Dog Diseases , Ehrlichia/isolation & purification , Ehrlichiosis/veterinary , Polymerase Chain Reaction/methods , Animals , Base Sequence , DNA Primers , Dogs , Ehrlichia/genetics , Ehrlichiosis/diagnosis , Genes, Bacterial , Luminescent Measurements , Male , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Sensitivity and SpecificityABSTRACT
136 fecal samples, collected from 47 dairy calves on a calf ranch and in a dairy herd in California, were tested for the presence of group A and group B rotaviruses by reverse transcription-polymerase chain reaction (RT-PCR). Samples were collected from each calf at days 1, 7 and 14. Within the 14 day period, 44 calves (94%) were positive for group A rotavirus and an unexpectedly high number of calves (38 calves, 81%) were positive for group B rotavirus. When these samples were examined by polyacrylamide gel electrophoresis (PAGE), rotavirus was found in 21 calves and all of them had group A electropherotype. Among 25 PAGE positive samples from 21 calves, 17 (68%) were of short electropherotype, 4 (28%) were of long electropherotype and 4 (28%) contained both short and long electropherotype rotaviruses. Group B and short and long electropherotype group A rotaviruses were found in both normal and diarrheic calves.
Subject(s)
Cattle Diseases/epidemiology , Feces/microbiology , Rotavirus Infections/veterinary , Rotavirus/isolation & purification , Animals , Animals, Newborn , Base Sequence , California/epidemiology , Cattle , Cattle Diseases/virology , DNA Primers/chemistry , DNA, Viral/analysis , Diarrhea/epidemiology , Diarrhea/veterinary , Diarrhea/virology , Electrophoresis, Polyacrylamide Gel/veterinary , Female , Molecular Sequence Data , Polymerase Chain Reaction/veterinary , Prevalence , Rotavirus/genetics , Rotavirus Infections/epidemiology , Rotavirus Infections/virology , Transcription, GeneticABSTRACT
A pair of primers designed from the sequence of genome segment 9 of group B rat rotavirus (IDIR) were employed to amplify genome segment 9 of a group B bovine rotavirus in a polymerase chain reaction (PCR) and to sequence the derived PCR products. A new pair of primers were synthesized from the obtained sequence data and used in a PCR detection assay for group B bovine rotavirus in fecal samples. In addition, another pair of primers were designed to produce a PCR-derived internal probe. This probe was used in a chemiluminescent hybridization to confirm the specificity and to increase the sensitivity of the assay. This assay could detect 0.1 fg of target double-stranded RNA. It was specific to group B bovine rotavirus and did not detect group B rat (IDIR) and porcine rotaviruses, group A bovine (NCDV), simian (SA-11), equine (H-2), porcine (OSU), human (DS-1), deer, and avian rotaviruses, coronavirus, or other enteric organisms tested in this study.
Subject(s)
Cattle Diseases/virology , Feces/microbiology , Polymerase Chain Reaction/veterinary , Rotavirus Infections/veterinary , Rotavirus/isolation & purification , Animals , Base Sequence , Cattle , DNA Primers , Molecular Sequence Data , Polymerase Chain Reaction/methods , Rats , Rotavirus Infections/virology , Sensitivity and SpecificityABSTRACT
A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3' ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 x 10(4) viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 x 10(2) viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.
Subject(s)
Cattle Diseases/diagnosis , Feces/microbiology , Polymerase Chain Reaction/veterinary , Rotavirus Infections/veterinary , Rotavirus/isolation & purification , Animals , Base Sequence , Cattle , Cell Line , DNA Primers , Enzyme-Linked Immunosorbent Assay/methods , Kidney , Macaca mulatta , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA, Double-Stranded/analysis , RNA, Viral/isolation & purification , Rotavirus/genetics , Rotavirus Infections/diagnosis , Sensitivity and SpecificityABSTRACT
To facilitate detection of active bluetongue virus (BTV) infection, a polymerase chain reaction (PCR) protocol was developed. The BTV reverse transcriptase PCR (RT-PCR) is a 1-tube reaction and involves chemical denaturation of the double-stranded viral RNA target, a complementary DNA (cDNA) synthesis step, and PCR amplification of the cDNA. BTV RT-PCR using primers derived from highly conserved genome segment 10 results in a 251-base pair (bp) product. BTV RNA from all USA prototype serotypes 2, 10, 11, 13, and 17; a wide spectrum of USA BTV field isolates including serotypes 10, 11, 13, and 17; and a spectrum of Israeli field isolates including serotypes 2, 4, 6, 10, and 16 were detected by BTV RT-PCR. With agarose gels, the 251-bp product was detected from as little as 100 fg-1 pg of BTV RNA, which is equivalent to 5 x 10(3)-5 x 10(4) viral particles or 5 x 10(2)-5 x 10(3) infectious units. With dot blot hybridization, specific PCR product was detected from as little as 1 fg of BTV RNA, which is equivalent to 50 viral particles, or 5 infectious units. This level of sensitivity is comparable to that of virus isolation. The BTV RT-PCR using primers derived from genome segment 10 can detect a wide spectrum of USA and Israeli BTV serotypes and has potential for detection of infection by the BTV serogroup. Application of this BTV PCR to clinical samples is in progress.
Subject(s)
Bluetongue virus/isolation & purification , Polymerase Chain Reaction/methods , RNA, Bacterial/genetics , Base Sequence , Bluetongue virus/genetics , Molecular Sequence Data , RNA , Sensitivity and SpecificityABSTRACT
The double-stranded (ds) RNA genome segment 4 of bluetongue virus (BTV) serotype 2 was cloned and used as a serogroup-specific complementary (c) DNA probe for BTV diagnosis. A cDNA representing a 60% copy of genome segment 4 BTV-2 prototype was produced. The specificity of the cDNA probe was determined by hybridizing this probe to a northern blot of dsRNA (separated by polyacrylamide gel electrophoresis) of plaque-purified BTV-2 prototype. This cDNA probe was then used to hybridize to the RNA samples. Because the probe hybridized to all BTV samples but not to epizootic hemorrhagic disease virus samples, it appears to be a group-specific probe that could be used in BTV diagnosis.