Cloning and expression of equine NF-kB2

Equine infectious anemia virus (EIAV) is a macrophage-tropic retrovirus that causes persistent disease in horses and ponies. In addition to its structural proteins, EIAV encodes four regulatory/accessory genes, tat, rev, ttm, and S2. It has been documented EIAV S2 gene expression is essential for disease expression of EIAV. Using a yeast two-hybrid assay, it was shown that S2 protein interacts with human NF-KB2. NF-KB2 plays a key role in the alternative or non-canonical NF-KB pathway. In order to determine if the interaction of S2 with NF-KB2 might be relevant to equine disease, a cDNA representing full length equine NF-KB2 was generated in our laboratory using PCR and rapid amplification of cDNA ends. To our knowledge this is the first time that equine NF-KB2 cDNAs have been recovered and characterized. The sequence of equine NF-KB2 was 95% homologous to human overall, however a major difference was found in the ankyrin repeat region where protein-protein interactions occur. Two splice variants of equine NF-KB2 were found that correspond to splice variants of human NF- KB2. We tested the interaction of EIAV S2 and equine NF-KB2 using the yeast two hybrid system (Y2H) and co-immunoprecipitation. Unfortunately we were not able to detect an interaction between EIAV S2 and equine NF-KB2 in either system. Despite this result, NF-KB2 is an important component in the immune response so we examined its expression in equine macrophages. Moreover we were interested to know if EIAV might affect expression levels of equine NF-KB2, as NF-KB2 is a target of other viruses. Hence, the expression level of equine NF-KB2 was measured in uninfected and infected primary equine monocyte- derived macrophage (eMDM). Using quantitative PCR we determined that equine NF-KB2 gene expression is decreased in eMDM after 3 days post plating, about the time that monocytes start to differentiate into mature macrophages. However EIAV infection of eMDM upregulated the expression level of NF-KB2

Molecular Detection and Characterization of Avian Bornavirus

Proventricular dilatation disease (PDD) was first recognized during an outbreak among captive macaws in the late 1970s. The disease, also known as proventricular dilatation syndrome or macaw wasting disease can occur in any psittacine but the most commonly affected birds are macaws, cockatoos and conures. The disease causes inflammation of the central, peripheral and autonomic nervous systems, as well as weight loss associated with regurgitation and the passage of undigested food in the feces. Although a viral etiology for PDD has been suspected for almost 40 years, the etiologic agent of the disease was unknown until lately. Recently we cultured a novel bornavirus from brain tissue from birds clinically diagnosed with PDD. This finding supports data from other groups who, in 2008, identified bornavirus sequences among birds suffering from PDD. It was reported that more 60 percent of PDD affected birds were infected with the new virus, designated avian bornavirus (ABV). ABV is a negative sense, single stranded RNA virus related to Borna disease virus (BDV). ABV isolates differ dramatically from BDV isolates in their level of genetic variation. Using polymerase chain reaction (PCR) assays, we were able to detect ABV in feces and tissue of PDD birds. We also detected ABV shedding from clinically healthy birds housed in aviaries with no history of the disease. We also determined the complete genome sequences of eight North American ABV isolates. Genotyping indicates that the majority of North American ABV isolates are genotype 4. We found one ABV, genotype 1, which is the first complete sequencing of this genotype. Moreover, we found ABV genotype 2, isolated from an apparently healthy cockatiel with no PDD clinical signs. In order to investigate whether this genotype is avirulent, the virus was grown in duck embryo fibroblasts and inoculated into two adult cockatiels by the oral and intramuscular routes. One bird developed clinical signs on day 33 and was euthanized on day 36. The second challenged bird developed clinical signs on day 41 and was euthanized on day 45. On necropsy, the proventriculus of both birds was slightly enlarged and microscopic examination showed lesions consistent with PDD in the brain, spinal cord, heart, adrenal gland and intestine. A control, uninoculated cockatiel was apparently healthy when euthanized on day 50. ABV2 is now the second ABV genotype to be formally shown to cause PDD

Complete Genome Sequence of Avian Bornavirus Genotype 1 from a Macaw with Proventricular Dilatation Disease

Avian bornaviruses (ABV) were first detected and described in 2008. They are the etiologic agents of proventricular dilatation disease (PDD), a frequently fatal neurologic disease of captive parrots. Seven ABV genogroups have been identified worldwide from a variety of sources, and that number may increase as surveillance for novel bornaviruses continues. Here, we report the first complete sequence of a genogroup 1 avian bornavirus (ABV1)

Use of Avian Bornavirus Isolates to Induce Proventricular Dilatation Disease in Conures

The fulfillment of Koch’s postulates shows that the virus causes proventricular dilatation disease in parrots