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Fulltext Effects of Thermal Manipulation on mRNA Regulation of Response Genes Regarding Improvement of Thermotolerance Adaptation in Chickens during Embryogenesis

Ramiah SK, Balakrishnan KN, Subramaniam Y, Iyasere OS, Idrus Z

Animals (Basel), 2022 Nov 29;12(23).
PMID: 36496875 DOI: 10.3390/ani12233354

The phenomenon of increasing heat stress (HS) among animals is of particular significance when it is seen in economically significant industries, such as poultry. Due to the identification of the physiological, molecular, and genetic roots of HS responses in chickens, a substantial number of studies have focused on reducing the effects of HS in poultry through environmental management, dietary manipulation, and genetic alterations. Temperature manipulation (TM) during embryogenesis has been claimed to increase the thermal tolerance and well-being of chickens without affecting their capacity for future growth. There has been little investigation into the vulnerability of the epigenome involving TM during embryogenesis, although the cellular pathways activated by HS have been explored in chickens. Epigenetic changes caused by prenatal TM enhance postnatal temperature adaption and produce physiological memory. This work offers a thorough analysis that explains the cumulative impact of HS response genes, such as genes related to heat shock proteins, antioxidants, and immunological genes, which may aid in the enhanced adaptability of chickens that have undergone thermal manipulation during their embryonic stages.
The application of naked DNA plasmid (DrZP3) and recombinant adenovirus (Ad-rZP3) in rat animal model to determine comparative efficacy of ZP3-Immunocontraceptive vaccines

Mohd-Lila MA, Yee LK, Cen LS, Bala JA, Balakrishnan KN, Allaudin ZN, et al.

Microb Pathog, 2019 Sep;134:103572.
PMID: 31163251 DOI: 10.1016/j.micpath.2019.103572

The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.
Fulltext Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

Balakrishnan KN, Abdullah AA, Bala JA, Jesse FFA, Abdullah CAC, Noordin MM, et al.

Virol J, 2020 Oct 27;17(1):164.
PMID: 33109247 DOI: 10.1186/s12985-020-01436-5

BACKGROUND: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy.
METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.
RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P
Contagious ecthyma: how serious is the disease worldwide?

Lawan Z, Bala JA, Bukar AM, Balakrishnan KN, Mangga HK, Abdullah FFJ, et al.

Anim Health Res Rev, 2021 06;22(1):40-55.
PMID: 34016216 DOI: 10.1017/S1466252320000018

Contagious ecthyma (CE) is an infectious disease of small ruminants caused by a parapoxvirus of family Poxviridae subfamily Chordopoxvirinae. The disease is obviously distinguished by an establishment of scabby lesions and ulcerative formation on less hairy areas including muzzle, ears, nostril, and sometimes on genitalia. The disease is endemic in sheep and goats. The virus is transmissible to other ruminants and is a public health concern in humans. Although the disease is known as self-limiting, it may cause a significant economic threat and financial losses due to lower productivity in livestock production. Information with regard to the risk of the disease and epidemiology in most parts of the world is underreported. This paper aims to provide relevant information about the epidemiology of CE in selected regions of Europe, South America, North America, Asia, Africa, and Australia. An in-depth comprehension of virus infection, diagnoses, and management of the disease will enable farmers, researchers, veterinarians, abattoir workers, health personnel, and border controllers to improve their measures, skills, and effectiveness toward disease prevention and control, toward reducing unnecessary economic loss among farmers. A herd health program for significant improvement in management and productivity of livestock demands a well planned extension program that ought to encourage farmers to equip themselves with adequate skills for animal healthcare.
The re-emerging of orf virus infection: A call for surveillance, vaccination and effective control measures

Bala JA, Balakrishnan KN, Abdullah AA, Mohamed R, Haron AW, Jesse FFA, et al.

