Electronic microarray assays for avian influenza and Newcastle disease virus.

Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

A microsphere immunoassay for detection of antibodies to avian influenza virus.

A microsphere immunoassay (MIA) was developed for the detection of serum antibodies to avian influenza virus. A recombinant influenza A nucleoprotein expressed in baculovirus was conjugated to microspheres and incubated with antibodies. High median fluorescent intensities (MFIs) were obtained with a monoclonal antibody and positive chicken sera. Chickens were inoculated with 10 strains of avian influenza virus representing different subtypes, including high and low pathogenic H5 and H7 subtypes. Three hundred and fifty-four samples from experimentally infected chickens and controls were tested with a competitive ELISA (cELISA) and the MIA. MFIs were converted to positive/negative (PN) ratios. The results of both tests, as percentage inhibition and PN ratio, showed a high correlation (R2 = 0.77). From the comparison data, a ratio of > or =4.5 was selected as the cut-off value for positivity in the MIA. Using this cut-off value, the sensitivity and specificity of the MIA relative to the cELISA when all discordant experimental samples were retested was 99.3 and 93.1%, respectively. The relative specificity increased to 94.7% when additional negative sera (n = 68) were tested. The MIA may be useful for surveillance testing and as a screening test for flocks infected with low pathogenic avian influenza virus and could be expanded for simultaneous detection of antibodies against other avian infectious disease agents.

A one-step multiplex real-time RT-PCR for detection and typing of bovine viral diarrhea viruses.

A one-step multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SmartCycler technology and TaqMan probes was developed for detection and typing of bovine viral diarrhea viruses (BVDV). Common primers and type-specific (BVDV1 and BVDV2) TaqMan probes were designed in the 5'-untranslated region of the viral genome. The real-time assay was able to detect 10-100 TCID50 of virus, with correlation coefficient (r2) values of 0.998 and 0.999 for BVDV1 and BVDV2, respectively. The assay accurately typed 54 BVDV strains and field isolates and specificity of the TaqMan probes was further demonstrated by the lack of reactivity with the closely related Pestiviruses, classical swine fever virus and border disease virus. The assay was also shown to have high reproducibility. When the assay was compared with virus isolation for bovine serum samples, there was full agreement between the tests. Thus, the one-step real-time RT-PCR assay appears to be a rapid, sensitive, and specific test for detection and typing of BVDV.

A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses.

An oligonucleotide suspension microarray (Luminex microsphere system) was developed for detection and differentiation of animal pestiviruses: classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV1 and BVDV2), and border disease virus (BDV). Species-specific and pestivirus-common oligonucleotide probes were designed to the 5' UTR region and conjugated to individual color-coded Luminex carboxy beads (probe beads). Target pestivirus sequences were amplified by asymmetric PCR using a biotinylated reverse primer and a forward and reverse primer ratio of 1:5. The biotinylated products were hybridized to eight probe beads in a multiplex assay and analyzed using streptavidin conjugated to a fluorescent reporter molecule. The assay was able to detect and differentiate all 40 strains of CSFV, BVDV1, BVDV2 and BDV tested. The analytical sensitivity was determined to be 0.2-10 TCID50/ml. The major advantages of the DNA-microsphere suspension microarray, as a low density array, are its ease of handling and ability to simultaneously detect and type multiple infectious agents.

Microarray-based detection and typing of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is the most economically important veterinary pathogen because of its highly infectious nature and the devastating effects the virus has on the livestock industry. Rapid diagnostic methods are needed for detection and typing of FMDV serotypes and differentiation from other viruses causing vesicular diseases. We developed a microarray-based test that uses a FMD DNA chip containing 155 oligonucleotide probes, 35-45 base pair (bp) long, virus-common and serotype-specific, designed from the VP3-VP1-2A region of the genome. A set of two forward primers and one reverse primer were also designed to allow amplification of approximately 1100 bp of target sequences from this region. The amplified target was labelled with Alexa-Fluor 546 dye and applied to the FMD DNA chip. A total of 23 different FMDV strains representing all seven serotypes were detected and typed by the FMD DNA chip. Microarray technology offers a unique capability to identify multiple pathogens in a single chip.

Phylogeny and antigenic relationships of three cervid herpesviruses.

