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.
The aim of this study was to evaluate the effects of fortification and nano-size reduction on calcium absorption and bioavailability of milk powder formula in sham, ovariectomized, and ovariectomized-osteoporosis rats as a menopause and menopause-osteoporosis model. Skim milk powder and skim milk powder fortified with calcium citrate and the suitable doses of inulin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and vitamins D3, K1, and B6 were formulated based on the North American and Western European recommended dietary allowances. Optimization on cycle and pressure of high-pressure homogenizer was done to produce nano-fortified milk powder. In vivo study demonstrated that fortification and calcium citrate nano-fortified milk powder increased absorption and bioavailability of calcium, as well as bone stiffness and bone strength in sham, ovariectomized, and ovariectomized-osteoporosis rats. This study successfully developed an effective fortified milk powder for food application.
Two Malaysian very virulent infectious bursal disease virus (vvIBDV) strains UPM0081 and UPM190 (also known as UPMB00/81 and UPM04/190, respectively) isolated from local IBD outbreaks were serially passaged 12 times (EP12) in specific pathogen free (SPF) chicken embryonated eggs (CEE) by chorioallantoic membrane (CAM) route. The EP12 isolate was further adapted and serially propagated in BGM-70 cell line up to 20 passages (P20). Characteristic cytopathic effects (CPEs) were subtly observed at P1 in both isolates 72 hours postinoculation (pi). The CPE became prominent at P5 with cell rounding, cytoplasmic vacuoles, granulation, and detachment from flask starting from day 3 pi, up to 7 days pi with titers of 109.50 TCID50/mL and log109.80 TCID50/mL for UPM0081 and UPM190, respectively. The CPE became subtle at P17 and disappeared by P18 and P19 for UPM0081 and UPM190, respectively. However, the presence of IBDV was confirmed by immunoperoxidase, immunofluorescence, and RT-PCR techniques. Phylogenetic analysis showed that these two isolates were of the vvIBDV. It appears that a single mutation of UPM190 and UPM0081 IBDV isolates at D279N could facilitate vvIBDV strain adaptability in CEE and BGM-70 cultures.
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.
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.
Very virulent infectious bursal disease virus (vvIBDV) targets B lymphocytes in the bursa of Fabricius (BF), causing immunosuppression and increased mortality rates in young birds. There have been few studies on the host immune response following vvIBDV infection at different inoculum doses in chickens with different genetic backgrounds. In this study, we characterized the immune responses of specific-pathogen-free (SPF) chickens and Malaysian red jungle fowl following infection with vvIBDV strain UPM0081 at 103.8 and 106.8 times the 50% embryo infectious dose (EID50). The viral burden, histopathological changes, immune cell populations, and expression of immune-related genes were measured and compared between infected and uninfected bursa at specific intervals. The populations of KUL1+, CD3+CD4+ and CD3+CD8+ cells were significantly increased in both types of chickens at 3 dpi, and there was significant early depletion of IgM+ B cells at 1 dpi in the red jungle fowl. vvIBDV infection also induced differential expression of genes that are involved in Th1 and pro-inflammatory responses, with groups receiving the higher dose (106.8 EID50) showing earlier expression of IFNG, IL12B, IL15, IL6, CXCLi2, IL28B, and TLR3 at 1 dpi. Although both chicken types showed equal susceptibility to infection, the red jungle fowl were clinically healthier than the SPF chickens despite showing more depletion of IgM+ B cells and failure to induce IFNB activation. In conclusion, high-dose vvIBDV infection caused an intense early host immune response in the infected bursa, with depletion of IgM+ B cells, bursal lesions, and cytokine expression as a response to mitigate the severity of the infection.
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.
An experiment was conducted to determine the effects of combining both pleasant and unpleasant contacts with human beings on physiology and behavior of broiler chickens. Birds were subjected to the following treatments: (i) received no physical or visual contact with humans (control); (ii) from d 1 to 28, chicks were individually stroked gently for 30 s once daily (PL); (iii) from d 1 to 28, chicks were picked up individually, suspended by both legs, exposed to recorded noise, and swung gently for 15 s once daily (UNPL); (iv) from d 1 to 14 and from d 15 to 28, chicks were subjected to PL and UNPL, respectively (PL-UNPL); and (v) from d 1 to 14 and from d 15 to 28, chicks were subjected to UNPL and PL, respectively (UNPL-PL). On d 42, birds from each treatment group were road-transported for 3 h. Heat shock protein (hsp) 70 expression, plasma levels of corticosterone, serum creatine kinase concentration, heterophil/lymphocyte ratios (HLR), and tonic immobility duration were determined pre- and posttransit. There were significant (P < 0.05) duration of transportation × human contact treatment interactions for HLR and hsp 70 density. Following transit, the PL chicks had significantly (P < 0.05) lower HLR and greater hsp 70 density than the other groups. The corticosterone of PL and UNPL chicks were lower than their control, PL-UNPL, and UNPL-PL counterparts. The PL and PL-UNPL treatments were effective in shortening tonic immobility duration significantly (P < 0.05). Except for UNPL-PL, the serum creatine kinase activity of PL was significantly lower than the other groups. In conclusion, subjecting birds to pleasant human contact reduced stress and fear reactions to transportation by enhancing the ability to express hsp 70 in the brain. Unpleasant human contact had adverse effect on the birds' response to transportation. Early age pleasant experience with humans failed to negate the adverse effects of subsequent unpleasant contact.
