Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein.

Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.

Role of chicken melanoma differentiation-associated gene 5 in induction and activation of innate and adaptive immune responses to infectious bursal disease virus in cultured macrophages.

The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 10(7) plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-ß (IFN-ß) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2', 5'-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1ß (IL-1ß) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 µM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-ß, PKR, OAS, Mx, IL-1ß, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-ß, PKR, OAS, Mx, IL-1ß, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-ß and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus.

Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-protein-expressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 × 10(2) times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p < 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-α, IFN-ß, IRF-1, IRF-10, IL-1ß, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-γ, IL-12(p40), IL-18, GM-CSF, GATA-3, p53, glucose transporter-2 and glucose transporter-3, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. Taken together, the results indicate that the bursal transcriptome involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport, except for granzyme K and CD8, was not differentially expressed in DNA-vaccinated chickens protected from IBDV challenge.

Genotyping of turkey coronavirus field isolates from various geographic locations in the Unites States based on the spike gene.

Turkey flocks have experienced turkey coronaviral enteritis sporadically in the United States since the 1990s. Twenty-four field isolates of turkey coronavirus (TCoV) from multiple states in the United States were recovered from 1994 to 2010 to determine the genetic relationships among them. The entire spike (S) gene of each TCoV isolate was amplified and sequenced. Pairwise comparisons were performed using the Clustal W program, revealing 90.0% to 98.4% sequence identity in the full-length S protein, 77.6% to 96.6% in the amino terminus of the S1 subunit (containing one hypervariable region in S1a), and 92.1% to 99.3% in the S2 subunit at the deduced amino acid sequence level. The conserved motifs, including two cleavage recognition sequences of the S protein, two heptad repeats, the transmembrane domain, and the Golgi retention signal were identified in all TCoV isolates. Phylogenetic analysis based on the full-length S gene was used to distinguish North American TCoV isolates from French TCoV isolates. Among the North American TCoV isolates, three distinct genetic groups with 100% bootstrap support were observed. North Carolina isolates formed group I, Texas isolates formed group II, and Minnesota isolates formed Group III. The S genes of 24 TCoV isolates from the United States remained conserved because they contained predominantly synonymous substitutions. The findings of the present study suggest endemic circulation of distinct TCoV genotypes in different geographic locations.

Chicken melanoma differentiation-associated gene 5 (MDA5) recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity.

The objective of the present study was to determine if chicken melanoma differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus (IBDV) infection to initiate and amplify an innate immune response in the chicken MDA5 (chMDA5) signaling pathway. Chicken embryo fibroblast DF-1 cells were infected with IBDV LP1 at a multiplicity of infection (MOI) of 0.5 or 10. In addition, knockdown and overexpression of chMDA5 were performed by transfecting DF-1 cells with chMDA5-targeting small interfering RNA (siRNA) or chMDA5-expressing DNA. The transfected cells were infected with IBDV LP1 at an MOI of 10. Cell culture supernatants and lysates were collected at 2, 8, 16 and 24 hours postinfection (hpi) for IBDV titer determination and RNA extraction, respectively. IBDV RNA loads and mRNA expression levels of chicken MDA5, interferon-ß (IFN-ß) promoter stimulator 1 (IPS-1), interferon regulatory factor-3 (IRF-3), IFN-ß, double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase (OAS), myxovirus resistance gene (Mx), and major histocompatibility complex class I (MHC class I) were determined by real-time RT-PCR. The IBDV titer increased up to 1.4 × 10(7) plaque-forming units (PFU)/mL at 24 hpi, and the IBDV RNA load reached 464 ng/µL at 24 hpi. The mRNA expression levels of chicken MDA5, IRF-3, IFN-ß, PKR, OAS, Mx and MHC class I in IBDV-infected DF-1 cells exhibited significant (p < 0.05) upregulation up to 906-, 199-, 26,310-, 12-, 66,144-, 64,039- and 33-fold, respectively. Expressed chMDA5 from transfection and double-stranded RNA from IBDV infection were localized or colocalized in the cytoplasm of DF-1 cells at 16 hpi. When chMDA5 was knocked down in DF-1 cells, IBDV titers and RNA loads were significantly higher (p < 0.05) than those in DF-1 cells without chMDA5 knockdown at 24 hpi. The expression levels of chicken MDA5, IRF-3, IFN-ß and MHC class I in chMDA5-knockdown DF-1 cells were significantly lower (p < 0.05) at 16 and 24 hpi. DF-1 cells overexpressing chMDA5 by transfection with chMDA5 expressing DNA had significantly lower (p < 0.05) IBDV titers and RNA loads at 16 and 24 hpi and showed significantly higher (p < 0.05) expression of chicken MDA5, IRF-3, IFN-ß, PKR, OAS, Mx and MHC class I at 2 hpi. The results indicated that chicken MDA5 recognized IBDV infection and that this interaction resulted in the activation of chMDA5-related innate immune genes and upregulation of chicken MHC class I.

