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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.

Eurycoma longifolia or Tongkat Ali (family: Simaroubaceae) has the potential to be utilised as an antimicrobial and antiparasitic agent that correlated with its traditional use to treat jaundice, malaria, antiseptic agent, and many more. This review is aimed at systematically sieving through articles regarding the antimicrobial and antiparasitic activity of E. longifolia. A total of 123 studies have been found using suitable keywords and manually searched from previous studies through the four databases. After title screening and abstract examination, 56 articles were excluded due to duplication and not meeting the acceptance criteria. 67 articles were assessed on full-text accessibility, 31 studies remained, and this number decreased to 20 articles after a careful examination of the full-text articles. Among the 20 articles selected, 17 articles proved the potential of E. longifolia as an antimicrobial and antiparasitic agent efficiently. 2 selected articles showed partial positive results, which specified specific microorganisms tested. In contrast, another 1 article gave a completely negative result. As for the conclusion, current studies highlighted by this review may shed light on the future direction of studies concerning E. longifolia as a novel antimicrobial and antiparasitic agent. However, more research should be done in the future focusing on the efficiency of E. longifolia for veterinary medicine utilisation.

Intraepithelial lymphocytes (IELs) provide the first line of immunological defense after the invasion of the intestine by a pathogen. To understand the changes of IEL response in chickens, we measured the population of different subsets of avian IELs at different time points after primary inoculation of Newcastle disease virus (NDV) lentogenic strain (LaSota) and subsequent challenge with NDV velogenic strain- genotypes VII and VIII. Furthermore, NDV shed after each treatment was quantified. Specific-pathogen-free chickens were randomly divided into six groups of chickens, one to six, inoculated with phosphate buffered saline; NDV lentogenic strain (LaSota); genotype VII (GVII); LaSota and challenged with GVII (LSGVII); genotype VIII (GVIII); and group of LaSota and challenged with GVIII (LSGVIII). The chickens were euthanized at 12, 36, and 60 h postchallenge. Immunophenotyping of CD25

Astroviruses have been associated with enteric and extra-intestinal disorders in many animal species, including chickens. Here, we describe the detection and characterisation of chicken astrovirus (CAstV) in broilers and its seroprevalence in broiler breeder flocks. Based on PCR protocol, viral confirmation was carried out on clinical tissue samples from broiler chickens suffering from uneven growth and poor performance. The tissues were molecularly detected for CAstV with differential diagnostic testing against the Newcastle disease virus, infectious bronchitis virus, avian nephritis virus, avian rotavirus, fowl adenovirus and avian reovirus. Polymerase gene-based phylogenetic analyses of the twenty samples detected positive for CAstV indicate they belong to Group I and are related to strains from the US, UK, India and Poland. From these 20 samples, CastV could be isolated from 3 samples upon inoculation in 5-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECE); virus-infected embryos showed dwarfing, haemorrhages, oedema and gelatinous lesions at harvest. The enzyme-linked immunosorbent assay (ELISA) results revealed a high prevalence of antibodies against CAstV amongst the broiler breeder flocks tested. It is the first study that describes the detection and prevalence of CAstV in broiler chickens and broiler breeder flocks in Malaysia.

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 was0.7 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.

