{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T13:44:33Z","timestamp":1790603073300,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,10,2]],"date-time":"2021-10-02T00:00:00Z","timestamp":1633132800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001407","name":"Department of Biotechnology, Ministry of Science and Technology, India","doi-asserted-by":"publisher","award":["BT\/PR21576\/ADV\/90\/249\/2016"],"award-info":[{"award-number":["BT\/PR21576\/ADV\/90\/249\/2016"]}],"id":[{"id":"10.13039\/501100001407","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Newcastle disease virus (NDV) strain R2B, with an altered fusion protein cleavage site, was used as a viral vector to deliver the immunogenic genes VP2 and VP1 of chicken infectious anaemia virus (CIAV) to generate a bivalent vaccine candidate against these diseases in chickens. The immunogenic genes of CIAV were expressed as a single transcriptional unit from the NDV backbone and the two CIA viral proteins were obtained as separate entities using a self-cleaving foot-and-mouth disease virus 2A protease sequence between them. The recombinant virus (rR2B-FPCS-CAV) had similar growth kinetics as that of the parent recombinant virus (rR2B-FPCS) in vitro with similar pathogenicity characteristics. The bivalent vaccine candidate when given in specific pathogen-free chickens as primary and booster doses was able to elicit robust humoral and cell-mediated immune (CMI) responses obtained in a vaccination study that was conducted over a period of 15 weeks. In an NDV and CIAV ELISA trial, there was a significant difference in the titres of antibody between vaccinated and control groups which showed slight reduction in antibody titre by 56 days of age. Hence, a second booster was administered and the antibody titres were maintained until 84 days of age. Similar trends were noticed in CMI response carried out by lymphocyte transformation test, CD4+ and CD8+ response by flow cytometry analysis and response of real time PCR analysis of cytokine genes. Birds were challenged with virulent NDV and CIAV at 84 days and there was significant reduction in the NDV shed on the 2nd and 4th days post challenge in vaccinated birds as compared to unvaccinated controls. Haematological parameters comprising PCV, TLC, PLC and PHC were estimated in birds that were challenged with CIAV that indicated a significant reduction in the blood parameters of controls. Our findings support the development and assessment of a bivalent vaccine candidate against NDV and CIAV in chickens.<\/jats:p>","DOI":"10.3390\/v13101985","type":"journal-article","created":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T01:59:47Z","timestamp":1633917587000},"page":"1985","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Newcastle Disease Virus Vectored Chicken Infectious Anaemia Vaccine Induces Robust Immune Response in Chickens"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0477-6829","authenticated-orcid":false,"given":"Madhan Mohan","family":"Chellappa","sequence":"first","affiliation":[{"name":"Recombinant DNA Laboratory, Division of Veterinary Biotechnnology, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7205-4599","authenticated-orcid":false,"given":"Sohini","family":"Dey","sequence":"additional","affiliation":[{"name":"Recombinant DNA Laboratory, Division of Veterinary Biotechnnology, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dinesh Chandra","family":"Pathak","sequence":"additional","affiliation":[{"name":"Recombinant DNA Laboratory, Division of Veterinary Biotechnnology, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asmita","family":"Singh","sequence":"additional","affiliation":[{"name":"Recombinant DNA Laboratory, Division of Veterinary Biotechnnology, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0483-848X","authenticated-orcid":false,"given":"Narayan","family":"Ramamurthy","sequence":"additional","affiliation":[{"name":"Recombinant DNA Laboratory, Division of Veterinary Biotechnnology, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7641-5067","authenticated-orcid":false,"given":"Saravanan","family":"Ramakrishnan","sequence":"additional","affiliation":[{"name":"Immunology Section, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6348-0724","authenticated-orcid":false,"given":"Asok Kumar","family":"Mariappan","sequence":"additional","affiliation":[{"name":"Division of Pathology, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7469-4752","authenticated-orcid":false,"given":"Kuldeep","family":"Dhama","sequence":"additional","affiliation":[{"name":"Division of Pathology, Indian Veterinary Research Institute, Bareilly 243122, UP, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0955-3010","authenticated-orcid":false,"given":"Vikram N.","family":"Vakharia","sequence":"additional","affiliation":[{"name":"Institute of Marine and Environmental Technology, University of Maryland Baltimore County, Baltimore, MD 21202, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1007\/BF01310522","article-title":"Morphological characterization of chicken anaemia agent (CAA)","volume":"109","author":"Gelderblom","year":"1989","journal-title":"Arch. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3131","DOI":"10.1128\/jvi.65.6.3131-3139.1991","article-title":"Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle","volume":"6","author":"Noteborn","year":"1991","journal-title":"J. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1080\/03079459508419046","article-title":"Chicken anaemia virus infection: Molecular basis of pathogenicity","volume":"24","author":"Noteborn","year":"1995","journal-title":"Avian Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3073","DOI":"10.1099\/0022-1317-79-12-3073","article-title":"Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope","volume":"79","author":"Noteborn","year":"1998","journal-title":"J. Gen.Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"13814","DOI":"10.1128\/JVI.02552-12","article-title":"Complete genome sequence of Newcastle disease virus mesogenic vaccine strain R2B from India","volume":"86","author":"Chellappa","year":"2012","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1007\/s11262-017-1433-3","article-title":"Rescue of a recombinant Newcastle disease virus strain R2B expressing green fluorescent protein","volume":"3","author":"Chellappa","year":"2017","journal-title":"Virus Genes"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Dey, S., Chellappa, M.M., Gaikwad, S., Kataria, J.M., and Vakharia, V.N. (2014). Genotype characterization of commonly used Newcastle disease virus vaccine strains of India. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0098869"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.micpath.2018.03.038","article-title":"Generation and evaluation of a recombinant Newcastle disease virus strain R2B with an altered fusion protein cleavage site as a vaccine candidate","volume":"118","author":"Yadav","year":"2018","journal-title":"Microb. Pathog."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Saikia, D.P., Yadav, K., Pathak, D.C., Ramamurthy, N., D\u2019Silva, A.L., Marriappan, A.K., Ramakrishnan, S., Vakharia, V.N., Chellappa, M.M., and Dey, S. (2019). Recombinant Newcastle disease virus (NDV) expressing sigma C protein of avian reovirus (ARV) protects against both ARV and NDV in chickens. Pathogens, 8.","DOI":"10.3390\/pathogens8030145"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"108890","DOI":"10.1016\/j.vetmic.2020.108890","article-title":"Newcastle disease virus vectored rabies vaccine induces strong humoral and cell mediated immune responses in mice","volume":"251","author":"Debnath","year":"2020","journal-title":"Vet. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1080\/030794500750047126","article-title":"Circoviruses: Immunosuppressive threats to avian species: A review","volume":"29","author":"Todd","year":"2000","journal-title":"Avian Pathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1916","DOI":"10.1016\/j.vaccine.2015.02.066","article-title":"Assessing the efficacy of an inactivated chicken anemia virus vaccine","volume":"33","author":"Zhang","year":"2015","journal-title":"Vaccine"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/j.cimid.2010.11.006","article-title":"Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV","volume":"34","author":"Moeini","year":"2011","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1186\/1743-422X-8-119","article-title":"Improving the potency of DNA vaccine against Chicken Anemia Virus (CAV) by fusing VP1 protein of CAV to Marek\u2019s Disease Virus (MDV)Type-1 VP22 protein","volume":"8","author":"Moeini","year":"2011","journal-title":"Virol. J."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.vaccine.2014.11.020","article-title":"Development of a DNA vaccine for chicken infectious anemia and its immunogenicity studies using high mobility group box 1 protein as a novel immunoadjuvant indicated induction of promising protective immune responses","volume":"33","author":"Sawant","year":"2015","journal-title":"Vaccine"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1016\/j.vetimm.2015.08.002","article-title":"Development of a subunit vaccine containing recombinant chicken anemia virus VP1 and pigeon IFN-\u03b3","volume":"167","author":"Shen","year":"2015","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6707868","DOI":"10.1155\/2017\/6707868","article-title":"Genetic analysis of two chicken infectious anemia virus variants-related Gyrovirus in stray mice and dogs: The First Report in China, 2015","volume":"2017","author":"Fang","year":"2017","journal-title":"Biomed. Res. Int."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"763","DOI":"10.1016\/0264-410X(94)00034-K","article-title":"Immunogenic and protective properties of chicken anaemia virus proteins expressed by baculovirus","volume":"13","author":"Koch","year":"1995","journal-title":"Vaccine"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Tseng, T.Y., Liu, Y.C., Hsu, Y.C., Chang, P.C., Hsieh, M.K., Shien, J.H., and Ou, S.C. (2019). Preparation of chicken anemiavirus (CAV) virus-Like particles and chicken interleukin-12 for vaccine development using a baculovirus expression system. Pathogens, 8.","DOI":"10.3390\/pathogens8040262"},{"key":"ref_20","first-page":"283","article-title":"Simultaneous expression of chicken anaemia virus proteins VP1 and VP2 in silkworms","volume":"17","author":"Xiao","year":"2001","journal-title":"Sheng Wu Gong Cheng Xue Bao"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.micpath.2003.07.003","article-title":"Role of fusion protein cleavage site in the virulence of Newcastle disease virus","volume":"36","author":"Panda","year":"2004","journal-title":"Microb. Pathog."},{"key":"ref_22","first-page":"493","article-title":"A simple method of estimation of fifty per cent end-points","volume":"27","author":"Reed","year":"1938","journal-title":"Am. J. Hyg."},{"key":"ref_23","unstructured":"Saif, Y.M. (2008). Newcastle disease other avian paramyxovirus and pneumovirus infection. Diseases of Poultry, Blackwell Publishing. [12th ed.]."