Studies have shown that infectious bursal disease virus (IBDV) infects lymphoid cells, mainly B cells and macrophages. This study was aimed to examine the involvement of chicken splenic-derived dendritic cells (ch-sDCs) in specific-pathogen-free chickens following inoculation with IBDV vaccine strain (D78) and a very virulent (vv) strain (UPM0081). Following IBDV infection, enriched activated ch-sDCs were collected by using the negative selection method and were examined based on morphology and immunophenotyping to confirm the isolation method for dendritic cells (DCs). The presence of IBDV on enriched activated ch-sDCs was analyzed based on the immunofluorescence antibody test (IFAT), flow cytometry, and quantitative real-time PCR (RT-qPCR) while the mRNAs of several cytokines were detected using RT-qPCR. The isolated ch-sDCs resembled typical DC morphologies found in mammals by having a veiled shape and they grew in clusters. Meanwhile, the expression of DC maturation markers, namely CD86 and MHCII, were increased at day 2 and day 3 following vvIBDV and vaccine strain inoculation, respectively, ranging from 10% to 40% compared to the control at 2.55% (P < 0.05). At day 3 postinfection, IBDV VP3 proteins colocalized with CD86 were readily detected via IFAT and flow cytometry in both vaccine and vvIBDV strains. In addition, enriched activated ch-sDCs were also detected as positive based on the VP4 gene by RT-qPCR; however, a higher viral load was detected on vvIBDV compared to the vaccine group. Infection with vaccine and vvIBDV strains induced the enriched activated ch-sDCs to produce proinflammatory cytokines and Th1-like cytokines from day 3 onward; however, the expressions were higher in the vvIBDV group (P < 0.05). These data collectively suggest that enriched activated ch-sDCs were permissive to IBDV infection and produced a strong inflammatory and Th1-like cytokine response following vvIBDV infection as compared to the vaccine strain.
Besides the vaccine strains, the Malaysian variant (MV) and QX-like are the predominant IBVs detected on commercial poultry farms. These two virus strains are distinct based on genomic and pathogenicity studies. In this study, we determined the sequence of the S1 gene and compared the pathogenicity of serial passage 70 (P70) of Malaysian QX-like (QX/P70) and MV (MV/P70) strains with that of their respective wild-type viruses. The nucleotide and amino acid sequences of the complete S1 genes of QX/P70 and MV/P70 showed 1.4 to 1.6% and 3.0 to 3.3% variation, respectively, when compared to the wild-type virus. Most of the mutations were insertions and substitutions in the hypervariable regions (HVRs), primarily in HVR 3. Furthermore, selection pressure analysis showed that both viruses are under purifying selection. A pathogenicity study in specific-pathogen-free (SPF) chickens showed a reduction in respiratory and kidney lesions in chickens inoculated with MV/P70, but not with QX/P70, when compared to the respective wild-type viruses. However, MV/P70 is still pathogenic and can cause ciliary damage. In conclusion, the MV IBV strain is more responsive than the QX-like IBV strain following the attenuation process used for the development of a live attenuated IBV vaccine.
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).
Infectious bronchitis viruses (IBVs) circulating in Malaysia are classified into two groups as Malaysian QX-like and variant strains. In this study, the pathogenicity of IBS130/2015 (QX-like) and IBS037A/2014 (variant) IBVs in 1-day-old and 30-day-old specific pathogen free (SPF) chickens was characterized. Both strains caused respiratory and kidney infections based on immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR) and a ciliostasis study; however, the results showed that the QX-like strain was more pathogenic, caused higher mortality and showed higher tissue tropism for the kidney than the variant strain. In contrast, despite causing low or no mortality depending on the age of the infected chickens, the Malaysian variant strain showed high tissue tropism for the respiratory tract compared with the QX-like strain. IHC and qPCR indicated the presence of both IBV strains in the epithelial lining of villi in the jejunum and the caecal tonsil; however, no pathological changes were detected in these organs. Both the Malaysian QX-like and variant IBV strains are able to infect the respiratory tract and kidney of chickens irrespective of age.
