ABSTRACT
Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G2254 constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G2254 nucleotide but with an A2254 nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A2254 and or G2254 strain was detected with real time PCR, and was confirmed with a rapid pyrosequencing technique. Pyrosequencing was useful in at least 2 cases where real time PCR was equivocal in determining the infecting EHV-1 strain type. The strain with G2254 mutation was detected in 9.4% of 21 studied abortion cases, and in 86.6% of 15 neurologic cases.
Subject(s)
Abortion, Spontaneous/virology , Herpesviridae Infections/veterinary , Herpesvirus 1, Equid/genetics , Horse Diseases/virology , Pathology, Molecular/methods , Polymorphism, Genetic , Spinal Cord Diseases/virology , Animals , Herpesviridae Infections/complications , Herpesviridae Infections/virology , Herpesvirus 1, Equid/isolation & purification , Horses , Real-Time Polymerase Chain Reaction/methods , Retrospective Studies , Sequence Analysis, DNA/methods , Tissue FixationABSTRACT
BACKGROUND: Highly pathogenic H5N1 avian influenza (AI) poses a grave risk to human health. An important aspect of influenza control is rapid diagnosis. OBJECTIVES: This study describes the efficiency of AI-RNA extraction utilizing silica-based magnetic beads with robotics and its detection with an influenza A matrix gene real-time RT-PCR from tracheal swabs, and compares it to virus isolation and manual spin column extractions. STUDY DESIGN: Analytical sensitivity was assessed by performing dilution analysis and detection of H2N2 AI viral RNA. Diagnostic sensitivity and specificity was assessed by analyzing tracheal swabs collected from H7N2 infected and uninfected chickens. RESULTS: Both manual and robotic extractions detected AI virus at 1log(10)EID(50)/ml. Diagnostic sensitivity and specificity of matrix gene detection with the automated extraction method for chicken tracheal swab specimens was similar to that of virus isolation and the manual extraction method. There were only three discordant results among 212 tested specimens. CONCLUSION: The main advantages of automated robotic viral nucleic acid extraction are high throughput processing; hands-free operation; and reduction in human and technical error. This study demonstrates successful detection of influenza A virus with magnetic beads utilizing the Qiagen MagAttract cell kit on a BioRobot M48 platform.
Subject(s)
Influenza A Virus, H5N1 Subtype/isolation & purification , Influenza in Birds/diagnosis , Polymerase Chain Reaction/methods , Animals , Automation , Chick Embryo , Chickens/virology , Influenza A Virus, H5N1 Subtype/genetics , Magnetics , Microspheres , RNA, Viral/isolation & purification , Robotics , Sensitivity and Specificity , Trachea/virologyABSTRACT
Influenza viruses bind host cells following an interaction between the viral hemagglutinin (HA) protein and host cell sialylated glycoproteins and glycolipids. Differences in binding affinities of the HAs for different types of sialic acid linkages (alpha2-3 vs. alpha2-6) contribute to determining the host range of an influenza virus. The ability of an avian influenza virus HA to bind the human form of the receptor may be one requirement for an avian virus to propagate in the human population. In this paper, we describe the characterization of the HA from an H2N2 virus isolated from a Pennsylvania chicken farm in 2004. Sequence analysis revealed that this HA is a member of the Eurasian clade, and receptor binding studies show that it maintains its specificity for the avian influenza virus alpha2-3 linked sialic acid receptor.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H2N2 Subtype/chemistry , Receptors, Cell Surface/metabolism , Receptors, Virus/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chickens/virology , Influenza A Virus, H2N2 Subtype/immunology , Molecular Sequence Data , Pennsylvania , PhylogenyABSTRACT
The expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the brains of cattle with natural rabies was studied. Increased expression of eNOS was detected in neurons of the brain stem and Purkinje cells of cerebellum. By contrast, iNOS was diffusely localized in the cytoplasm of affected neurons, and some inflammatory cells were positive. eNOS and rabies antigen were co-localized in inclusion bodies (Negri bodies) in neurons. The specific localization of eNOS, but not iNOS, in the Negri bodies suggests that eNOS is involved in the formation of rabies virus inclusion bodies.
Subject(s)
Cattle Diseases/metabolism , Gene Expression , Neurons/metabolism , Nitric Oxide Synthase/metabolism , Rabies/veterinary , Animals , Brain/metabolism , Cattle , Immunohistochemistry , Inclusion Bodies, Viral/metabolism , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Rabies/metabolismABSTRACT
West Nile virus (WNV) RNA was quantified in WNV infected crows and horses with the help of a real-time reverse transcriptase-PCR assay. A 5' nuclease assay, based on NS5 gene detection with a fluorescent probe was used for quantifying WNV RNA using formalin fixed paraffin embedded tissue specimens. Quantitative detection of WNV RNA showed the presence of a higher amount of the viral RNA in crow tissues compared to equine tissues and these results correlated well with the detection of WNV antigen by immunostaining. In crows, the highest amount of virus was seen in the intestine and in horses in the brain.