Elk herpesvirus (ElkHV) from North American elk (wapiti, Cervus elaphus nelsoni) is a recently identified alphaherpesvirus related to bovine herpesvirus-1 (BHV-1). In this study, we determined its relationship with European cervid herpesviruses: cervid herpesvirus-1 (CerHV-1) from red deer and rangiferine herpesvirus (RanHV) from reindeer. For phylogenetic analysis, genes for the gC and gD proteins of these viruses were sequenced. These genes demonstrated an extremely high GC content (76-79%). Genetically, ElkHV was found to be closely related to CerHV-1 and both viruses are more closely related to BHV-1 than to RanHV. Antigenically, the same relationships were found. ElkHV shares common neutralizing epitopes with both CerHV-1 and RanHV. A total of 10 epitopes were defined on the gB, gC and gD proteins of these viruses, including a shared neutralizing epitope on gD. The results indicate that ElkHV and CerHV-1 have diverged from a common ancestor virus. Cervid herpesviruses may be useful in determination of evolutionary rates of change for alphaherpesvirus genes.

Mapping of two antigenic domains on the NS3 protein of the pestivirus bovine viral diarrhea virus.

The immunodominant NS3 (p80) protein of the pestivirus bovine viral diarrhea virus (BVDV) functions as a serine protease and a RNA helicase. To identify antigenic domains of the BVDV NS3, a panel of monoclonal antibodies (mAbs) was tested against fragments of the protein expressed in E. coli. Two large overlapping NS3 fragments, A (amino acids [aa] 1-434) and B (aa 368-683) which together contain all NS3 sequences, were used to screen mAbs for reactivity. Two mAbs, 21.5.8 and 1.11.3, were reactive to fragment A (in ELISA only) and one mAb, 20.10.6, was reactive to fragment B (in ELISA and Western blotting). Further mapping demonstrated that the smallest fragment mAbs 21.5.8 and 1.11.3 bound to was comprised of aa 205-369 (domain A). In Western blotting, the smallest fragment reactive with mAb 20.10.6 was comprised of aa 368-549 (domain B). However, in indirect ELISA, mAb 20.10.6 also demonstrated high reactivity to a smaller fragment comprising aa 368-512 (domain B'). This indicated that the epitope of mAb 20.10.6 was conformational and not linear. Blocking ELISAs using these mAbs and type 1 and type 2 BVDV antisera demonstrated that an immunodominant region of the NS3 protein in cattle is defined by aa 205-549.

Bovine viral diarrhea virus in alpaca: abortion and persistent infection.

An alpaca herd in eastern Ontario experienced vague signs of illness, including anorexia and lethargy in 9 animals, 2.5 months after the addition of a chronically ill cria and his dam to the farm. Subsequently 2 alpaca had early pregnancy loss; one aborted at 5.5 months gestation and the other at 7 months gestation. Seventeen were found to have serum antibody to bovine viral diarrhea virus (BVDV), with highest titers to BVDV type 1. The fetus that was aborted at 5.5 months gestation, 3 months after the clinical outbreak, was found to be positive for BVDV on immunohistochemical staining, and noncytopathic BVDV type 1b was isolated. Of the 13 cria born alive that season, a single male underweight alpaca cria, born 9 months after the clinical illnesses, was infected with BVDV type 1b. The cria was positive for BVDV at birth, at 3 and 26 days of age and continued to be positive for noncytopathic BVDV using virus isolation, nested reverse transcription PCR, antigen detection ELISA, and immunohistochemical staining until euthanasia at 46 days of age. The cria remained serum antibody negative to both BVDV type 1 and type 2. A diagnosis of persistent infection was made. This is the first report describing persistent infection with BVDV in an alpaca cria.

Effect on hematopoietic tissue of experimental infection of calves with noncytopathic type 2 bovine viral diarrhea virus.

To investigate the hematologic abnormalities observed with noncytopathic type 2 bovine viral diarrhea virus (ncpBVDV-2), calves 6 to 8 mo old were inoculated with an isolate of either high virulence (HV24515) or low virulence (LV11Q); control animals received the same volume of uninfected cell-culture supernatant. Peripheral blood neutrophil, lymphocyte, and platelet counts decreased in all the virus-inoculated calves but were significantly lower and remained decreased longer in the calves given HV24515. For each isolate, a decrease in the number of mature myeloid cells in the bone marrow coincided with the development of neutropenia, but the depletion persisted significantly longer (4 to 6 d) in the calves given HV24515. In the bone marrow of calves given LV11Q, the number of proliferating myeloid cells increased in proportion to the decrease in the number of mature myeloid cells. In the calves inoculated with HV24515, BVDV antigen was observed in bone marrow cells when the peripheral blood counts were lowest. Megakaryocytes were the predominant cell type exhibiting positive BVDV staining; myeloid cells rarely stained positively. Viral antigen was not observed in the bone marrow of calves given LV11Q. These experiments demonstrated that ncpBVDV-2 isolates of both high and low virulence caused decreased leukocyte and platelet counts, but only the high-virulence HV24515 isolate caused a delay in the production of myeloid proliferating cells. The delay may contribute to the ability of certain ncpBVDV-2 isolates to induce severe disease.