In spite of the available information on the role of natural killer (NK) cells in several viral infections, the interactions between chicken intraepithelial-NK (IEL-NK) cells and Newcastle disease virus (NDV) are poorly understood. In this study, we investigated these interactions following the inoculation of chickens with NDV vaccine strain LaSota and subsequent challenge with velogenic NDV (vNDV) genotype VII (GVII) and VIII (GVIII), through quantification of IEL-NK cell's apoptosis and expression profiling of its surface receptors. Specific-pathogen-free chickens were randomly divided into six groups, as follows: one group of an uninfected control, one group infected with NDV LaSota, two groups each infected with either GVII or GVIII, and two groups inoculated with NDV LaSota and challenged with either GVII (LaSota-genotype VII [LSGVII]) or GVIII (LaSota-genotype VIII [LSGVIII]). Avian intraepithelial lymphocytes (IEL) were isolated from the duodenal loops, and CD3- cells were characterized. Immunophenotyping and apoptosis analysis of CD3-/CD25+/CD45+IEL NK cells were conducted using a flow cytometer. In addition, a gene expression study was conducted using real-time quantitative PCR. Data were analyzed using two-way analysis of variance. The results showed that vNDV GVII or GVIII caused apoptosis of IEL-NK cells; however, following inoculation of LSGVII or LSGVIII, the effect of vNDV GVII and GVIII to cause a reduction in the population of viable IEL-NK cells was significantly reduced. Furthermore, the expression profiles of activating receptors CD69, NK-lysin, and IFN-γ, were generally upregulated in chickens inoculated with LSGVII or LSGVIII. In contrast, B-NK, an inhibitory receptor, was downregulated in these treatment groups. In NDV GVII- and GVIII-challenged groups, however, B-NK was upregulated, whereas the other receptors were generally downregulated. The findings of this study showed that NDV vaccine strain LaSota may prevent apoptosis and cause upregulation of activating receptors of chicken IEL-NK cells in velogenic virus-challenged settings.Perfiles de expresión de genes relacionados con la inmunidad y estudio de apoptosis de células asesinas naturales intraepiteliales aviares en pollos inoculados con la cepa vacunal del virus de la enfermedad de Newcastle (NDV) y desafiados con de Newcastle virulento. A pesar de la información disponible sobre el papel de las células asesinas naturales (NK) en varias infecciones virales, se conoce poco acerca de las interacciones entre las células NK intraepiteliales de pollo (IEL-NK) y el virus de la enfermedad de Newcastle (NDV). En este estudio, investigamos estas interacciones luego de la inoculación de pollos con la cepa vacunal LaSota y con el desafío posterior con los genotipo VII (GVII) y VIII (GVIII) velogénico de NDV (vNDV), mediante la cuantificación de la apoptosis de las células IEL-NK y los perfiles de expresión de sus receptores de superficie. Los pollos libres de patógenos específicos se dividieron aleatoriamente en seis grupos, de la siguiente manera: un grupo de control no infectado, un grupo infectado con LaSota, dos grupos cada uno infectado con GVII o GVIII, y dos grupos inoculados con LaSota y desafiados con ya sea el genotipo GVII (LaSota-genotipo VII [LSGVII]) o con el genotipo GVIII (LaSota-genotipo VIII [LSGVIII]). Se aislaron células NK intraepiteliales de pollo de las asas duodenales y se caracterizaron las células CD3-. La inmunofenotipificación y el análisis de apoptosis de las células NK CD3-/CD25+/CD45+IEL se realizaron utilizando citometría de flujo. Además, se realizó un estudio de expresión de genes mediante PCR cuantitativa en tiempo real. Los datos se analizaron utilizando un análisis de varianza de dos vías. Los resultados mostraron que el virus de Newcastle genotipos GVII o GVIII causaron apoptosis de células NK intraepiteliales; sin embargo, después de por los tratamientos LaSota-genotipo VII o LaSota-genotipo VIII, el efecto de del virus virulento de Newcastle GVII y GVIII para provocar una reducción en la población de células NK intraepiteliales viables se redujo significativamente. Además, los perfiles de expresión de los receptores activadores CD69, NK-lisina e IFN-γ generalmente aumentaron en pollos inoculados con los tratamientos LaSota-genotipo VII o LaSota-genotipo VIII. Por el contrario, B-NK, que es un receptor inhibidor, se reguló a la baja en estos grupos de tratamiento. Sin embargo, en los grupos expuestos a los virus de Newcastle genotipos GVII y GVIII, el gene B-NK estaba regulado al alza, mientras que los otros receptores generalmente estaban regulados a la baja. Los hallazgos de este estudio mostraron que la cepa vacunal LaSota puede prevenir la apoptosis y causar una regulación al alza de los receptores activadores de las células NK intraepiteliales de pollo en entornos expuestos al virus velogénico.

This study was conducted to inactivate Salmonella enteriditis phage types (SE pt) and to determine the safety and efficacy of inactivated SE pt in chickens. SE pt 1, 3A, 6A, 7, and 35 were inactivated and inoculated (0.20 mL) in 124 chickens divided into 6 groups (CV1, CV3A, CV6A, CV7, CV35, and CV0 as a control). Sampling was conducted on day 14 after inoculation (pi). Eight chickens from each group were separated on day 14 pi for oral challenge with 0.20 mL/chicken (1010 cfu/mL) SE pt 6A and designated CV1C, CV3AC, CV6AC, CV7C, CV35C, and CV0C as control chickens. On days 7 and 14 postchallenge (pc), 4 chickens from every group were sacrificed for sampling. There was no significant difference in the body weight between different groups. In challenged groups, there was no significant association between different tissues and isolation of Salmonella on days 7 and 14 pc. There was significance (p

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.

