Attenuation and molecular characterization of fowl adenovirus 8b propagated in a bioreactor and its immunogenicity, efficacy, and virus shedding in broiler chickens.

Background and Aim: Live-attenuated vaccines are the most successful type of vaccine and could be useful in controlling fowl adenovirus (FAdV) 8b infection. This study aimed to attenuate, molecularly characterize, and determine the immunogenicity, efficacy, and challenge virus shedding in broiler chickens. Materials and Methods: The FAdV 8b isolate (UPM08136) was passaged onto chicken embryo liver (CEL) cells until attenuation. We sequenced and analyzed the hexon and fiber genes of the passage isolates. The attenuated bioreactor-passage isolate was inoculated into 1-day-old broiler chickens with (attenuated and inactivated) and without booster groups and challenged. Body weight (BW), liver weight (LW), liver: body weight ratio (LBR), FAdV antibody titers, T-lymphocyte subpopulation in the liver, spleen, and thymus, and challenge virus load and shedding were measured. Results: Typical cytopathic effects with novel genetic changes on CEL cells were observed. The uninoculated control-challenged (UCC) group had significantly lower BW and higher LW and LBR than the inoculated groups. A significantly higher FAdV antibody titer was observed in the challenged non-booster and attenuated booster groups than in the UCC group. T cells in the spleen and thymus of the liver of inoculated chickens were higher than uninoculated control group levels at all-time points and at different times. A significantly higher FAdV challenge virus load was observed in the liver and shedding in the cloaca of UCC chickens than in non-booster chickens. Conclusion: The FAdV 8b isolate was successfully attenuated, safe, and immunogenic. It reduces virus shedding and is effective and recommended as a vaccine against FAdV infection in broiler chickens.

Efficacy, immunogenicity, and virus shedding in broiler chickens inoculated with live attenuated fowl adenovirus serotype 8b propagated a bioreactor.

Background: Fowl adenovirus (FAdV) 8b causes huge economic losses in the poultry industry worldwide. Attenuated FAdV 8b could be useful in preventing FAdV infections globally and scale-up obstacles could be solved by bioreactor technology. Aim: This study was carried out to attenuate the FAdV 8b isolate, propagate it in a bioreactor, molecularly characterize the passage isolates, and determine the immunogenicity, efficacy, and shedding of the virus of chickens. Methods: FAdV serotype 8b (UPM11142) isolate was passaged on chicken embryo liver (CEL) cells until attenuation and propagated in a bioreactor (UPM11142P20B1). Hexon and fiber genes of the isolates were sequenced and analyzed. UPM11142P20B1 was administered to 116-day-old broiler chickens divided into four groups, A (control), B (non-booster), C (booster with UPM11142P20B1), and D (booster with inactivated UPM11142P5B1). Eight chickens from each group were challenged. Body weight (BW) and liver weight (LW), liver: BW ratio (LBR), FAdV antibody titer, T lymphocyte sub-populations in the liver, spleen and thymus; and challenge virus load in the liver and shedding in cloaca were measured at weekly intervals. Results: The isolate caused typical cytopathic effects on CEL cells typical of FAdV. Novel molecular changes in the genes occurred which could be markers for FAdV 8b attenuation. BW, LW, and LBR were similar among groups throughout the trial but the uninoculated control-challenged group (UCC) had significantly higher LBR than the inoculated and challenged groups at 35 dpi. Non-booster group had higher FAdV antibodies at all time points than the uninoculated control group (UCG); and the challenged booster groups had higher titer at 35 dpi than UCC. T lymphocytes increased at different time-points in the liver of inoculated chickens, and in the spleen and thymus as well, and was higher in the organs of inoculated challenged groups than the UCC. There was a significantly higher challenge virus load in the liver and cloaca of UCC chickens than in the non-booster chickens. Conclusion: UPM11142P20B1 was safe, efficacious, significantly reduced shedding, and is recommended as a candidate vaccine in the prevention and control of FAdV 8b infections in broiler chickens.

TaqMan probe-based qPCR method for specific detection and quantification of fowl adenovirus 8b challenge from chickens inoculated with live attenuated or inactivated virus.

