Construction Of Recombinant Marek’s Disease Viruses Expressing The Glycoproteins D Or E Of Infectious Laryngotracheitis Virus

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). This disease leads to serious economic loss due to increased morbidity, retarded growth and reduced egg production in poultry industries worldwide. The control of the disease is based on live-attenuated vaccines. In terms of domestic and overseas research, recent ILT epizootics is related with live-attenuated vaccines, especially to chicken embryo origin (CEO) vaccine strains that persisted in the field and regained virulence. This situation has encouraged the development of a safe and effective new vaccine to prevent and control ILT.In this research, we firstly amplified gD and gE gene from the ILTV Anhui-2011-2DNA, then the transfer vector pLTR-EGFP-gD/gE which contained the homologous sequence to the IRS-US junction region of the CVI988/Rispens genome, long terminal repeat (LTR) promoter of reticuloendotheliosis virus (REV), green fluorescent protein (EGFP) gene and ILTV gD or gE gene was constructed. The recombinant viruses were produced by calcium phosphate-mediated transfection of secondary CEFs with the pLTR-EGFP-gD/gE transfer vector and CVI988/Rispens genomic DNA, and then screened and purified with fluorescently label. The EGFP gene of rMDV-EGFP-gD/gE genomic DNA was excised with Cre recombinase. These new genomic DNAs were used for subsequent transfection experiments, and the final recombinant viruses rMDV-gD and rMDV-gE were acquired after screening and purifying. The glycoproteins D and E (gE) expressed by the recombinant viruses, rMDV-gD and rMDV-gE, respectively, have been demonstrated by Western blot. In this study, we evaluated the protection efficacy of rMDV-gD and rMDV-gE against both ILTV Anhui-2011-2and vvMDV RBIB by vaccinating1-day-old SPF chickens. The protection against ILT was evaluated on morbidity, mortality, clinical symptoms, histopathological alterations, weight gain after challenge and serology, by setting rMDV-gD vaccined challenged group (rMDV-gD), rMDV-gE vaccined challenged group (rMDV-gE), CEO vaccined challenged group (CEO), non-vaccinated challenged group (NV-ch, positive control) and non-vaccinated, non-challenged group (NV-Nch, negative control). The morbidity and mortality of chickens vaccined with rMDV-gD (35%,5%) was significantly lower (p<0.05) than the NV-ch group (100%,36.8%), respectively. The mortality of chickens vaccined with rMDV-gE (19%) had no significantly differences (p>0.05) compared with the NV-ch group. The average body weight gain was recorded between42(1day pre-challenge) and52days of age (10days post challenge, d.p.c). Among the vaccinated groups, those that received the CEO vaccine showed the lowest weight gain (0.113kg), but no differences observed compared with the other groups (p>0.05).-Antibody titers tested by ELISA showed rarely correlation with protection efficacy. The histopathological alterations in the larynx-trachea and lung of the rMDV-gD vaccinated group were slighter than the CEO and rMDV-gE vaccinated groups. Furthermore, the experiments of recombinant viruses against with MD showed the rMDV-gD and rMDV-gE vaccined groups provided full protection as well as CVI988/Rispens vaccined group when challenged with vvMDV RB1B strain. Overall this study demonstrates that the recombinant virus of rMDV-gD is a suitable candidate vaccine strain of ILTV.