Background Infectious bursal disease (IBD) is an economically very important issue to the poultry industry and it is one of the major threats to the nation’s food security. The pathogen, a highly pathogenic strain of a very virulent IBD virus causes high mortality and immunosuppression in chickens. The importance of understanding the underlying genes that could combat this disease is now of global interest in order to control future outbreaks. We had looked at identified novel genes that could elucidate the pathogenicity of the virus following infection and at possible disease resistance genes present in chickens. Results A set of sequences retrieved from IBD virus-infected chickens that did not map to the chicken reference genome were de novo assembled, clustered and analysed. From six inbred chicken lines, we managed to assemble 10,828 uni-transcripts and screened 618 uni-transcripts which were the most significant sequences to known genes, as determined by BLASTX searches. Based on the differentially expressed genes (DEGs) analysis, 12 commonly upregulated and 18 downregulated uni-genes present in all six inbred lines were identified with false discovery rate of q-value Conclusion Our data revealed the commonly up- and downregulated novel uni-genes identified to be immune- and extracellular binding-related, respectively. Besides, these novel findings are valuable contributions in improving the current existing integrative chicken transcriptomics annotation and may pave a path towards the control of viral particles especially towards the suppression of IBD and other infectious diseases in chickens.

Chitosan is a biodegradable, non-toxic polysaccharide that is extensively studied as a biocompatible vector for gene and drug delivery. However, the fabrication of chitosan nanoparticle (CNP) is usually encountered with a wide size distribution and poor particle stability, which unfortunately limits their role for certain biological applications. This study reports the synthesis and characterisation of CNPs under optimised conditions. The CNPs were synthesised via ionic gelation process utilizing tripolyphosphate (TPP) as a cross-linking agent. The particle size and morphology of samples were subsequently evaluated using dynamic light scattering (DLS), electron microscopy and Fourier-transform infrared spectroscopy (FTIR). Findings arising from this study showed the optimised nanoparticles exhibited spherical shaped CNPs with a size range from 4 to 25nm which lays the foundation for further applications.

The main aim of our study was to explore the genome sequence of the inclusion body hepatitis associated Fowl adenovirus serotype 8b (FAdV-8b) UPM04217 and to study its genomic organisation. The nucleotide sequence of the whole genome of FAdV-8b UPM04217 was determined by using the 454 Pyrosequencing platform and the Sanger sequencing method. The complete genome was found to be 44,059 bp long with 57.9% G + C content and shared 97.5% genome identity with the reference FAdV-E genome (HG isolate). Interestingly, the genome analysis using ORF Finder, Glimmer3 and FGENESV predicted a total of 39 open reading frames (ORFs) compared to the FAdV-E HG that possessed 46 ORFs. Fourteen ORFs located within the central genomic region and 16 ORFs located within the left and right ends of the genome were assigned as being the high protein-coding regions. The fusion of the small ORFs at the right end terminal specifically in ORF22 and ORF33 could be the result of gene truncation in the FAdV-E HG. The frame shift mutation in ORF25 and other mutations in ORF13 and ORF17 might have lead to the emergence of genes that could have different functions. Besides, one of the minor capsid components, pVI, in FAdV-8b UPM04217 shared the highest similarity of 93% with that of FAdV-D, while only 47% similarity was found with FAdV-E. From the gene arrangement layout of the FAdV genome, FAdV-8b UPM04217 showed intermediate evolution between the FAdV-E HG and the FAdV-D although it was apparently more similar to the FAdV-E HG. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13337-019-00530-9) contains supplementary material, which is available to authorized users.

