Chicken Embryo Fibroblast Cell Cultures Research Articles

Molecular characterization and immunopathological investigation of Avian reticuloendotheliosis virus in breeder flocks in Egypt

BackgroundReticuloendotheliosis virus (REV) is an oncogenic immunosuppressive retrovirus that infects different kinds of avian species; posing significant economic losses to the poultry industry worldwide.MethodsIn Egypt, there is an unidentified disease associated with the runting-stunting syndrome with neoplasia, suspected to be REV, that has been continuously monitored in several breeder flocks. To diagnose and analyze REV by cell cultures, enzyme-linked immunosorbent assay (ELISA), histopathological investigation, the polymerase chain reaction (PCR) test, and sequencing analysis, 200 blood samples, and 50 tissue specimens were collected. The current study targets the occurrence and genetic characteristics of a viral neoplastic disease, resembling REV infection, circulating in breeder flocks from 2022 to 2023 in the Ismailia, El-Sharqia, and El-Dakahliya governorates.ResultHere, REV was isolated on chicken embryo fibroblast cell culture; exhibiting cell aggregation, rounding, and cell detachments. Collectively, only 70 serum samples were positive for anti‐REV antibodies with seroprevalence rates of 35% based on the ELISA test. The histopathological observation demonstrated lymphoreticular tumors in the liver, spleen, and other examined organs. The immunohistochemical staining method confirmed the REV-positive signals in all examined organs (liver, kidney, spleen, bursa, ovaries) except for the heart. The PCR assay of the LTR gene assessed 370 base pairs with only 5 positive samples with a percentage of 16.6%. Three positive samples were further sequenced and submitted to the Genbank under accession numbers (PP763709, PP763710, PP763711). Phylogenetic analysis of the REV-LTR gene showed that our three isolates (Sharquia-1-REV, Ismilia-2-REV, Mansoura-3-REV) are REV subtype III which predominantly circulated in breeders in Egypt. These three isolates are highest similar to American, Chinese, and Taiwanese REV reference strains, and other Egyptian strains with nucleotide identity percentages of 100%, 99%, and 99%; respectively, and on the amino acid identity level were with (99–100%), (98%, 99%), (99%, 100%); respectively.ConclusionsThis study established that REV infection was extensively distributed in the breeders and became one of the causes of the clinical outbreaks of tumors, raising awareness of REV as the causative agent of avian oncogenic disease in Egypt.

Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Molecular characterization of the chicken embryo fibroblast cell culture propagated Indian isolate of duck enteritis virus and its bioinformatics analysis

Molecular characterization of the chicken embryo fibroblast cell culture propagated Indian isolate of duck enteritis virus and its bioinformatics analysis

First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

Pathological and Molecular Characterization of a Duck Plague Outbreak in Southern China in 2021.

Duck plague (DP) is a highly contagious viral disease in ducks caused by the duck plague virus (DPV). The DPV, a member of Herpesviridae, poses a severe threat to the waterfowl farming industry worldwide. In this study, we reported a recent outbreak of DPV in domestic laying ducks at 310 days of age from southern China in December 2021. The gross lesion, histopathologic examination, molecular detection, and genetic characterization studies of DPV are described here. As a result, gross lesions such as an enlarged congestive spleen and liver were observed. Liver with vacuolar degeneration and small vacuoles and spleen with hemosiderosis were remarkable microscopic findings. Our results suggested that the liver had the highest viral load, followed by the trachea, pancreas, kidney, brain, spleen, and heart. In addition, DPV was successfully isolated in chicken embryo fibroblast cell culture and designated as DP-GD-305-21. The UL2, UL12, UL41, UL47, and LORF11 genes of DP-GD-305-21 shared a high nucleotide homology with the Chinese virulent (CHv) strain and the Chinese variant (CV) strain. In conclusion, this study reports the isolation and molecular characterization of DPV from a recent outbreak in southern China. Our results contributed to the understanding of the pathological and molecular characterization of currently circulating DPV in China.

Isolation and Molecular Detection of Feline Infectious Peritonitis Virus.