Background: Fowl adenovirus (FAdV) 8b and other serotypes cause inclusion body hepatitis (IBH) in chickens. Specific detection of aetiologic serotype in mixed infection and vaccine failure could be difficult. Aim: The objective of this study was to develop a TaqMan probe-based qPCR method for the detection and quantification of the FAdV 8b challenge virus. Methods: Forty-eight broiler chickens inoculated with live attenuated or inactivated FAdV 8b strains at day 1 of age either with or without booster at day 14 post-inoculation were used. The chickens were challenged with a pathogenic strain of FAdV 8b at day 28 of age. Liver and cloacal swabs were collected on days 7 and 14 post-challenge. Primers and probes were designed, specificity confirmed, and used to carry out qPCR amplification. Results: The assay amplified the FAdV DNA challenge virus, but not that of the live attenuated virus. It could detect FAdV 8b DNA as low as 0.001 ng/µl in liver and cloacal swab samples. Copy numbers obtained indicate virus load and shedding. Conclusions: It shows that a selective detection of FAdV 8b within serotype is possible. It can be useful for rapid detection and diagnosis of the disease, virus quantification and differentiation within species, determination of vaccination failure, and efficacy especially the virus load in the target organ and shedding.

Efficacy, humoral, and cell-mediated immune response of inactivated fowl adenovirus 8b propagated in chicken embryo liver cells using bioreactor in broiler chickens.

Background and Aim: Fowl adenovirus (FAdV) 8b causes inclusion body hepatitis, resulting in major economic losses globally among chickens. The objectives were to inactivate FAdV 8b isolate propagated in chicken embryo liver (CEL) cells using a stirred tank bioreactor (UPM08136P5B1) and determine the humoral and cell-mediated immune response, efficacy, and virus shedding in broiler chickens. Materials and Methods: The FAdV 8b isolate UPM08136P5B1 was inactivated using binary ethyleneimine, adjuvanted with Montanide 71VG, inoculated into day-old broiler chickens in a booster group (BG) and non-booster group (NBG), and challenged with a pathogenic FAdV 8b strain. Clinical signs, gross lesions, body weight (BW), liver: body weight ratio, FAdV antibody titer using enzyme-linked immunosorbent assay, and histopathological changes were recorded. The CD3+, CD4+, and CD8+ T-lymphocyte profiles of the liver, spleen, and thymus using flow cytometry, and viral load in liver and cloacal shedding using quantitative polymerase chain reaction were evaluated. Results: Chickens in the challenged control group (CCG) exhibited mild clinical signs, gross lesions, and histopathological changes, which were absent in the inoculated groups, and had lower BW and higher liver BW ratio than chickens in the unchallenged control group (UCG); BG and NBG on 35- and 42-days post-inoculation (DPI). Chickens in NBG and BG had higher antibodies than UCG on 7, 21, 35, and 42 DPI. The challenged BG and NBG produced higher antibodies than the CCG on 35 DPI. T-lymphocytes were higher among the inoculated groups than UCG in the liver, spleen, and thymus. Inoculated challenged groups recorded higher CD3+, CD4+, and CD8+ T-lymphocytes on 35 and 42 DPI than CCG. The challenged control group had a significantly higher viral load in the liver than challenged that in BG on 35 DPI and BG and NBG on 42 DPI. The challenged control group had significantly higher challenge FAdV shedding than challenged inoculated groups on 35 and NBG on 42 DPI. Conclusion: UPM08136P5B1 was successfully inactivated and mixed with Montanide 71VG. The inactivated vaccine candidate that induced humoral and cellular immunity was effective, reduced FAdV load in the liver, and shedding in the cloaca, and could be useful against FAdV 8b infections in chickens.

A recent perspective on fiber and hexon genes proteins analyses of fowl adenovirus toward virus infectivity-A review.