Vaccination is an essential component in controlling infectious bursal disease (IBD), however, there is a lack of information on the genetic characteristics of a recent infectious bursal disease virus (IBDV) that was isolated from IBD vaccinated commercial flocks in Malaysia. The present study investigated 11 IBDV isolates that were isolated from commercial poultry farms. The isolates were detected using reverse transcription-polymerase chain reaction (RT-PCR) targeting the hypervariable region (HVR) of VP2. Based on the HVR sequences, five isolates (IBS536/2017, IBS624/2017, UPM766/2018, UPM1056/2018, and UPM1432/2019) were selected for whole-genome sequencing using the MiSeq platform. The nucleotide and amino acid (aa) sequences were compared with the previously characterized IBDV strains. Deduced aa sequences of VP2HVR revealed seven isolates with 94–99% aa identity to very virulent strains (genogroup 3), two isolates with 97–100% aa identity to variant strains (genogroup 2), and two strains with 100% identity to the vaccine strain (genogroup 1) of IBDV. The phylogenetic analysis also showed that the isolates formed clusters with the respective genogroups. The characteristic motifs 222T, 249K, 286I, and 318D are typical of the variant strain and were observed for UPM1219/2019 and UPM1432/2019. In comparison, very virulent residues such as 222A, 249Q, 286T, and 318G were found for the vvIBDV, except for the UPM1056/2018 strain with a A222T substitution. In addition, the isolate has aa substitutions such as D213N, G254D, S315T, S317R, and A321E that are not commonly found in previously reported vvIBDV strains. Unlike the other vvIBDVs characterized in this study, UPM766/2018 lacks the MLSL aa residues in VP5. The aa tripeptides 145/146/147 (TDN) of VP1 were conserved for the vvIBDV, while a different motif, NED, was observed for the Malaysian variant strain. The phylogenetic tree showed that the IBDV variant clustered with the American and Chinese variant viruses and are highly comparable to the novel Chinese variants, with 99.9% identity. Based on the sequences and phylogenetic analyses, this is the first identification of an IBDV variant being reported in Malaysia. Further research is required to determine the pathogenicity of the IBDV variant and the protective efficacy of the current IBD vaccines being used against the virus.

Inclusion body hepatitis (IBH) is considered one of the re-emerging diseases of avian virus that causes economic damage worldwide. IBH is caused by different serotypes of fowl adenovirus (FAdV), and most of the FAdV cases in Malaysia are related to the serotype 8b. The objective of this study was to determine the molecular markers of UPMT27 Malaysian FAdV isolate and to identify the evolutionary relationship through the phylogenetic approach. Propagation of the isolate was made in embryonated chicken eggs and chicken embryo liver (CEL cells) before it was subjected to viral DNA extraction. Both the fiber and hexon genes of the isolate were amplified and sequenced. The sequences were aligned with the published FAdV sequences. The results showed 100% identity between UPMT27 and the previous Malaysian isolates. A phylogenetic study showed that UPMT27 was closely related to the previous Malaysian isolates. Interestingly, the substitution of the amino acids was consistent between the Malaysia isolates of both fiber protein at positions 72 (Serine –serine), 101 (Alanine -alanine), 125 (Glycine-glycine), and hexon protein 85 (Serine-serine) 160 (Glutamate- glutamate) and 205 (Alanine-alanine) respectively. It appeared that the amino acid variations were the indicators for genetic diversity. Thus, these findings provide information on the evolutionary relationship between FAdV subtypes for IBH prevention.

Infectious bronchitis (IB) is an economically important disease of poultry that also serve as model for the understanding of other coronaviruses associated diseases. IB is considered as a major challenge to the poultry industry worldwide as a result of its effect on egg production, weight gain as well as mortality. Different IBV genotypes continue to emerge, thus, the need for broad based vaccines to curb the disease. Based on bioinformatic data obtained in this study, sets of monovalent (either M41 or CR88) and bivalent DNA vaccines encoding the S1 glycoprotein from two different strains namely, M41 and CR88 were developed. The candidate vaccine was further encapsulated with a chitosan-saponin nanoparticle with the view to enhance its immunogenicity. Following in vitro characterization of the constructs, the vaccine candidates were tested in specific pathogen free (SPF) chickens. Analysis of humoral responses revealed a significant increase in anti-IBV antibody after immunization with the bivalent DNA plasmid (pBudCR88-S1/M41-S1). Likewise, cell mediated immune (CMI) response was significantly higher in vaccinated groups as compared to the unvaccinated chickens. Vaccinated chickens exhibited milder clinical signs as well as tracheal and kidney lesion scores following virus challenge as compared to the control groups. Additionally, encapsulation of the bivalent DNA vaccine with chitosan-saponin nanoparticles was found to improve protection against challenge with IBV strains M41 and CR88 as revealed by a significant reduction (p, Highlights • DNA vaccine could offer promising advantage against infectious bronchitis in poultry. • Vaccination with IBV S-1 gene based DNA vaccine leads to improved antibody and T cell responses. • Encapsulation of the vaccine with chitosan and Saponin enhances the immune response and abrogated the need for multiple booster administration. • The vaccine offered protection to vaccinated chickens as revealed by reduction in oropharyngeal and cloacal virus shedding as well as reduced tracheal and kidney lesion score following challenge.