An influenza A virus hemagglutinin (HA) epitope inserted in and expressed from several loci of the infectious bursal disease virus genome induces HA-specific antibodies.

The N-terminus of infectious bursal disease virus (IBDV) VP5 has been shown to be capable of tolerating the insertion of small epitopes. The objective of the present study was to determine if IBDV genomic sites, including the 5' end of vp5, could carry an influenza A virus hemagglutinin (HA) epitope. HA-expressing IBDVs were generated when the HA epitope was fused to the N-terminus of VP5 (HA5-IBDV) or VP4 (HA4-IBDV) or the C-terminus of VP1 (1HA-IBDV). Viral titers obtained after co-transfection with cDNA from the ha-containing segment and the complementary genomic segment were 1.3 × 10(4), 3.7 × 10(3) and 3.8 × 10(4) pfu/ml for HA5-IBDV, HA4-IBDV and 1HA-IBDV, respectively. The HA tag expression remained stable after 10 passages when the tag gene was inserted into the vp4 and vp1 genes. HA-IBDVs did not cause pathogenicity in specific-pathogen-free (SPF) chickens. However, only HA4-IBDV and 1HA-IBDV induced HA-specific antibodies, which were measured by ELISA with a maximum optical density (OD) value of 0.701 and 0.769, respectively, at 24 days after infection. Thus, IBDV can potentially be employed as a bivalent viral vector when the epitope is fused with VP4 or VP1.

Identification and characterization of a neutralizing-epitope-containing spike protein fragment in turkey coronavirus.

Little is known about the neutralizing epitopes in turkey coronavirus (TCoV). The spike (S) protein gene of TCoV was divided into 10 fragments to identify the antigenic region containing neutralizing epitopes. The expression and antigenicity of S fragments was confirmed by immunofluorescence antibody (IFA) assay using an anti-histidine monoclonal antibody or anti-TCoV serum. Polyclonal antibodies raised against expressed S1 (amino acid position 1 to 573 from start codon of S protein), 4F/4R (482-678), 6F/6R (830-1071), or Mod4F/Epi4R (476-520) S fragment recognized native S1 protein and TCoV in the intestines of TCoV-infected turkey embryos. Anti-TCoV serum reacted with recombinant 4F/4R, 6F/6R, and Mod4F/Epi4R in a western blot. The results of a virus neutralization assay indicated that the carboxyl terminal region of the S1 protein (Mod4F/Epi4R) or the combined carboxyl terminal S1 and amino terminal S2 protein (4F/4R) possesses the neutralizing epitopes, while the S2 fragment (6F/6R) contains antigenic epitopes but not neutralizing epitopes.

Infectious bursal disease DNA vaccination conferring protection by delayed appearance and rapid clearance of invading viruses.

The present study was undertaken to determine the kinetics of viral load and immune response in protection against infectious bursal disease virus (IBDV) by DNA vaccination. Chickens were DNA-vaccinated and challenged with IBDV one week after the third vaccination. Tissues were collected at 12 hours postinfection (HPI), 1 day postinfection (DPI), 3, 5, 7 and 10 DPI. The vaccinated chickens had less viral RNA, with delayed appearance and shorter duration in the bursa of Fabricius, spleen, and cecal tonsil than the challenged control chickens. Their ELISA and neutralizing antibody titers were decreased at 12 HPI and significantly lower (P < 0.05) than those in the challenged control chickens at later time points. Their spleen IFNγ expression was up-regulated compared to that in the DNA-vaccinated chickens without IBDV challenge. These results indicate that DNA vaccination confers protection against IBDV challenge by delayed appearance and rapid clearance of the invading viruses.

Detection of antibodies against Leptospira serovars via microscopic agglutination tests in dogs in the United States, 2000-2007.