Fowl adenovirus (FAdV) is a double-stranded DNA virus with a non-enveloped structure comprising three major proteins known as hexon, penton, and fiber. Molecular analysis which emphasizes on hexon and fiber proteins is currently the major focus of curiosity for FAdV antigenicity and pathogenicity. Recently, disease outbreaks associated with FAdV infections such as inclusion body hepatitis, hepatitis hydropericardium syndrome, and gizzard erosion, were commonly reported and continue to increase worldwide. Studies on the virulence gene of the virus were intensively conducted to provide a better understanding on the role of these major capsid proteins in the development of a safe and effective vaccine against the disease in the poultry industry. This paper highlights the variations of the fiber and hexon genes, their importance in genotypes and serotypes differentiation, and infectivity between FAdV strains. It appears that the L1 loop of hexon and the knob of fiber genes are the infectivity markers for FAdV infection. The fiber-2 protein plays a major role in FAdV pathogenicity than the hexon protein, while the fiber-1 protein is important for viral replication and assembly, regardless of virulence capability instead of infectivity. The hexon protein plays a major role in virus infectivity and tissue tropism. These findings could further enhance the knowledge of FAdV strains' classification and evolution, diagnosis, and strategies to prevent and control FAdV infection and outbreaks in chicken farms.

Exploring the Prospects of Engineered Newcastle Disease Virus in Modern Vaccinology.

Many traditional vaccines have proven to be incapable of controlling newly emerging infectious diseases. They have also achieved limited success in the fight against a variety of human cancers. Thus, innovative vaccine strategies are highly needed to overcome the global burden of these diseases. Advances in molecular biology and reverse genetics have completely restructured the concept of vaccinology, leading to the emergence of state-of-the-art technologies for vaccine design, development and delivery. Among these modern vaccine technologies are the recombinant viral vectored vaccines, which are known for their incredible specificity in antigen delivery as well as the induction of robust immune responses in the vaccinated hosts. Although a number of viruses have been used as vaccine vectors, genetically engineered Newcastle disease virus (NDV) possesses some useful attributes that make it a preferable candidate for vectoring vaccine antigens. Here, we review the molecular biology of NDV and discuss the reverse genetics approaches used to engineer the virus into an efficient vaccine vector. We then discuss the prospects of the engineered virus as an efficient vehicle of vaccines against cancer and several infectious diseases of man and animals.

Bursal transcriptome profiling of different inbred chicken lines reveals key differentially expressed genes at 3 days post-infection with very virulent infectious bursal disease virus.

Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.

Induction of a robust immune response against avian influenza virus following transdermal inoculation with H5-DNA vaccine formulated in modified dendrimer-based delivery system in mouse model.

This study was aimed to evaluate the immunogenicity of recombinant plasmid deoxyribonucleic acid (DNA), pBud-H5-green fluorescent protein (GFP)-interferon-regulatory factor (IRF)3 following delivery using polyamidoamine (PAMAM) dendrimer and transactivator of transcription (TAT)-conjugated PAMAM dendrimer as well as the effect of IRF3 as the genetic adjuvant. BALB/c mice were vaccinated transdermally with pBud-H5-GFP, PAMAM/pBud-H5-GFP, TAT-PAMAM/pBud-H5-GFP, and TAT-PAMAM/pBud-H5-GFP-IRF3. The expression analysis of H5 gene from the blood by using quantitative real-time reverse transcriptase polymerase chain reaction confirmed the ability of PAMAM dendrimer as a carrier for gene delivery, as well as the ability of TAT peptide to enhance the delivery efficiency of PAMAM dendrimer. Mice immunized with modified PAMAM by TAT peptide showed higher hemagglutination inhibition titer, and larger CD3+/CD4+ T cells and CD3+/CD8+ T cells population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2, IL-15, IL-12, IL-6, and tumor necrosis factor-α compared with those immunized with native PAMAM. These results suggest that the function of TAT peptide as a cell-penetrating peptide is able to enhance the gene delivery, which results in rapid distribution of H5 in the tissues of the immunized mice. Furthermore, pBud-H5-GFP co-expressing IRF3 as a genetic adjuvant demonstrated the highest hemagglutination inhibition titer besides larger CD3+/CD4+ and CD3+/CD8+ T cells population, and strong Th1-like cytokine responses among all the systems tested. In conclusion, TAT-PAMAM dendrimer-based delivery system with IRF3 as a genetic adjuvant is an attractive transdermal DNA vaccine delivery system utilized to evaluate the efficacy of the developed DNA vaccine in inducing protection during challenge with virulent H5N1 virus.