Large volume production of vaccine virus is essential for prevention and control of viral diseases. The objectives of this study were to propagate Fowl adenovirus (FAdV) isolate (UPM08136) in chicken embryo liver (CEL) cells adapted to Cytodex&trade, 1 microcarriers using stirred tank bioreactor (STB) and molecularly characterize the virus. CEL cells were prepared and seeded onto prepared Cytodex&trade, 1 microcarriers and incubated first in stationary phase for 3 h and in STB at 37 &deg, C, 5% CO2, and 20 rpm for 24 h. The CEL cells were infected with FAdV isolate (UPM08136) passage 5 (UPM08136CELP5) or passage 20 (UPM08136CELP20) and monitored until cell detachment. Immunofluorescence, TCID50, sequencing, alignment of hexon and fiber genes, and phylogenetic analysis were carried out. CEL cells were adapted well to Cytodex&trade, 1 microcarriers and successfully propagated the FAdV isolates in STB with virus titer of 107.5 (UPM08136CELP5B1) and 106.5 (UPM08136CELP20B1) TCID50/mL. These isolates clustered with the reference FAdV serotype 8b in the same evolutionary clade. The molecular characteristics remained unchanged, except for a point substitution at position 4 of the hexon gene of UPM08136CELP20B1, suggesting that propagation of the FAdV isolate in STB is stable and suitable for large volume production and could be a breakthrough in the scale-up process.

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.

Background The predominant infectious bronchitis virus (IBV) strains detected in chickens in Malaysia are the Malaysian variant (MV) and QX-like, which are associated with respiratory distress, nephropathy, and high mortality. On the other hand, the antigenic relatedness and efficacy of IBV vaccines against these 2 field IBV strains are not well characterized. Objectives This study aimed to determine the antigen relatedness and efficacy of different IB vaccine strains against a challenge with MV and QX-like strains. Methods The antigen relatedness and the ability of different IB vaccine strains in conferring protection against MV and QX-like were assessed based on the clinical signs, macroscopic lesions, and ciliary activity. Results The MV strain IBS037A/2014 showed minor antigenic subtype differences with the vaccine virus Mass H120 and 4/91 strains but showed major antigenic subtype differences with the K2 strain. The Malaysian QX-like strain IBS130/2015 showed major antigenic subtype differences with the MV strain IBS037A/2014 and the vaccine strains except for K2. Chickens vaccinated once with Mass (H120) or with non-Mass (4/91 and K2) developed antibody responses with the highest antibody titer detected in the groups vaccinated with H120 and 4/91. The mean ciliary activities of the vaccinated chickens were between 56 to 59% and 48 to 52% in chickens challenged with IBS037A/2014 and IBS130/2015, respectively. The vaccinated and challenged birds showed mild to severe lesions in the lungs and kidneys. Conclusions Despite the minor antigenic subtype differences, a single inoculation with Mass or non-Mass vaccines could not protect against the MV IBS037A/2014 and QX-like IBS130/2015.