Feline coronavirus (FCoV) is an enveloped single-stranded RNA virus, affecting wild and domestic cats. Feline infectious peritonitis viruses (FIPV) variants of FCoV cause fatal peritonitis affecting approximately 5% of FCoV infected animals. The present study aimed to detect and isolate the feline infectious peritonitis virus for the first time in Iraq. In this study, 50 samples (fecal swab and peritoneal fluid) were collected from suspected pet cats from different areas of Baghdad, Iraq. The very suitable age was under two years old. Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) was used to detect Feline infectious peritonitis in infected collected samples by the amplification of spike protein (S). The result of real-time RT-PCR revealed that out of 50 samples from suspected cats, 10 samples were positive for FIPV. Moreover, 10 positive samples by real-time RT-PCR were used for the isolation of the virus in chicken embryo fibroblast cell culture. Subsequently, the isolated virus was detected by real-time RT-PCR and then by conventional RT-PCR, followed by electrophoresis.

Detection and Isolation of Camelpox Virus in Wasit Province, Iraq.

Camels are susceptible to a variety of infectious diseases, such as trypanosomiasis, anthrax, hemorrhagic septicemia, brucellosis, mange, and pox, which can also affect other farm animal species. Camelpox is one of the most infectious skin diseases, which is caused by the Camelpox virus (CPV), a member of the Orthopoxvirus genus in the Poxviridae family. This study mainly aimed to detect and isolate CPV affecting camels in Wasit province, Iraq. Initially, the study focused principally on clinical manifestations of the disease in affected animals. Afterward, 110 skin samples were collected from infected animals under strict aseptic conditions. They were to be subjected to inoculation into the local embryonated chicken eggs to isolate the virus from the chicken embryo fibroblast cell culture. Finally, the isolates were confirmed using the molecular polymerase chain reaction (PCR) assay. Camelpox virus isolates appeared as several necrotic pock lesions on the Chorioallantoic Membrane (CAM) with cell clustering and sloughing or detachment from the monolayer. Molecular testing was conducted using the PCR assay by targeting the ATIP gene at 881bp. The findings demonstrated that all the investigated isolates were poxvirus positive. In addition, it was found that Camelpox disease is significantly endemic in Wasit province, Iraq. The visualization of the characteristic features of the virus also revealed it can easily adapt to replication in the CAM and cell culture.

Epidemiology and laboratory diagnosis of very virulent infectious bursal disease virus in vaccinated chickens in Khartoum, Sudan.

Background:Infectious Bursal Disease (IBD, Gumboro disease) has become more severe than in early outbreaks in the 1980s. The present research aims to study the epidemiology of IBD in Khartoum state and compare some commonly used laboratory techniques for diagnosis.Method:We collected epidemiological data from 30 farms that showed signs suggestive of IBD, estimated the morbidity and mortality rates, and interviewed the owners about the type and the doses of the used vaccines. We collected bursas of Fabricius for virus assays and histopathology. Samples positive in the agar gel immunodiffusion (AGID) test were inoculated onto chicken embryo fibroblast cell culture and embryonated chicken eggs. Twenty-two-day-old chicks were infected experimentally with three selected isolates, and morbidity and mortality rates were compared.Results:The results showed that 70% of outbreaks occurred between 6 and 8 weeks of age, and the mean mortality rate was 51%. Epidemiologic, clinical, gross, and histopathological findings were characteristic of the severe disease caused by the very virulent IBDvirus (vvIBDV). The farms that used intermediate or the intermediate plus vaccines had lowered mortality compared with the farms that used intermediate vaccines. The AGID was found more sensitive than the counter-immuno-electrophoresis (CIEP) since it detected 83.4% of the IBDV antigen in the samples while the CIEP detected 66.7% of the samples. The reverse transcriptase polymerase chain reaction (RT-PCR) was found to be rapid, specific, and was more sensitive detecting 100% of the tested samples. Virus isolation in embryonated eggs and cell culture was not successful.Conclusion:A vvIBDV is responsible for the recent outbreaks of the disease in Sudan, resulting in a mean high mortality rate of 51%, even in vaccinated flocks. The RT-PCR and AGID are the best methods for laboratory confirmation.

Pathogenicity of field strain of fowl aviadenovirus serotype 11 isolated from chickens with inclusion body hepatitis in Morocco.