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Dey, S., Chellappa, M.M., Pathak, D.C., Gaikwad, S., Yadav, K., Ramakrishnan, S., and Vakharia, V.N. (2017). Newcastle disease virus vectored bivalent vaccine against virulent infectious bursal disease and Newcastle disease of chickens. Vaccines, 5.","DOI":"10.3390\/vaccines5040031"},{"key":"ref_25","unstructured":"Campbell, T.W. (1995). Avian Haematology and Cytology, Iowa State University Press. [2nd ed.]."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1016\/j.micpath.2016.05.003","article-title":"Impact of virus load on immunocytological and histopathological parameters during clinical chicken anemia virus (CAV) infection in poultry","volume":"96","author":"Wani","year":"2016","journal-title":"Microb. Pathog."},{"key":"ref_27","unstructured":"Bancroft, J.D., and Cook, H.C. (1999). Manual of Histological Techniques and Their Diagnostic Applications, Churchill Livingstone."},{"key":"ref_28","first-page":"9296520","article-title":"Scoring system for lesions induced by different strains of Newcastle disease virus in chicken","volume":"9","author":"Hussein","year":"2018","journal-title":"Vet. Med. Int."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1099\/0022-1317-71-4-819","article-title":"Purification and biochemical characterization of chicken anaemia agent","volume":"71","author":"Todd","year":"1990","journal-title":"J. Gen. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1219","DOI":"10.1099\/vir.0.000067","article-title":"Development of a novel thermostable Newcastle disease virus vaccine vector for expression of a heterologous gene","volume":"96","author":"Wen","year":"2015","journal-title":"J. Gen. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1111\/cas.13468","article-title":"Newcastle disease virus co-expressing interleukin 7 and interleukin 15 modified tumor cells as a vaccine for cancer immunotherapy","volume":"109","author":"Xu","year":"2018","journal-title":"Cancer Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"8197","DOI":"10.1073\/pnas.0602461103","article-title":"Newcastle disease virus expressing H5 hemagglutinin gene protects chickens against Newcastle disease and avian influenza","volume":"103","author":"Veits","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1016\/j.vaccine.2016.12.038","article-title":"Newcastle disease virus vectored infectious laryngotracheitis vaccines protect commercial broiler chickens in the presence of maternally derived antibodies","volume":"35","author":"Yu","year":"2017","journal-title":"Vaccine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"11951","DOI":"10.1038\/s41598-018-30356-2","article-title":"A recombinant Newcastle Disease Virus (NDV) expressing S protein of infectious bronchitis virus (IBV) protects chickens against IBV and NDV","volume":"8","author":"Shirvani","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1590\/S1516-635X2003000100001","article-title":"Avian cytokines in health and disease","volume":"5","author":"Wigley","year":"2003","journal-title":"Braz. J. Poultry Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1959","DOI":"10.1007\/s00705-020-04669-5","article-title":"Immune effect of a Newcastle disease virus DNA vaccine with IL-12 as a molecular adjuvant delivered by electroporation","volume":"165","author":"Xie","year":"2020","journal-title":"Arch. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/j.vetmic.2011.02.047","article-title":"Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein","volume":"151","author":"Su","year":"2011","journal-title":"Vet. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1093\/abbs\/gmz158","article-title":"IFN-\u03b3 establishes interferon-stimulated gene-mediated antiviral state against Newcastle disease virus in chicken fibroblasts","volume":"52","author":"Yang","year":"2020","journal-title":"Acta Biochim. Biophys. Sin."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1007\/s00580-018-2883-2","article-title":"Chicken infectious anemia: Emerging viral disease of poultry-an overview","volume":"28","author":"Orakpoghenor","year":"2019","journal-title":"Comp. Clin. Path."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1080\/03079459108418756","article-title":"Chicken anemia agent: A review","volume":"20","author":"McNulty","year":"1991","journal-title":"Avian Pathol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/S0145-305X(99)00076-2","article-title":"Immunopathogenesis of chicken anemia virus infection","volume":"24","author":"Adair","year":"2000","journal-title":"Dev. Comp. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1007\/s13337-018-0445-5","article-title":"Pathological and molecular investigation of velogenic viscerotropic Newcastle disease outbreak in a vaccinated chicken flocks","volume":"29","author":"Mariappan","year":"2018","journal-title":"Virus Dis."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1016\/j.jcpa.2012.09.011","article-title":"Histopathological alterations in immune organs of chickens and ducks after experimental infection with virulent 9a5b Newcastle disease virus","volume":"149","author":"Anis","year":"2013","journal-title":"J. Comp. Pathol."},{"key":"ref_44","unstructured":"Karel, A.S., Bernd, K., and Pete, K. (2014). Avian Immunology, Elsevier Academic Press. [2nd ed.]. Chapter 16-Avian Immunosuppressive Diseases and Immunoevasion."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/10\/1985\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:08:59Z","timestamp":1760166539000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/10\/1985"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,2]]},"references-count":44,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["v13101985"],"URL":"https:\/\/doi.org\/10.3390\/v13101985","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,2]]}}}