To gain insights into the role of CD3-/28.4+ intraepithelial lymphocytes-natural killer (CD3-/28.4+IEL-NK) cells during infectious bursal disease virus (IBDV) infection, characterisation of the cells was performed following infection with different strains of the virus. In vitro treatment with IL-18 or ionomycin/PMA successfully stimulated and activated the cells via a significant increase in the expression of CD69, B-Lec, CHIR-AB1 and NK-lysin. Similarly, chickens infected with the vaccine strain of IBDV also up-regulated the expression of CD69, B-Lec, CHIR-AB1 and NK-lysin in CD3-/28.4+ IEL-NK cells up to 3 days post infection (dpi) and down-regulated the expression of the inhibitory receptor B-NK at 3 dpi. On the contrary, infection with the very virulent IBDV (vvIBDV) strain lead to a reduced activation of the cells by down-regulating the expression of the CD69, CHIR-AB1 and NK-lysin especially at 1 dpi. These findings altogether demonstrate the differential activation of CD3-/28.4+IEL-NK cells in chicken following infection with the vaccine or very virulent strains of IBDV. The study therefore provides an important clue into the differential pathogenesis of IBDV infection in chicken. Further studies are however required to determine the functional importance of these findings during IBDV vaccination and infection.

Summary 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.

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.

Infectious bronchitis virus (IBV) is one of the major poultry pathogens of global importance. However, the prevalence of IBV strains in Malaysia is poorly characterized. The partial genomic sequences (6.8 kb) comprising the S-3a/3b-E-M-intergenic region-5a/5b-N gene order of 11 Malaysian IBVs isolated in 2014 and 2015 were sequenced using next-generation sequencing technology. Phylogenetic and pairwise sequence comparison analysis showed that the isolated IBVs are divided into two groups. Group 1 (IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, and IBS142/2015) shared 90%-95% nucleotide and deduced amino acid similarities to the QX-like strain. Among these isolates, IBS142/2015 is the first IBV detected in Sarawak state located in East Malaysia (Borneo Island). Meanwhile, IBV isolates in Group 2 (IBS037A/2015, IBS037B/2015, IBS051/2015, and IBS180/2015) were 91.62% and 89.09% identical to Malaysian variant strain MH5365/95 (EU086600) at nucleotide and amino acid levels, respectively. In addition, all studied IBVs were distinctly separate from Massachusetts (70%-72% amino acid similarity) and European strains including 793/B, Italy-02, and D274 (68%-73% amino acid similarity). Viruses in Group 1 have the insertion of three amino acids at positions 23, 121, and 122 of the S1 protein and recombinant events detected at nucleotide position 4354-5864, with major parental sequence derived from QX-like (CK-CH-IBYZ-2011) and a minor parental sequence derived from Massachusetts vaccine strain (H120). This study demonstrated coexistence of the IBV Malaysian variant strain along with the QX-like strain in Malaysia.

The poultry industry faces challenge amidst global food security crisis. Infectious bronchitis is one of the most important viral infections that cause huge economic loss to the poultry industry worldwide. The causative agent, infectious bronchitis virus (IBV) is an RNA virus with great ability for mutation and recombination; thus, capable of generating new virus strains that are difficult to control. There are many IBV strains found worldwide, including the Massachusetts, 4/91, D274, and QX-like strains that can be grouped under the classic or variant serotypes. Currently, information on the epidemiology, strain diversity, and global distribution of IBV has not been comprehensively reported. This review is an update of current knowledge on the distribution, genetic relationship, and diversity of the IBV strains found worldwide.

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 andlog109.80 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.

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens.

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 emphasis on hexon and fiber proteins is currently the major focus of curiosity for FAdV antigenicity and pathogenicity. Recently, disease outbreaks associated with FAdV infection such as inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HHS), 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 highlighted on variation 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, whilst 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 on the knowledge of FAdV strains classification and evolution, diagnosis, and strategies to prevent and control FAdV infection and outbreaks in chicken farms.