Outbreaks of inclusion body hepatitis have emerged in Morocco since 2013 and has resulted in significant economic losses to poultry farms. Three isolates of the causative virus, Fowl adenonovirus (FAdV)were characterized from chickens with IBH, but their pathogenicity has never been investigated. In this work, the pathogenicity of an isolate FAdV 11 (MOR300315 strain) was evaluated by inoculating a group of 40 SPF chickens at 3 days of age by oral route. A group of 40 chicks injected with phosphate-buffered saline solution was used as a control group. The infected chickens showed decreased weight gain from 3dpi. Necropsy displayed pallor and enlargement in liver, swelling and slight hemorrhage in kidney and spleen at 6 dpi. Histopathological changes were mainly characterized by severe and extensive hepatic necrosis associated with the presence of basophilic intra-nuclear inclusion bodies within hepatocytes. The FAdV was reisolated in chicken embryo fibroblast cell culture from liver tissue homogenate of infected chicken from 3 to 6 dpi. Viral DNA was detected by PCR in liver, kidney, spleen and cloacal swabs from 3 to 13 dpi. Antibody response against inoculated FAdV was appeared from 9 dpi. These results confirmed that the FAdV 11 strain is pathogenic in chicken. This study is the first experimental infection of FAdV 11 in chicken in Morocco, which increase our understanding of its pathogenicity in chickens and indicate that preventive measures against FAdV infection in poultry farms should be implemented in Morocco.

Attenuation of a recombinant Marek's disease virus lacking the meq oncogene and evaluation on its immune efficacy against Marek's disease virus

SC9-2 is a recombinant Marek's disease virus (MDV) strain lacking the meq oncogene. Previous study demonstrated that SC9-2 virus provides good protection against challenge with a very virulent MDV rMd5, but it induces immunosuppressive effects in specific pathogen-free (SPF) chickens. In the present study, SC9-2 was serially passaged on chicken embryo fibroblast (CEF) cell cultures. The pathogenicity and immune efficacy of SC9-2/10th and SC9-2/40th against rMd5 were evaluated. Animal experimental results showed that SC9-2/10th and SC9-2/40th showed no lethality or tumorigenicity in SPF chickens. Body weight of chickens inoculated with SC9-2/40th were significantly higher than that of the chickens inoculated with SC9-2/10th but lower than that of the uninoculated controls. The severity of bursa and thymus atrophy (BTA) and spleen enlargement in SC9-2/40th-inoculated chickens were also weaker than the SC9-2/10th-inoculated ones but stronger than the uninoculated controls. Chickens inoculated with SC9-2/40th and SC9-2/10th showed similar antibody levels induced by H9N2 subtype avian influenza virus/Newcastle disease virus inactivated vaccines, both of which were lower than the uninoculated controls. Replication of SC9-2/40th was significantly lower than SC9-2/10th in feather follicle epithelium (FFE) of infected chickens. The immune protection index of SC9-2/40th was also lower than that of SC9-2/10th, but the difference was not significantly, and both of which were significant higher than that of the commercial MDV vaccine CVI988/Rispens. The results of our studies demonstrated that SC9-2/40th showed weaker severity of BTA, spleen enlargement, and body weight loss and lower replication level in FFE than SC9-2/10th in SPF chickens. However, SC9-2/40th was able to confer better immune protection as compared with CVI988/Rispens vaccination in SPF chickens. In conclusion, serially attenuation of SC9-2 in CEFs reduced the lymphoid organ atrophy and replication in SPF chickens, and the immune protective efficacy of attenuated viruses was still superior than CVI988/Rispens.

Isolation and characterization of fowl aviadenovirus serotype 11 from chickens with inclusion body hepatitis in Morocco.