OBJECTIVE: To use results of microscopic agglutination tests (MATs) conducted at a commercial veterinary diagnostic laboratory to determine temporal and demographic distributions of positive serologic test results for leptospirosis in dogs and identify correlations among results for various Leptospira serovars. DESIGN: Serosurvey. STUDY POPULATION: MAT results for 33,119 canine serum samples submitted to a commercial veterinary diagnostic laboratory from 2000 through 2007. PROCEDURES: Electronic records of MAT results for dogs were obtained from a veterinary diagnostic laboratory. Seropositivity for antibodies against Leptospira serovars was determined by use of a cutoff titer of >or=1:1,600 to reduce the possible impact of postvaccinal antibodies on results. Correlations between results for all possible pairs of serovars were calculated by ordinal ranking of positive (>or=1:100) antibody titer results. RESULTS: 2,680 samples (8.1%; 95% confidence interval [CI], 7.8% to 8.4%) were seropositive for antibodies against Leptospira serovars. The highest percentage of positive MAT results was for the year 2007 (10.2%; 95% CI, 9.5% to 10.9%) and for the months of November and December during the study period. Antibodies were most common against serovars Autumnalis, Grippotyphosa, Pomona, and Bratislava. Seroprevalence of leptospirosis was lowest for dogs>10 years of age but was similar across other age strata. CONCLUSIONS AND CLINICAL RELEVANCE: Leptospirosis can affect dogs of small and large breeds and various ages. Although an increase in proportions of positive MAT results was evident in the fall, monthly and annual variations suggested potential exposure in all months. Because of the limitations of MAT results and the limited number of serovars used in the test, bacterial culture should be used to identify infective Leptospira serovars.

Real-time PCR, compared to liquid and solid culture media and ELISA, for the detection of Mycobacterium avium ssp. paratuberculosis.

A goal of Johne's disease control programs is to accurately detect Mycobacterium avium ssp. paratuberculosis (MAP) infected cattle as quickly as possible to reduce disease transmission. A newly introduced real-time PCR provides results rapidly, but its accuracy in the field has not been evaluated. Fecal and serum samples collected from dairy cows in northern Indiana were used to estimate the sensitivity and specificity of a newly licensed real-time PCR test for direct fecal detection of Mycobacterium avium ssp. paratuberculosis (MAP). Results of the real-time PCR were evaluated in parallel with solid and liquid media culture systems and a serum ELISA for detection of MAP antibodies to determine the accuracy of the real-time PCR and the tests' potential usefulness in the field. A total of 143 samples were tested by all four methods. Using prior published estimates for sensitivity and specificity of each of the tests and Bayesian methodology, the sensitivity and specificity of the real-time PCR test was estimated to be 0.60 and 0.97, respectively. The accuracy of real-time PCR (0.90) was comparable to both solid (0.91) and liquid (0.93) culture. Because real-time PCR accuracy is comparable to standard culture methods, it is a useful new test. In addition, test results are obtained as rapidly as an ELISA, but are more accurate than the ELISA (0.82). This makes real-time PCR an attractive test and should shorten the quarantine period required for new purchases of unknown MAP-status animals into herds participating in an MAP control program.

Antimicrobial susceptibility and resistance of chicken Escherichia coli, Salmonella spp., and Pasteurella multocida isolates.

Escherichia coli, Salmonella species, and Pasteurella multocida are the major bacterial pathogens isolated from poultry. Difference in susceptibility to antibiotics by microorganisms has become a major factor in drug choice and success of treatment. Great concerns have been raised regarding emerging antimicrobial resistance among bacteria that may result in unpredictable antimicrobial susceptibility and failure of therapy. The primary objective of the present study was to determine the levels of antimicrobial susceptibility/resistance of E. coli, Salmonella species, and P. multocida isolated from diseased chickens. A total of 445 E. coli isolates, 387 Salmonella spp. isolates, and 80 P. multocida isolates from diseased chickens during the period ranging from 2001 to 2003 were obtained. Minimal inhibitory concentrations of 14 antimicrobial agents against each bacterial isolate were determined using a microbroth dilution assay described by the Clinical Laboratory Standards Institute. Resistance of E. coli isolates measured as follows: 98.20% were resistant to tilmicosin, 79.33% to tetracycline, 51.46% to spectinomycin, 44.04% to gentamicin, and 40% to ampicillin. Resistance to tetracycline was found in 72.61% of Salmonella spp. isolated, followed by resistance to spectinomycin (68.48%), ampicillin (63.57%), gentamicin (63.31%), and ticarcillin (61.76%). The resistance rate of P. multocida isolates to all antimicrobials tested was less than 5%, except for tetracycline (6.25%). In summary, E. coli and Salmonella isolates were sensitive to ceftiofur and fluoroquinolones but were resistant to other antimicrobials tested, while P. multocida isolates remained sensitive to all the antimicrobial agents tested in a 3-yr analysis.

Complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus.

The complete nucleotide sequence of polyprotein gene 1 and the assembled full-length genome sequence are presented for turkey coronavirus (TCoV) isolates 540 and ATCC. The TCoV polyprotein gene encoded two open reading frames (ORFs), which are translated into two products, pp1a and pp1ab, the latter being produced via -1 frameshift translation. TCoV polyprotein pp1a and pp1ab were predicted to be processed to 15 non-structure proteins (nsp2-nsp16), with nsp1 missing. ClustalW analysis revealed 88.99% identity and 96.99% similarity for pp1ab between TCoV and avian infectious bronchitis virus (IBV) at the amino acid level. The whole genome consists of 27,749 nucleotides for 540 and 27,816 nucleotides for ATCC, excluding the poly(A) tail. A total of 13 ORFs were predicted for TCoV. Five subgenomic RNAs were detected from ATCC-infected turkey small intestines by Northern blotting. The whole genome sequence had 86.9% identity between TCoV and IBV, supporting that TCoV is a group 3 coronavirus.

Turkey coronavirus non-structure protein NSP15--an endoribonuclease.

UNLABELLED: Turkey coronavirus (TCoV) polyprotein was predicted to be cleaved into 15 non-structural proteins (nsp2 to nsp16), but none of these nsps have been characterized. TCoV nsp15 consists of 338 residues and shares 40% sequence similarity to U-specific Nidovirales endoribonuclease (NendoU) of severe acute respiratory syndrome coronavirus. OBJECTIVE: The purpose of the present study was to characterize TCoV nsp15. METHODS: The TCoV nsp15 gene was cloned into pTriEX1 and expressed as a C-terminal His-tagged recombinant protein in BL21 (DE3). The recombinant nsp15 was purified by Ni-NTA resin. Synthetic RNA substrates were used to determine the substrate specificity of the TCoV nsp15. RNA zymography was used to determine the active form of the nsp15. RESULTS: The TCoV nsp15 did not cleave DNA but degraded total cellular RNA. The TCoV nsp15 cleaved single-stranded (ss) RNA at the uridylate site. The TCoV nsp15 cleaved hairpin RNA, pRNA, and double-stranded RNA (dsRNA) of infectious bursal disease virus very slowly, implying that dsRNA is not a good substrate for the TCoV nsp15. No divalent metal ion was required for in vitro enzymatic activity of the TCoV nsp15. The active form of the TCoV nsp15 was a homohexamer and disulfide bond was essential for the enzymatic activity. CONCLUSION: The TCoV nsp15 is a NendoU but has some characteristics different from other NendoU.

Hypertrophic osteopathy associated with mycotic pneumonia in two juvenile elk (Cervus elaphus).

Two yearling bull elk (cervus claphus) from the same farm developed anorexia, weight loss, and lameness. On physical examination, both elk were thin and showed diffuse swelling of all lower limbs. Radiographs of the lower limbs showed periosteal thickening of the distal extremities, consistent with hypertrophic osteopathy. Thoracic radiographs indicated the presence of pulmonary nodules. Cytologic evaluations of tracheal washes on both elk were consistent with inflammation. Acid-fast stains on both samples were negative. Because of the poor prognosis, both elk were euthanized. At necropsy, the carpal, metacarpal, tarsal, and metatarsal bones, as well as the radius, ulna, and tibia had thickening of cortical bone. There were multiple encapsulated nodules throughout the lungs, lymph nodes, and kidney, and smaller nodules in the myocardium. On microscopic examination, these nodules contained myriads of hyphae, and immunohistochemistry for Aspergillus sp. was strongly positive. Aspergillus fumigatus was isolated from affected tissue in 1 elk. Necropsy findings in both elk were consistent with disseminated fungal granulomas and periosteal hyperostosis. This case presents the first description of hypertrophic osteopathy in elk. The source of infection was undetermined, but inhalation of spores from contaminated feed or bedding was suspected.

Systemic nocardiosis in a reindeer (Rangifer tarandus tarandus).

Systemic infection by Nocardia asteroides was diagnosed in a reindeer (Rangifer tarandus tarandus). At necropsy, the animal had enlarged tracheobronchial lymph nodes, pleural and peritoneal effusions, and numerous, multifocal to coalescing, yellow, firm nodules with inspissated pus in the lung, pleura, omentum, liver, heart, adrenal glands, and left kidney. Microscopically, the nodules consisted of foci of pyogranulomatous inflammation. Microscopic lesions were present in the grossly affected organs as well as spleen and brain. Sections stained with Gram and modified Fite-Faraco histochemical stains had numerous gram-positive, variably acid fast, beaded and branching filamentous organisms in the necrotic centers. N. asteroides was isolated from the lung, bronchial lymph nodes, liver, and left kidney. To the authors' knowledge, this is the first report of systemic N. asteroides infection in a reindeer.

Nocardia tenerifensis genome identification in a cutaneous granuloma of a cat.

An adult Domestic Longhair cat developed a subcutaneous mass in its tail. Histologically, this mass consisted of ill-defined pyogranulomas centered around aggregates of gram-positive, acid-fast filamentous bacteria, consistent with Nocardia. Due to the lack of fresh samples, DNA was extracted from formalin-fixed paraffin-embedded tissue sections and subjected to polymerase chain reaction amplification and DNA sequencing of 16S ribosomal RNA gene encompassing Nocardia sp.-specific sequences. Sequences analyzed using the GenBank database revealed 99.5% homology with Nocardia spp. and had the highest sequence homology of 98.2% with Nocardia tenerifensis among Nocardia spp. To the authors' knowledge, this is the first report of detection of N. tenerifensis genome associated with cutaneous nocardiosis in an animal.

Molecular detection and differentiation of infectious bursal disease virus.

Vaccination of hens, with the subsequent maternal immunity imparted to chicks, is the primary means of controlling infectious bursal disease virus (IBDV). Effective vaccination depends on rapid and accurate diagnosis of the subtype present in a flock because vaccines based on the classic subtype of IBDV can fail to protect against challenge with a variant subtype. This review describes the various methods available to detect and differentiate between IBDV subtypes. Serotype 1 IBDV causes economically significant immunosuppressive disease in young chickens. Within serotype 1, two subtypes, classic and variant, can be differentiated by the virus neutralization assay. Antigen capture enzyme-linked immunosorbent assay (AC-ELISA) with MAbs has been successful at differentiating the very virulent IBDV phenotype (vvIBDV) from less pathogenic types. More rapid and sensitive molecular diagnostic methods based on reverse transcription-polymerase chain reaction (RT-PCR) for amplification of the IBDV VP2 gene have been a major focus of investigation in recent years. Conventional RT-PCR has been useful in detecting IBDV serotypes and, to a lesser extent, differentiating IBDV subtypes. One of the approaches has been the use of SspI and NgoM IV restriction enzymes, for restriction endonuclease (RE) analysis of RT-PCR products (RT-PCR-RE) and BstNI and MboI for restriction fragment length polymorphism (RFLP) analysis (RT-PCR-RFLP) to find unique banding patterns associated with antigenic variation within the variable region of the IBDV VP2 protein. However, these approaches were ultimately found to be unreliable because subtypes could not be consistently distinguished with restriction enzymes. These limitations led to studies in differentiating subtypes by detection of single nucleotide differences in sequence through real-time RT-PCR or DNA sequencing of RT-PCR products. Conventional RT-PCR, amplifying the VP2 hypervariable region, in combination with DNA sequencing of the PCR product, can differentiate classic, variant, and vvIBDV strains because variant and vvIBDV have characteristic nucleotide and amino acid substitutions. Real-time RT-PCR, targeting different regions of the IBDV genome, including VP1, VP2, and VP4 genes, in conjunction with melting-curve analysis is being investigated as a promising tool for molecular diagnosis of IBDV infection. These methods potentially allow for more rapid, sensitive, and specific detection and differentiation of IBDV classic, very virulent, and variant subtypes.

A free VP3 C-terminus is essential for the replication of infectious bursal disease virus.

Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3' noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3' NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication.

Hepatic yersiniosis in a cougar (Felis concolor).

A cougar (Felis concolor) was diagnosed with hepatic yersiniosis by bacterial culture and histopathology. The animal had a 2-week history of anorexia and jaundice before its death. Grossly, the liver exhibited caseo-necrotic foci. Histopathologically, there was necrotizing and suppurative hepatitis, with large numbers of intralesional gram-negative coccobacilli. Additional hepatic lesions included central vein thrombosis, lymphoplasmacytic portal hepatitis, and capsulitis. Yersinia pseudotuberculosis coccobacilli were isolated in pure culture from the hepatic lesions. Because the hepatic lesions in this animal resemble those of other zoonotic diseases, such as plague and tularemia, veterinarians and laboratory personnel who handle samples should take adequate safety precautions. This report is the first to describe the pathology associated with hepatic yersiniosis in a cougar.