Microb Pathog, 2018 Jul;120:55-63.
PMID: 29709684 DOI: 10.1016/j.micpath.2018.04.057

Orf disease is known to be enzootic among small ruminants in Asia, Africa, and some other parts of the world. The disease caused by orf virus is highly contagious among small ruminant species. Unfortunately, it has been neglected for decades because of the general belief that it only causes a self-limiting disease. On the other hand, in the past it has been reported to cause huge cumulative financial losses in livestock farming. Orf disease is characterized by localized proliferative and persistent skin nodule lesions that can be classified into three forms: generalized, labial and mammary or genitals. It can manifest as benign or malignant types. The later type of orf can remain persistent, often fatal and usually causes a serious outbreak among small ruminant population. Morbidity and mortality rates of orf are higher especially in newly infected kids and lambs. Application of antibiotics together with antipyretic and/or analgesic is highly recommended as a supportive disease management strategy for prevention of subsequent secondary microbial invasion. The presence of various exotic orf virus strains of different origin has been reported in many countries mostly due to poorly controlled cross-border virus transmission. There have been several efforts to develop orf virus vaccines and it was with variable success. The use of conventional vaccines to control orf is a debatable topic due to the concern of short term immunity development. Following re-infection in previously vaccinated animals, it is uncommon to observe the farms involved to experience rapid virus spread and disease outbreak. Meanwhile, cases of zoonosis from infected animals to animal handler are not uncommon. Despite failures to contain the spread of orf virus by the use of conventional vaccines, vaccination of animals with live orf virus is still considered as one of the best choice. The review herein described pertinent issues with regard to the development and use of potential effective vaccines as a control measure against orf virus infection.
Identification and comparison of RCMV ALL 03 open reading frame (ORF) among several different strains of cytomegalovirus worldwide

Balakrishnan KN, Abdullah AA, Bala J, Abba Y, Sarah SA, Jesse FFA, et al.

Infect Genet Evol, 2017 10;54:81-90.
PMID: 28642159 DOI: 10.1016/j.meegid.2017.06.020

BACKGROUND: Rat cytomegalovirus ALL-03 (Malaysian strain) which was isolated from a placenta and uterus of a house rat, Rattus rattus diardii has the ability to cross the placenta and infecting the fetus. To further elucidate the pathogenesis of the Malaysian strain of Rat Cytomegalovirus ALL-03 (RCMV ALL-03), detailed analysis on the viral genome sequence is crucial.
METHODS: Genome sequencing of RCMV ALL-03 was carried out in order to identify the open reading frame (ORF), homology comparison of ORF with other strains of CMV, phylogenetic analysis, classifying ORF with its corresponding conserved genes, and determination of functional proteins and grouping of gene families in order to obtain fundamental knowledge of the genome.
RESULTS: The present study revealed a total of 123 Coding DNA sequences (CDS) from RCMV ALL-03 with 37 conserved ORF domains as with all herpesvirus genomes. All the CDS possess similar function with RCMV-England followed by RCMV-Berlin, RCMV-Maastricht, and Human CMV. The phylogenetic analysis of RCMV ALL-03 based on conserving genes of herpes virus showed that the Malaysian RCMV isolate is closest to RCMV-English and RCMV-Berlin strains, with 99% and 97% homology, respectively. Similarly, it also demonstrated an evolutionary relationship between RCMV ALL-03 and other strains of herpesviruses from all the three subfamilies. Interestingly, betaherpesvirus subfamily, which has been shown to be more closely related with gammaherpesviruses as compared to alphaherpesviruses, shares some of the functional ORFs. In addition, the arrangement of gene blocks for RCMV ALL-03, which was conserved among herpesvirus family members was also observed in the RCMV ALL-03 genome.
CONCLUSION: Genomic analysis of RCMV ALL-03 provided an overall picture of the whole genome organization and it served as a good platform for further understanding on the divergence in the family of Herpesviridae.
Velogenic newcastle disease virus tissue tropism and pathogenesis of infection in chickens by application of in situ PCR, immunoperoxase staining and HE staining

Hussein EA, Hair-Bejo M, Omar AR, Arshad SS, Hani H, Balakrishnan KN, et al.

Microb Pathog, 2019 Apr;129:213-223.
PMID: 30771470 DOI: 10.1016/j.micpath.2019.02.017

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.
Immediately early 2 (IE-2) and DNA polymerase SiRNA as virus-specific antiviral against novel transplacental cytomegalovirus strain ALL-03 in vitro

Balakrishnan KN, Abdullah AA, Bala JA, Jesse FFA, Abdullah CAC, Noordin MM, et al.

Infect Genet Evol, 2021 06;90:104783.
PMID: 33640483 DOI: 10.1016/j.meegid.2021.104783

OBJECTIVE: This study investigated the suitability of siRNA targeting specific genes that cause inhibition of virus replication in vitro especially for the virus that capable of crossing placenta and we employed a novel transplacental rat cytomegalovirus that mimics infection in human.
METHODS: Six unique siRNAs with three each targeting different regions of IE2 (ie2a, ie2b and ie2c) and DNA polymerase (dpa, dpb and dpc) were prepared and tested for antiviral activities. The efficacy as an antiviral was determined in in-vitro by measuring TCID50 virus titer, severity of virus-induced cytopathic effect (CPE), intracellular viral genome loads by droplet digital PCR, the degree of apoptosis in siRNA-treated cells and relative expression of viral mRNA in infected Rat Embryo Fibroblast (REF) cells.
FINDINGS: Remarkably, the siRNAs: dpa, dpb and IE2b, significantly reduced virus yield (approximately >90%) compared to control group at day 18 post infection (p.i). Changes in CPE indicated that DNA polymerase siRNAs were capable of protecting cells against CMV infection at day 14 p.i with higher efficiency than GCV (at the concentration of 300 pmol). Gene expression analysis revealed a marked down regulation of the targeted DNA polymerase gene (73.9%, 96.0% and 90.7% for dpa, dpb and dpc siRNA, respectively) and IE2 gene (50.8%, 49.9% and 15.8% for ie2a, ie2b and ie2c siRNA, respectively) when measured by RT-qPCR. Intracellular viral DNA loads showed a significant reduction for all the DNA polymerase siRNAs (dpa: 96%, dpb: 98% and dpc:92) compared to control group (P
Fulltext An association of Orf virus infection among sheep and goats with herd health programme in Terengganu state, eastern region of the peninsular Malaysia

Bala JA, Balakrishnan KN, Abdullah AA, Adamu L, Noorzahari MSB, May LK, et al.

BMC Vet Res, 2019 Jul 18;15(1):250.
PMID: 31319873 DOI: 10.1186/s12917-019-1999-1

BACKGROUND: Orf virus causes a scabby skin lesions which decreases productivity in small ruminants. The unknown status of this disease in the eastern region of Peninsular Malaysia warrants a study to determine sero-prevalence of orf with regards to farmers' compliance level towards the Herd Health Program (HHP) programme.
RESULTS: Out of 504 animals, 115 were positive for Orf-virus antibodies. An overall prevalence rate of 22.8% indicated a high prevalence of orf disease in this region. It was observed that 25.1% (92/367) of goats were positive and 16.8% (23/137) of sheep sero-converted for Orf virus antibody. Several factors were measured for their possible association with prevalence of Orf virus infection. The prevalence was higher in LY farm, JC breed, kid and female animals, and in the presence of disease lesion. Chi-square analysis showed a significant association of three risk factors which are species, age and sex of the animals (P 0.05). Farms surveyed usually practised intensive management system, keeping animals in the shade at all time, due to limited availability of suitable land as a free-range grazing area. An interview with small holder farmers revealed a lack of awareness of the main goals of herd health programme. An overall compliance level of 42.7% was observed for all HHP parameters. Among the 14 main components of HHP modules, animal identification had recorded highest compliance level (84.62%) while milking management recorded the least compliance (- 82.69%). That explained why there was a high sporadic prevalence of Orf infection in this region.
CONCLUSION: Good herd health supervision is a rehearsal target to prevent an outbreak and the spread of diseases thus reduces economic losses among farmers. Therefore, a good herd health programme should be in place, in order to prevent and control disease transmission as well as to improve herd immunity.
Fulltext Dermatopathology of Orf Virus (Malaysian Isolates) in Mice Experimentally Inoculated at Different Sites with and without Dexamethasone Administration

Bala JA, Balakrishnan KN, Abdullah AA, Kimmy T, Abba Y, Bin Mohamed R, et al.

