This study was carried out to describe clinical, gross and histopathological findings in the respiratory tract in chickens infected intranasally with A96 strain of infectious laryngotracheitis virus (ILTV). In addition, the presence of ILTV antigens in formalin-fixed and paraffin-embedded larynx and trachea tissues was investigated with the immunoperoxidase (IP) method in the infected chickens. At various days of viral infection, nares, larynx, trachea, lungs and air sacs tissue samples of the infected chickens were obtained and fixed with formalin and embedded in paraffin. The cross sections were stained with hematoxylineosin, and the larynx and trachea sections were also stained with the IP method. Mild rales and gasping were observed in only 4 of 35 chickens. The virus caused mild inflammatory changes in the respiratory tract. It was shown that clinical, gross and histopathological findings were not specific for differential diagnosis of the disease. However, ILTV antigens were detected by the IP method in formalin-fixed and paraffin-embedded larynx and trachea sections. These results revealed that the study use of the IP method might be useful for the diagnosis of ILTV infections with non-specific lesions.
Animals ; Chickens ; Hemorrhage/pathology ; Herpesviridae Infections/*pathology ; Herpesvirus 1, Gallid/isolation & purification/*pathogenicity ; Immunohistochemistry ; Larynx/blood supply/pathology ; Mucous Membrane/pathology ; Virulence

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect (CPE) was examined daily, and the MEQ expression in MDV-infected microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR (qPCR) and qRT-PCR, respectively. The transcripts of Toll-like receptor (TLR) mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the virus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro (P < or = 0.05/0.001). The transcriptions of TLR15 and TLR1LB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection (dpi), and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens (P < or = 0.01/0.001), while CVI988/Rispens induced more TLR1LB transcript than YL040920 (P < or = 0.001).
Animals ; Brain ; metabolism ; virology ; Chickens ; