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.

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.

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.

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.