I. Aini

Pathogenicity of SspI-positive infectious bursal disease virus and molecular characterization of the VP2 hypervariable region

The pathogenicity of four isolates of infectious bursal disease virus (IBDV) that have restriction fragment length polymorphism patterns of very virulent IBDV (vvIBDV), based on the presence of SspI and TaqI sites in the VP2 hypervariable region, was studied in specific pathogen free chickens. Chickens inoculated with isolates 92/04, 94/B551 and 97/61 developed severe clinical signs with a high mortality ranging from 70 to 80%, whereas the 94/273 isolate caused 10% mortality. Regardless of the isolates, significant differences were noted in the bursal lesion scores and bursa:body weight ratio index in the infected groups in comparison with the control groups. However, the presence of lesions in non-bursal tissues, muscles, thymus and at the junction of the proventriculus and gizzard were found only in the 92/04, 97/61 and 94/B551 isolates. Restriction fragment length polymorphism and sequence analysis of the VP2 hypervariable region indicated that all the isolates can be classified as vvIBDV based on the presence of SspI and TaqI sites at nucleotide positions 1011 and 833, respectively. In addition, all the isolates had amino acid substitutions at P222A, V256I and L294I, which are characteristic for vvIBDV isolated from different parts of the world. All the isolates except 94/273 also had a StyI site at nucleotide position 888. The absence of a StyI site in this isolate was associated with amino acid substitution at 254 from G to S. The 94/273 also had an amino acid substitution at position 270 from A to E, which is variable in the STC, Cu1 and OH strains. The presence of amino acid substitutions from G254S andA270E in SspI- and TaqI-positive vvIBDV strains is very uncommon and has not been reported previously. These amino acid variations might have caused the 94/273 to become less virulent in specific pathogen free chickens and resemble a classical virulent IBDV strain.

Sequence analysis of both genome segments of two very virulent infectious bursal disease virus field isolates with distinct pathogenicity.

Summary.The deduced amino acid sequences of segment A and B of two very virulent Infectious bursal disease virus (vvIBDV) isolates, UPM94/273 and UPM97/61 were compared with 25 other IBDV strains. Twenty amino acid residues (8 in VP1, 5 in VP2, 2 in VP3, 4 in VP4, 1 in VP5) that were common to vvIBDV strains were detected. However, UPM94/273 is an exceptional vvIBDV with usual amino acid substitutions. The differences in the divergence of segment A and B indicated that the vvIBDV strains may have been derived from genetic reassortment of a single ancestral virus or both segments have different ability to undergo genetic variation due to their different functional constraints.

Pathogenicity and Molecular Analysis of an Infectious Bursal Disease Virus Isolated from Malaysian Village Chickens

Abstract The characteristics of the pathogenic infectious bursal disease virus (IBDV) that infected avian species other than commercial chickens were largely unknown. In this study, by using in vivo and molecular methods, we had characterized an IBDV isolate (named 94268) isolated from an infectious bursal disease (IBD) outbreak in Malaysian village chickens—the adulterated descendant of the Southeast Asian jungle fowl (Gallus bankiva) that were commonly reared in the backyard. The 94268 isolate was grouped as the very virulent IBDV (vvIBDV) strain because it caused severe lesions and a high mortality rate in village chickens (>88%) and experimentally infected specific-pathogen-free chickens (>66%). In addition, it possessed all of the vvIBDV molecular markers in its VP2 gene. Phylogenetic analysis using distance, maximum parsimony, and maximum likelihood methods revealed that 94268 was monophyletic with other vvIBDV isolates and closely related to the Malaysian vvIBDV isolates. Given that the VP2 gene of 94268 isolate was almost identical and evolutionarily closely related to other field IBDV isolates that affected the commercial chickens, we therefore concluded that IBD infections had spread across the farm boundary. IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease.

Pathogenicity, sequence and phylogenetic analysis of Malaysian Chicken anaemia virus obtained after low and high passages in MSB-1 cells

Summary. Specific-pathogen-free (SPF) chickens inoculated with low passage Chicken anaemia virus (CAV), SMSC-1 and 3-1 isolates produced lesions suggestive of CAV infection. Repeated passages of the isolates in cell culture until passage 60 (P60) and passage 123 produced viruses that showed a significantly reduced level of pathogenicity in SPF chickens compared to the low passage isolates. Sequence comparison indicated that nucleotide changes in only the coding region of the P60 passage isolates were thought to contribute to virus attenuation. Phylogenetic analysis indicated that SMSC-1 and 3-1 were highly divergent, but their P60 passage derivatives shared significant homology to a Japanese isolate A2.

Reverse transcriptase-polymerase chain reaction-enzyme linked immunosorbent assay for rapid detection of avian influenza virus subtype H9N2

Summary.The performance of a simplified nucleoprotein (NP) and hemagglutinin-subtype-9 (H9) based reverse transcriptase-polymerase chain reaction-enzyme linked immunosorbent assay (RT-PCR-ELISA) for the detection of avian influenza virus (AIV) subtype H9N2 was compared to the standard the virus isolation method and serology testing using hemagglutination (HA) and hemagglutination inhibition (HI) tests. The H9-based RT-PCR-ELISA was 100% sensitive when compared to virus isolation method in detecting H9N2 from experimentally infected specific-pathogen-free (SPF) chickens. The NP- and H9-based RT-PCR-ELISA have a detection limit similar to the virus isolation method in detecting serially diluted tracheal swab samples obtained from chickens inoculated with H9N2. Both RT-PCR-ELISAs were also ten times more sensitive than agarose gel electrophoresis for the detection of PCR products. The result of this study demonstrate that the developed RT-PCR-ELISA is a simple and sensitive assay for the detection of type A influenza virus, particularly AIV subtype H9N2, in chickens.