The present study was conducted in order to isolate, identify and characterize fowl aviadenovirus associated with inclusion body hepatitis (IBH) in three poultry farms (two of broiler chickens and one of breeder broiler chickens) in Morocco during 2015. Liver samples collected from affected three poultry farms were examined by histopathological examination. Tissue samples showing necrosis of hepatocytes associated with basophilic intranuclear inclusion bodies were homogenized and submitted to FAdV isolation in chicken embryo fibroblast (CEF) cell cultures and in SPF embryonated eggs. The cytopathic effect (CPE) was observed in the second passage with swelling and rounding of infected cells. The inoculated embryos were hemorrhagic and showed hepatitis with the presence of basophilic intra-nuclear inclusion bodies within hepatocytes. The presence of the virus was confirmed by conventional polymerase chain reaction based on hexon gene from all investigated samples. Moreover, phylogenetic analysis of the hexon gene revealed that FAdVs isolated from different affected poultry belonged to FAdV 11 serotype of the D genotype group. This work is the first isolation in cell culture and SPF embryonated eggs of FAdV from Moroccan broilers and breeder broiler chickens with IBH.

Adaptation of wild strain of duck plague virus in cell culture systems

Duck virus enteritis (DVE) is an acute, contagious herpes viral disease of ducks, geese and swans and members of the family Anatidae under the order Anseriformes. As per the recent taxonomic classification by ICTV, DEV has been classified into the genus Mardivirus, subfamily Alpha-herpesvirinae of the family Herpesviridae. Vaccination is the only option for the prevention and control of Duck plague. In India vaccination is done with chicken embryo adapted live virus which has many shortcomings. So, in this present situation a safe and potent vaccine development is the need of the hour. The use of chicken embryo fibroblast and a certified cell line may, probably, be the best option to achieve this. Therefore, the present study was undertaken for adaptation and propagation of local strain of duck plague virus in various cell culture systems. During the study a wild strain of DPV (DP/As-Km/0019) which was isolated in Duck Embryo Fibroblast, available in the Department of Microbiology was selected. The selected wild strain was used for adaptation in various cell culture systems viz. Chicken embryo fibroblast cell culture (CEF), Vero cell line and QT-35 cell line. The virus was passaged up to 12th passage levels and the presence of viral antigen was demonstrated by appearance of cytopathic effect (CPE), by Polymerase Chain Reaction (PCR) and by Sandwich ELISA. PCR was able to detect virus from all the cell cultures from 5th passage onwards and virus titre was detected at 10th and 12th passage level by S-ELISA. It was observed that various cell culture systems can be a good candidate for propagation of DPV and further study is required to study its immunogenicity and its feasibility as a vaccine candidate.

Isolation and molecular detection of Fowl pox and Pigeon pox viruses for the development of live attenuated vaccine seeds from the local isolates

Avipox is a viral disease of fowl and pigeon which is characterized by proliferative and nodular lesions in the feather-free parts of the skin or fibro-necrotic and proliferating part in the mouth, esophagus, and mucous membrane of the upper respiratory tract. This investigation was carried out with an aim to isolate and molecular detection of Fowl pox virus (FPV) and Pigeon pox virus (PPV) for development of live attenuated vaccine seeds from the local virus isolates. In this study, nodular lesions were collected from seven pigeons and four chickens from different areas of Mymensingh in Bangladesh which were affected by pox. Viral inoculums were prepared and DNA materials were extracted for PCR-based identification of P4b genes. Detection of virus was confirmed by PCR following propagation into 9-11 days old embryonated chicken egg (ECE) and also chicken embryo fibroblast (CEF) cell culture All the field samples were found positive for FPV and PPV by PCRR. These field isolates were propagated and attenuated in duck embryo through CAM route and duck embryo fibroblast (DEF) cell culture for the development of live attenuated vaccine seeds. Attenuation of both FPV and PPV were successful in duck embryo through CAM route and duck embryo fibroblast (DEF) cell culture after serial six passages. Attenuation of the virus was confirmed by inoculation into experimental birds. Inoculation of attenuated FPV and PPV in chicken and pigeon respectively exhibited no pox lesions whereas control chicken and pigeon inoculated with field isolates develop nodular lesions. Both FPV and PPV were confirmed from both groups of birds by PCR. These attenuated local isolates of FPV and PPV could be used as potential vaccine candidates for the prevention and control of fowl pox and pigeon pox in Bangladesh.
 J. Bangladesh Agril. Univ. 17(2): 211–219, June 2019

Diagnosis of Human Papilloma virus in Colorectal Cancer and Inhibition by Gamma Radiation

Human Papillomavirus was is olated from different age groups from 40 upto 71 years for both gender. The samples were diagnosed by RT-PCR. Positive cases of human Papillomavirus were forty represent (63.49%) and twenty two represent (36.50%) negative cases. Isolation process of Human Papilloma virus was successfully performed on chicken embryo fibroblast cell culture for 4th passage for suspected samples obtained from (colon organ) as well as isolate from chick embryo [specific pathogen free (SPF). It was found that at the tissue culture infective dosage (TCID50/0.1 ml) was (107.8) of virus. The gamma irradiation that dosage used for inactivating of the purified virus which was extracted from allantoic fluid and tissue culture was (6.594) mSv/h. This revision intended to gauge the prevalence of HPV cannon and viral incorporation in colorectal tissues in imperative to estimate its presumed role in colorectal cancer and inhibition viral by gamma radiation.

In Vitro Antiviral Activity of Commercial Products of Herbal Extracts Against Highly Pathogenic Avian Influenza (H5N1) Virus

Three commercial products of herbal extracts (Amphiflu-Ex®, Fyto-immune Plus® and Viu-Vet®) were tested for antiviral activity against two Egyptian highly pathogenic avian influenza subtype H5N1 isolates (isolated form backyard poultry) and upon sequencing and phylogenetic analysis, they belonged to clade 2.2.1.2., This antiviral assay for the products done by using chicken embryo fibroblast (CEF) cell culture through cytopathic effect (CPE) inhibition assay in two different layouts to elucidate post-exposure and pre-exposure antiviral activity of the products. The Amphiflu-Ex® at conc. 50 µl/ml showed significant virucidal activity (100% inhibition) in post-exposure assay for both viral isolates which was further confirmed by quantitative real time RT-PCR method showed 100% reduction in virus titers for both isolates and further investigation done on The Amphiflu-Ex® on conc. 20 µl/ml, the virus titers were decreased by 2.72-2.8 logs (Log 10 virus titers) and on conc 10 µl/ml, the virus titers were decreased by 1-1.27 logs for both isolates, The maximum non-toxic concentration (MNTC) for the three products estimated by cytotoxicity assay was 50 µl/ml and TCID50 used for both isolates were 106 copy/100 µl.

Plaque formation of Quesland V4 lentogenic strain of Newcastle disease virus adapted in chick embryo fibroblast cells

Vaccination is one of the important methods for preventing and controlling of the Newcastle disease in Iran. Inactivated Newcastle disease vaccine is produced in the Razi Vaccine and Serum Research Institute, Iran, and is mainly used for vaccination against Newcastle disease. Clone vaccines are used in many countries nowadays. One of the ways for cloning a virus is propagation of the virus on cell cultures to obtain discrete plaques. In this study, monolayer chick embryo fibroblast (CEF) cell cultures were prepared by a standard method. Monolayer CEF cells were used for plaque production trial. Confluent monolayer CEF cells were in tissue culture plates and inoculated with avian paramyxovirus-1/chicken/Australia/Queensland/V4/1966 (QV4) (V4 seed of Newcastle vaccine) with 109.7 50 % egg infection dose. Various dilutions of the viruses were inoculated into monolayer CEF cell cultures that were supplemented with magnesium sulfate and trypsin and agar overlay medium. No plaques were observed on CEF tissue culture plates inoculated with V4 strain without adaptation. Eight discrete large and small plaques were obtained in the 10−5 dilution of adapted viruses (titer 3 × 107 plaque-forming units (PFU)/ml) with 2–4 mm diameter at 80 h post inoculation, and in the 10−7–10−10 dilution, there was no any plaque obtained. A dilution of 10−4 produced 28 plaques (1.75 × 108 PFU/ml) but not discrete plaque, and at 10−1–10−3 dilution, plaques were too much that they were not countable. Eight various plaques at 10−5 were collected and propagated for further investigations. The present assay is a useful tool for demonstration of discrete plaques using the adapted v4 of Newcastle disease virus within 3 days on monolayer CEF cell cultures covered with modified overlay media containing magnesium ions, diethylaminoethyl dextran, and ionoagar to ascertain the purity. This technique for plaque formation by v4 of Newcastle disease virus provides a method for establishing the purity of seed stock used in the production of vaccine.