Specific-pathogen-free (SPF) chickens infected with very virulent (vv) infectious bursal disease virus (IBDV) UPM94/273 developed lower pathogenicity compared to UPM97/61. Sequence analysis indicated that UPM94/273 is an exceptional vvIBDV. In this study, a SYBR Green I based real-time reverse transcriptase reaction assay was developed to measure viral RNA in the bursae of SPF chickens infected with IBDV. Specificity of the amplified products was confirmed by melting temperature analysis. A linear relationship was observed between the amount of input viral RNA and the threshold values for IBDV-specific product over five log10 dilutions. The viral RNA level following infection with UPM94/273 was significantly higher at day 1 and 2 post-inoculation (p.i.) compared to UPM97/61 infected chickens. However, chickens infected with UPM97/61 had significantly higher numbers of bursal cells undergoing apoptosis compared to UPM94/273 infected chickens. In both groups, the number of apoptotic cells and viral RNA levels peak at day 3 p.i. This study indicates that UPM97/61 and UPM94/273 have different efficiency of replication and percentage of apoptotic cells in bursae during the acute phase of IBDV infection.
Variant infectious bursal disease virus (vaIBDV) has been identified in various countries with significant economic losses. Recently, the first identification of a variant strain in Malaysia was reported. The pathogenicities of the Malaysian variant, UPM1432/2019, and very virulent infectious bursal disease virus (vvIBDV), UPM1056/2018 strains were comparatively evaluated in specific-pathogen-free (SPF) chickens based on gross and histopathological examinations and viral load. Four-week-old SPF chickens were randomly divided into three groups; group 1 served as the control, while groups 2 and 3 birds were challenged with the vaIBDV and vvIBDV, respectively. Three birds from each group were weighed, euthanized and necropsied at 2, 3, 4, 5, 7 and 21 days post-challenge (dpc). Unlike UPM1056/2018 group, birds from UPM1432/2019 group did not show clinical signs or death. UPM1056/2018 strain caused 11% mortality rate in the infected chickens. The bursal body index (BBIX) for UPM1432/2019- and UPM1056/2018-infected groups was <0.7 from 2 dpc and continued to decrease to 0.49 and 0.45, respectively, at 21 dpc. UPM1432/2019 strain was more persistent in the bursa than UPM1056/2018 strain. Both strains induced similar pathological lesions in SPF chicks. These results indicate that the Malaysian vaIBDV severely damaged the immune organs of chickens and was more persistent in bursal tissue than vvIBDV. The study provides insight into the pathogenicity of the variant strain as further study may be required to evaluate the efficacy of the currently available IBD vaccines in Malaysia against the strain. RESEARCH HIGHLIGHTSEmerging Malaysian variant IBDV caused severe bursal damage without mortality.Atypical vvIBDV induced bursal atrophy with inflammatory response and caused low mortality.Malaysian variant IBDV was more persistent in bursal tissue than vvIBDV.
The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.
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.
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.
SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C(t)) 18.19+/-3.63 and a melting temperature (T(m)) 86.0+/-0.28 degrees C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C(t) value 14.70+/-2.32 and T(m) 87.4+/-0.21 degrees C. The assay had a dynamic detection range which spans over a 5log(10) concentration range, 10(9)-10(5) copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45+/-0.31% and 1.30+/-0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.
'Gold standard' OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).
Previously we have shown that avian leukosis virus subgroup J (ALV-J) might be present in chicken flocks from Malaysia based on serological study and also on detection of tissue samples with myelocytic infiltration. In this study, the polymerase chain reaction was used to detect ALV-J sequences from archived frozen samples. Out of 21 tissue samples examined, 16 samples were positive for proviral DNA and four samples for ALV-J RNA. However, only nine samples were found positive for myelocytic infiltration. A total of 465 base pairs equivalent to positions 5305 to 5769 of HPRS-103 from each of the viral RNA positive samples were characterized. Sequence analysis indicated that the samples showed high identity (95.9 to 98.2%) and were close to HPRS-103 with identities between 97.4 and 99.3%. This study indicates that ALV-J-specific sequences can be detected by polymerase chain reaction from frozen tissue samples with and without myelocytic infiltration.