Sequence Analysis of Malaysian Infectious Bursal Disease Virus Isolate and the Use of Reverse Transcriptase Nested Polymerase Chain Reaction Enzyme-Linked Immunosorbent Assay for the Detection of VP2 Hypervariable Region

SUMMARY. The VP2 hypervariable region of P97/302 local infectious bursal disease virus (IBDV) isolate was amplified by the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and cloned. This region of P97/302 local isolate was sequenced and compared with eight other reported IBDV sequences. The result showed that P97/302 IBDV was most identical to the reported very virulent IBDV strains because it has amino acid substitutions at positions 222, 256, 294, and 299, which encode alanine, isoleucine, isoleucine, and serine, respectively. This region can be digested with restriction enzymes of Taq1, Sty1, Ssp1 but not with Sac1. The P97/302 isolate was then used for the optimization of RT nested PCR enzyme-linked immunosorbent assay (ELISA). The RT nested PCR ELISA was able to detect 10−4 dilution of the infected bursa homogenates and was 10 times more sensitive when compared with the agarose gel detection method. The RT nested PCR ELISA can detect up to 0.48 ng of the PCR product. The specificity of this nested PCR ELISA was also high (100%).

Pathogenicity of Fowl Adenovirus in Specific Pathogen Free Chicken Embryos

Inclusion body hepatitis (IBH) associated with fowl adenovirus (FAdV) infection has a worldwide distribution. The aim of the present study was to determine the pathogenicity of Malaysian FAdV serotype 9 (UPM04217) in specific pathogen free (SPF) embryonated chicken embryos. FAdV (titre 10(5.8)/ml) was inoculated into SPF embryonated chicken eggs (0.1 ml per egg) via the chorioallantoic membrane (CAM). There was 100% embryo mortality within 4-11 days post infection (dpi). The gross and microscopical lesions of the embryo were confined to the liver and were noted at 5, 7, 9 and 11 dpi. The liver was pale with multifocal areas of necrosis, fibrosis and haemorrhage. Microscopically, there was moderate to severe congestion and haemorrhage and severe and diffuse hepatocyte degeneration and necrosis, with intranuclear inclusion bodies (INIBs) and associated inflammation. Haemorrhage, congestion, degeneration, necrosis and hyperplasia of the CAM with INIBs were observed at 5, 7, 9 and 11 dpi. Varying degrees of congestion, haemorrhage, degeneration and necrosis were also observed in the yolk sac, kidney, spleen, heart and bursa of Fabricius. Ultrastructurally, numerous viral particles in the nucleus of hepatocytes were recorded at 7, 9 and 11 dpi, whereas at 5 dpi, fine granular and filamentous INIBs were observed. The INIBs in the CAM were present either as fine granular filamentous structures or as large viral inclusions. FAdV (UPM04217) is therefore highly pathogenic to SPF chicken embryos and the embryonic liver should be used for isolation and propagation of the virus.

Cloning and expression of highly pathogenic avian influenza virus full-length nonstructural gene in Pichia pastoris.

Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).

Immunochromatographic gold-based test strip for rapid detection of Infectious bursal disease virus antibodies.

The immunochromatographic assay is an alternative method for simple and rapid detection of Infectious bursal disease virus (IBDV) in chickens using colloidal gold—antibody conjugate. The whole-virus antigen of IBDV (UPM04190 isolate) and the high-affinity polyclonal antibodies directed against IBDV were blotted onto nitrocellulose membranes for test and control lines, respectively. Evaluation of the strip was performed using serum samples from experimentally and naturally infected chickens. The results showed that the test strip was more sensitive than the commercial enzyme-linked immunosorbent assay (ELISA) because it could detect a dilution factor up to 1:20,000 (250 ELISA units) for positive samples. It was also specific, in that it detected IBDV antibodies and did not cross-react with antibodies to other chicken viruses. The method was rapid (2 min) in both clinical and field environments with samples needing only a minimum amount (50 μl) of blood to produce an acceptable detection signal. The pen—site test strip proved successful in monitoring the immune status of chickens against the IBDV infection.

Sequence and phylogenetic analysis of the VP2 gene of very virulent infectious bursal disease virus isolates.

Previously we have shown that very virulent infectious bursal disease viruses (vvIBDV) that are SspI, TaqI and StyI positive (92/04, 97/61 and 94/B551) but not SspI and TaqI positive and StyI negative (94/273) cause high mortality, up to 80% in specific-pathogen-free chickens with significant damage of the bursal as well as nonbursal tissues. In this study, we sequenced the VP2 gene (1351 bp) of the 92/04, 94/273 and 94/B551 and compared them with other IBDV strains. All the isolates have the unique amino acid residues at positions 222A, 256I, 294I and 299S found in other vvIBDV strains. The deduced VP2 amino acids encoded by 92/04 is identical to the vvIBDV strains from Israel (IBDVKS), Japan (OKYM) and Europe (UK661), whereas the 94/273 and 94/B551 isolates have one to three amino acid substitutions. The 94/273 has two amino acid substitutions at positions 254 G to S and at 270 A to E that have not been reported before from vvIBDV strains. The 94/B551 also has one amino acid substitution at position 300 E to S, which is uncommon among other vvIBDV strains. However, phylogenetic analysis suggested that the isolates are very close to each other and all of them may have derived from the same origin as vvIBDV strains isolated from China, Japan and Europe. Even though antigenic index analysis of the 94/273 and 94/B551 indicated that the isolates are unique compared to other IBDV strains, their antigenic variation remain to be determined by monoclonal antibody study.