Simvastatin modulates cellular components in influenza A virus-infected cells.

Influenza A virus is one of the most important health risks that lead to significant respiratory infections. Continuous antigenic changes and lack of promising vaccines are the reasons for the unsuccessful treatment of influenza. Statins are pleiotropic drugs that have recently served as anti-influenza agents due to their anti-inflammatory activity. In this study, the effect of simvastatin on influenza A-infected cells was investigated. Based on the MTT cytotoxicity test, hemagglutination (HA) assay and qPCR it was found that simvastatin maintained cell viability and decreased the viral load significantly as compared to virus-inoculated cells. The expression of important pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6 and interferon-γ), which was quantified using ELISA showed that simvastatin decreased the expression of pro-inflammatory cytokines to an average of 2-fold. Furthermore, the modulation of actin filament polymerization was determined using rhodamine staining. Endocytosis and autophagy processes were examined by detecting Rab and RhoA GTPase protein prenylation and LC3 lipidation using western blotting. The results showed that inhibiting GTPase and LC3 membrane localization using simvastatin inhibits influenza replication. Findings of this study provide evidence that modulation of RhoA, Rabs and LC3 may be the underlying mechanisms for the inhibitory effects of simvastatin as an anti-influenza compound.

Prophylactic effect of herbal-marine compound (HESA-A) on influenza A virus infectivity.

BACKGROUND: Influenza virus is still a severe respiratory disease affecting human and other species. As conventional drugs are not recommended for long time because of side effects and drug resistance occurrence, traditional medication has been focused as alternative remedy. HESA-A is a natural compound from herbal-marine origin. Previous studies have reported the therapeutic properties of HESA-A on psoriasis vulgaris and different types of cancers and we also showed its anti-inflammatory effects against influenza A infection. METHODS: This study was designed to investigate the potential properties of HESA-A as prophylaxis or treatment. To investigate the prophylaxis or treatment activities of HESA-A, Madin-Darby Canine Kidney (MDCK) cells were exposed to HESA-A and influenza A virus in different manners of exposure and different time intervals. The results were evaluated by MTT and HA assays. RESULTS: It was found that HESA-A is much more effective against influenza cytopathic effects when it is applied for prophylaxis and also in concurrent treatment (p ≤ 0.05) but not in post-infection treatment (p ≥ 0.05). CONCLUSION: In conclusion, HESA-A is significantly effective against influenza replication in prophylaxis application affecting the virus penetration/adsorption to the cell without any toxic effect on the cell viability.

Transcriptional profiling of feline infectious peritonitis virus infection in CRFK cells and in PBMCs from FIP diagnosed cats.

BACKGROUND: Feline Infectious Peritonitis (FIP) is a lethal systemic disease, caused by the FIP Virus (FIPV); a virulent mutant of Feline Enteric Coronavirus (FECV). Currently, the viruses virulence determinants and host gene expressions during FIPV infection are not fully understood. METHODS: RNA sequencing of Crandell Rees Feline Kidney (CRFK) cells, infected with FIPV strain 79-1146 at 3 hours post infection (h.p.i), were sequenced using the Illumina next generation sequencing approach. Bioinformatic's analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench mapped both control and infected cell reads to 18899 genes out of 19046 annotated genes. Kal's Z test statistical analysis was used to analyse the differentially expressed genes from the infected CRFK cells. Real time RT-qPCR was developed for further transcriptional profiling of three genes (PD-1, PD-L1 and A3H) in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diseased cats. RESULTS: Based on Kal's Z-test, with False Discovery Rate (FDR) <0.05 and >1.99 fold change on gene expressions, a total of 61 genes were differentially expressed by both samples, where 44 genes were up-regulated and the remainder were down-regulated. Most genes were closely clustered together, suggesting a homogeneous expression. The majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and Th1 cell functions, and the regulation of apoptosis. Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data. CONCLUSION: The possible roles of these genes, and their importance in feline coronaviruses infection, are discussed.