Growth and Cytopathogenicity of a Field Isolate of Fowl Poxvirus in Chicken Embryo Fibroblast Cell Culture

Growth and Cytopathogenicity of a Field Isolate of Fowl Poxvirus in Chicken Embryo Fibroblast Cell Culture

Assessment of the pathogenicity of cell-culture-adapted Newcastle disease virus strain Komarov.

Newcastle disease vaccines hitherto in vogue are produced from embryonated chicken eggs. Egg-adapted mesogenic vaccines possess several drawbacks such as paralysis and mortality in 2-week-old chicks and reduced egg production in the egg-laying flock. Owing to these possible drawbacks, we attempted to reduce the vaccine virulence for safe vaccination by adapting the virus in a chicken embryo fibroblast cell culture (CEFCC) system. Eighteen passages were carried out by CEFCC, and the pathogenicity was assessed on the basis of the mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index, at equal passage intervals. Although the reduction in virulence demonstrated with increasing passage levels in CEFCC was encouraging, 20% of the 2-week-old birds showed paralytic symptoms with the virus vaccine from the 18th(final) passage. Thus, a tissue-culture-adapted vaccine would demand a few more passages by CEFCC in order to achieve a complete reduction in virulence for use as a safe and effective vaccine, especially among younger chicks. Moreover, it can be safely administered even to unprimed 8-week-old birds.

Detection of T- and B-cell Target Antigens of Fowlpox Virus Isolated from Backyard Chickens in India.

With the aim of assessing the antigenic characteristics of a circulating pool of fowlpox virus (FPV) that exists in the backyard poultry system in India, one of the field isolates generated was characterized by in vitro immunologic techniques. FPV was isolated from clinically positive fowlpox cases (n = 10) from the Jhargram (West Midnapur district) and Kakdwip (South 24 Pargana district) areas of West Bengal State, India. Initially, FPV-specific PCR was performed for confirmation of the samples. Isolation of FPV was done using embryonated chicken eggs and the choreoallantoic membrane route. Subsequently, FPV antigen was prepared from chicken embryo fibroblast cell culture-adapted field isolate. Biologic transmission of FPV was performed in Rhode Island red chickens experimentally to assess humoral and cell-mediated immune (CMI) responses. High level of anti-FPV antibodies were observed in test birds as assessed by indirect ELISA. Seroreactive polypeptides (B-cell antigens) of FPV antigen with molecular weights of 44.5, 66.5, 75, 90.5, and 99 kDa were detected by western blot analysis. Significant increases in CMI responses were observed in inoculated chickens as assessed by lymphocyte proliferation assay, cytotoxicity assay, and T-cell immunoblotting. The predominant T-cell antigen of FPV detected had a molecular weight of 66.5 kDa. The present study revealed the antigenic characteristics of FPV that exists in backyard poultry system in West Bengal for the first time, thus exploring the rationality of designing future T- and B-cell vaccines against fowlpox.

Regeneration of a recombinant infectious bursal disease virus having four amino acid substitutions in VP2 by reverse genetics

Infectious bursal disease virus (IBDV), a virus with a double-stranded, bi-segmented RNA genome, is an economically important pathogen of chickens. Recent understanding of the molecular biology of IBDV has implicated several amino acid residues in the capsid protein VP2 in pathogenicity and tissue culture adaptation. In the present study a recombinant strain of IBDV having four mutations in VP2 (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) has been generated using reverse genetics. Desired mutations were introduced in the VP2 gene of the cloned cDNA of genome segment A of a very virulent (vv) IBDV by site-directed mutagenesis. Capped RNA transcribed in vitro from cloned cDNA of the modified segment A and wild type segment B was co-transfected into chicken embryo fibroblast (CEF) cell culture. The recombinant virus, designated as BD- 3tcC, was rescued from the transfected cell culture and characterized in vitro. BD-3tcC retained all the four desired mutations and replicated with titres only slightly lower than those of CEF cell-culture-adapted wild-type IBDV. This recombinant strain can be used in future studies for understanding the biological significance of these four amino acid residues in VP2. DOI: http://dx.doi.org/10.3329/bvet.v31i1.22838 Bangl. vet. 2014. Vol. 31, No. 1, 12-19