J Pathog, 2018;2018:9207576.
PMID: 30155311 DOI: 10.1155/2018/9207576

Orf is a clinical manifestation of parapoxvirus infection often fatal in goats and sheep especially when they are under stress or influenced by unfavorable environment. This study investigated the pathogenicity of two Orf virus isolates (ORFV UPM1/14 and UPM2/14) and host response in mouse model by using different inoculation sites with/without prior exposure to dexamethasone. Treatments with dexamethasone served as an immunosuppressant that may mimic stress situation in affected animals. Groups of five mice were given intradermal injection of 0.2 mL of tissue culture infective dose 50 (TCID50) of UPM1/14 (Group 1) and UPM2/14 (Group 2) at the dorsum (Group 1A; Group 2A), ear pinna (Group 1B; Group 2B), and labial commissure (Group 1C; Group 2C). An inoculum 0.2 mL of UPM1/14 was administered to animals treated with dexamethasone (n=5; 5 mg/kg/day intraperitoneally) and nondexamethasone (n=5) groups at the dorsum, ear pinna, and labial commissure. No significant difference (p>0.05) was observed in the mean lesion scores among the groups of different inoculation sites or between dexamethasone-treated and nontreated groups. However, there was a significant difference (p<0.05) in the mean stratum thickness of affected skin following inoculation with UPM2/14 isolate at the ear pinna and labial commissure. Histopathology examination revealed keratosis, acanthosis, and ballooning degeneration in the skin of affected mice. Orf virus DNA was detected in the skin samples by targeting F1L and B2L virus-specific genes in polymerase chain reaction (PCR) assay. Intradermal inoculation with UPM1/14 or UPM2/14 isolate produced a mild skin lesion in mice, and there was no significant difference in orf disease manifestation despite variation of inoculation sites. Similarly, short-term dexamethasone administration gave no adverse effects on pathogenicity of orf virus isolates.
Identification of strain diversity and phylogenetic analysis based on two major essential proteins of Orf viruses isolated from several clinical cases reported in Malaysia

Bala JA, Balakrishnan KN, Jesse FFA, Abdullah AA, Noorzahari MSB, Ghazali MT, et al.

Infect Genet Evol, 2020 01;77:104076.
PMID: 31678648 DOI: 10.1016/j.meegid.2019.104076

There is a little information on the characterization of Orf virus strains that are endemic in Malaysia. The relationship between the severity of disease and the molecular genetic profile of Orf virus strains has not been fully elucidated. This study documented the first confirmed report of contagious ecthyma causing by Orf virus in goats from a selected state of eastern peninsular Malaysia. The disease causes significant debilitation due to the inability of affected animals to suckle which brings a great economic loss to the farmers. A total of 504 animals were examined individually to recognize the affected animals with Orf lesion. Skin scrapping was used to collect the scab material from the infected animals. The presence of Orf virus was confirmed by combination of methods including virus isolation on vero cells, identification by Transmission Electron Microscopy (TEM) and molecular technique using PCR and Sanger sequencing. The results showed the successful isolation of four Orf virus strains with a typical cytopathic effects on the cultured vero cells line. The morphology was confirmed to be Orf virus with a distinctive ovoid and criss cross structure. The phylogenetic analysis revealed that these isolated strains were closely related to each other and to other previously isolated Malaysian orf viruses. In addition these Orf virus strains were closely related to Orf viruses from China and India. This study provides more valuable insight in terms of genotype of Orf virus circulating in Malaysia.
Isolation and phylogenetic analysis of caprine Orf virus in Malaysia

Abdullah AA, Ismail MF, Balakrishnan KN, Bala JA, Hani H, Abba Y, et al.

Virusdisease, 2015 Dec;26(4):255-9.
PMID: 26645035 DOI: 10.1007/s13337-015-0278-4

Orf virus is a DNA virus that causes contiguous ecthyma in goat and sheep. Infection of animals with this virus cause high mortality in young animals resulting in huge economic losses. In this study, we investigated an outbreak of Orf in a goat farm in Malaysia. Samples were collected from infected animals and viral isolation was done using both LT and MDCK cell lines. Molecular detection was done by conventional PCR for specific primers; B2L and F1L genes and phylogenetic analysis was done on the sequence data obtained. Cytopathic effects (CPE) were observed in both cell lines after 3 days of inoculation and were 50 % by the sixth day. PCR showed positive bands for both B2L and F1L genes and phylogenetic analysis showed that the Malaysian strain had close homology to the Chinese and Indian Orf virus isolates. This study gives more insight into the existing Orf viral strains in Malaysia and their relationship with other strains globally.
Fulltext Complete Genome Sequence of Rat Cytomegalovirus Strain ALL-03 (Malaysian Strain)

Balakrishnan KN, Abdullah AA, Camalxaman SN, Quah YW, Abba Y, Hani H, et al.

Genome Announc, 2015;3(3).
PMID: 26044413 DOI: 10.1128/genomeA.00451-15

The complete genome sequence of the ALL-03 strain of rat cytomegalovirus (RCMV) has been determined. The RCMV genome has a length of 197,958 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. This strain is closely related to the English strain of RCMV in terms of genetic arrangement but differs slightly in size.

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