Attenuation of influenza virus infectivity with herbal-marine compound (HESA-A): an in vitro study in MDCK cells.

BACKGROUND: The influenza virus is still one of the most important respiratory risks affecting humans which require effective treatments. In this case, traditional medications are of interest. HESA-A is an active natural biological compound from herbal-marine origin. Previous studies have reported that the therapeutic properties of HESA-A are able to treat psoriasis vulgaris and cancers. However, no antiviral properties have been reported. METHODS: This study was designed to investigate the potential antiviral properties of HESA-A and its effects in modulating TNF-α and IL-6 cytokine levels. HESA-A was prepared in normal saline as a stock solution (0.8 mg/ml, pH = 7.4). Percentages of cell survival when exposed to different concentrations of HESA-A at different time intervals was determined by MTT assay. To study the potential antiviral activity of HESA-A, Madin-Darby Canine Kidney (MDCK) cells were treated with the effective concentration (EC50) of HESA-A (0.025 mg/ml) and 100 TCID50/0.1 ml of virus sample under different types of exposure. RESULTS: Based on the MTT method and hemagglutination assay (HA), HESA-A is capable of improving cell viability to 31% and decreasing HA titre to almost 99% in co-penetration exposures. In addition, based on quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), it was found that HESA-A causes decrements in TNF-α and IL-6 cytokine expressions, which was significant for TNF-α (p ≤ 0.05) but not for IL-6. CONCLUSION: In conclusion, HESA-A was effective against influenza infection through suppressing cytokine expression.

Descriptive distribution and phylogenetic analysis of feline infectious peritonitis virus isolates of Malaysia.

The descriptive distribution and phylogeny of feline coronaviruses (FCoVs) were studied in cats suspected of having feline infectious peritonitis (FIP) in Malaysia. Ascitic fluids and/or biopsy samples were subjected to a reverse transcription polymerase chain reaction (RT-PCR) targeted for a conserved region of 3'untranslated region (3'UTR) of the FCoV genome. Eighty nine percent of the sampled animals were positive for the presence of FCoV. Among the FCoV positive cats, 80% of cats were males and 64% were below 2 years of age. The FCoV positive cases included 56% domestic short hair (DSH), 40% Persian, and 4% Siamese cats. The nucleotide sequences of 10 selected amplified products from FIP cases were determined. The sequence comparison revealed that the field isolates had 96% homology with a few point mutations. The extent of homology decreased to 93% when compared with reference strains. The overall branching pattern of phylogenetic tree showed two distinct clusters, where all Malaysian isolates fall into one main genetic cluster. These findings provided the first genetic information of FCoV in Malaysia.

Sequence and phylogenetic analysis of Newcastle disease virus genotypes isolated in Malaysia between 2004 and 2005.

Sequence analysis of the fusion (F) gene of eight Malaysian NDV isolates showed that all the isolates were categorized as velogenic viruses, with the F cleavage site motif (112)R-R-Q-K-R(116) or (112)R-R-R-K-R(116) at the C-terminus of the F(2) protein and phenylalanine (F) at residue 117 at the N-terminus of the F(1) protein. Phylogenetic analysis revealed that all of the isolates were grouped in two distinct clusters under sub-genotype VIId. The isolates were about 4.8-11.7% genetically distant from sub-genotypes VIIa, VIIb, VIIc and VIIe. When the nucleotide sequences of the eight Malaysian isolates were compared phylogenetically to those of the old published local isolates, it was found that genotype VIII, VII, II and I viruses exist in Malaysia and caused sporadic infections. It is suggested that genotype VII viruses were responsible for most of the outbreaks in recent years.

Prevalence of feline coronavirus in two cat populations in Malaysia.