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

Masoumeh Firouzamandi,1,2 Hassan Moeini,3 Seyed Davood Hosseini,4 Mohd Hair Bejo,1 Abdul Rahman Omar,1,3 Parvaneh Mehrbod,3 Mohamed E El Zowalaty,5 Thomas J Webster,6 Aini Ideris1,3 1Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia; 2Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Iran; 3Laboratory of Vaccine and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia; 4Razi Vaccine and Serum Research Institute, Arak, Iran; 5Biomedical Research Center, Vice President Office for Research, Qatar University, Doha, Qatar; 6Department of Chemical Engineering, Northeastern University, Boston, MA,USA Abstract: Plasmid DNA (pDNA)-based vaccines have emerged as effective subunit vaccines against viral and bacterial pathogens. In this study, a DNA vaccine, namely plasmid internal ribosome entry site-HN/F, was applied in ovo against Newcastle disease (ND). Vaccination was carried out using the DNA vaccine alone or as a mixture of the pDNA and dextran-spermine (D-SPM), a nanoparticle used for pDNA delivery. The results showed that in ovo vaccination with 40 µg pDNA/egg alone induced high levels of antibody titer (P0.05). Higher antibody titer was observed in the group immunized with 40 µg pDNA/egg at 4 weeks postvaccination. Thefindings also showed that vaccination with 40 µg pDNA/egg alone was able to confer protection against Newcastle disease virus strain NDIBS002 in two out of seven SPF chickens. Although the chickens produced antibody titers 3 weeks after in ovo vaccination, it was not sufficient to provide complete protection to the chickens from lethal viral challenge. In addition, vaccination with pDNA/D-SPM complex did not induce high antibody titer when compared with naked pDNA. Therefore, it was concluded that DNA vaccination with plasmid internal ribosome entry site-HN/F can be suitable for in ovo application against ND, whereas D-SPM is not recommended for in ovo gene delivery. Keywords: Newcastle disease, DNA vaccine, in ovo vaccination, Newcastle disease virus, dextran-spermine nanoparticle, hemagglutinin and fusion

Genotype VII Newcastle disease viruses are associated with huge economic losses in the global poultry industry. Despite the intensive applications of vaccines, disease outbreaks caused by those viruses continue to occur frequently even among the vaccinated poultry farms. An important factor in the suboptimal protective efficacy of the current vaccines is the genetic mismatch between the prevalent strains and the vaccine strains. Therefore, in the present study, an effective and stable genotype-matched live attenuated Newcastle disease virus (NDV) vaccine was developed using reverse genetics, based on a recently isolated virulent naturally recombinant NDV IBS025/13 Malaysian strain. First of all, the sequence encoding the fusion protein (F) cleavage site of the virus was modified in silico from virulent polybasic (RRQKRF) to avirulent monobasic (GRQGRL) motif. The entire modified sequence was then chemically synthesized and inserted into pOLTV5 transcription vector for virus rescue. A recombinant virus termed mIBS025 was successfully recovered and shown to be highly attenuated based on OIE recommended pathogenicity assessment indices. Furthermore, the virus was shown to remain stably attenuated and retain the avirulent monobasic F cleavage site after 15 consecutive passages in specific-pathogen-free embryonated eggs and 12 passages in one-day-old chicks. More so, the recombinant virus induced a significantly higher hemagglutination inhibition antibody titre than LaSota although both vaccines fully protected chicken against genotype VII NDV induced mortality and morbidity. Finally, mIBS025 was shown to significantly reduce both the duration and quantity of cloacal and oropharyngeal shedding of the challenged genotype VII virus compared to the LaSota vaccine. These findings collectively indicate that mIBS025 provides a better protective efficacy than LaSota and therefore can be used as a promising vaccine candidate against genotype VII NDV strains.

Limited deep studies are available in the field of early stages of pathogenesis of Newcastle disease virus (NDV) infection and tissue tropism of NDV. In this study, 24 specific pathogen free (SPF) chickens of white leghorn breed were infected with Newcastle disease (ND) by intranasal administration of 10⁵ 50% EID50/0.1 mL of velogenic NDV (vNDV). A second group of 15 chickens were kept as a control group. Chickens were monitored every day to record clinical signs. Infected chickens were euthanized by cervical dislocation at successive times, namely at hours (hrs) 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). Whereas, control group chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Tissues of brain, trachea, lung, caecal tonsil, liver, kidney, spleen, heart, proventriculus, intestine, and thymus were collected, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. HS staining, immunoperoxidase staining (IPS) and in situ PCR were applied. It was concluded that at hr 2 pi, virus seemed to be inclined to trachea and respiratory tract. Meanwhile, it attacked caecal tonsils, intestine and bursa of Fabricus. While primary viraemia was ongoing, virus created footing in kidney and thymus. At hr 4 pi, proventriculus, liver, and spleen were attacked. However, at hr 6 pi, brain and heart were involved. Secondary viraemia probably started as early as hr 12 pi since all collected tissues were positive. Tissue tropism was determined in trachea, caecal tonsil, liver, bursa of Fabricius, intestine, proventriculus, lung, spleen, thymus, kidney, heart, and brain.