The prevalence of feline coronavirus (FCoV) was studied in two catteries in Malaysia. Rectal swabs or faecal samples were collected from a total of 44 clinically healthy Persian purebred and mix-breed cats. RNA extracted from the faecal material was subjected to a reverse transcription-polymerase chain reaction (RT-PCR) using primers flanking for a conserved region of the virus genome. The overall prevalence of FCoV infection was 84% and the infection rate was higher in Persian purebred cats (96%) than mix-breed cats (70%). There was no significant association between the age or gender of tested cats and shedding the virus. This study is the first PCR-based survey for FCoV in Malaysia and showed the ubiquitous presence of FCoV in Malaysian cat colonies.

Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus.

A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P<0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV.

Detection and characterization of chicken anemia virus from commercial broiler breeder chickens.

BACKGROUND: Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia (CIA). Study on the type of CAV isolates present and their genetic diversity, transmission to their progeny and level of protection afforded in the breeder farms is lacking in Malaysia. Hence, the present study was aimed to detect CAV from commercial broiler breeder farms and characterize CAV positive samples based on sequence and phylogenetic analysis of partial VP1 gene. RESULTS: A total of 12 CAV isolates from different commercial broiler breeder farms were isolated and characterized. Detection of CAV positive embryos by the PCR assay in the range of 40 to 100% for different farms indicated high level of occurrence of vertical transmission of viral DNA to the progeny. CAV antigen was detected in the thymus and in the bone marrow but not in spleen, liver, duodenum, ovary and oviduct by indirect immunoperoxidase staining. The 12 CAV isolates were characterized based on partial sequences of VP1 gene. Six isolates (MF1A, MF3C, M3B5, NF4A, P12B and P24A) were found to have maximum homology with previously characterized Malaysian isolate SMSC-1, four isolates (M1B1, NF3A, PYT4 and PPW4) with isolate BL-5 and the remaining two (NF1D and NF2C) have maximum homology both with isolates 3-1 and BL-5. Meanwhile, seven of the isolates with amino acid profile of 75-I, 97-L, 139-Q and 144-Q were clustered together in cluster I together with other isolates from different geographical places. The remaining five isolates with amino acid profile of 75-V, 97-M, 139-K and 144-E were grouped under cluster II. All the CAV isolates demonstrated omega values (Ka/Ks) of less than one (the values ranging from 0.07 to 0.5) suggesting the occurrence of purifying (negative) selection in all the studied isolates. CONCLUSION: The present study showed that CAV is widespread in the studied commercial broiler breeder farms. The result also indicated the occurrence of genetic variability in local CAV isolates that can be divided at least into two groups based on characteristic amino acid substitutions at positions 75, 97, 139 and 144 of the VP1 protein.

Base usage and dinucleotide frequency of infectious bursal disease virus.

Base usage and dinucleotide frequency have been extensively studied in many eukaryotic organisms and bacteria, but not for viruses. In this paper, a comprehensive analysis of these aspects for infectious bursal disease virus (IBDV) was presented. The analysis of base usage indicated that all of the IBDV genes possess equivalent overall nucleotide distributions. However when the base usage at each codon positions was analysed by using cluster analysis, the VP5 open reading frame (ORF) formed a different cluster isolated from the other genes. The unusual base usage of VP5 ORF may indicate that the gene was originated by the virus "overprinting strategy", a strategy in which virus may create novel gene by utilizing the unused reading frames of its existing genes. Meanwhile, the GC content of the IBDV genes and the chicken's coding sequences was comparable; suggesting the virus imitation of the host to increase its translational efficiency. The analysis of dinucleotide frequency indicated that IBDV genome had dinucleotide bias: the frequencies of CpG and TpA were lower and the TpG was higher than the expected. Classical methylation pathway, a process where CpG converted to TpG, may explain the significant correlation between the CpG deficiency and TpG abundance. "Principal component analysis of the dinucleotide frequencies" (DF-PCA) was used to analyse the overall dinucleotide frequencies of IBDV genome. DF-PCA on the hypervariable region and polyprotein (VPX-VP4-VP3) gene showed that the very virulent IBDV (vvIBDV) was segregated from other strains; which meant vvIBDV had a unique dinucleotide pattern. In summary, the study of base usage and dinucleotide frequency had unravelled many overlooked genomic properties of the virus.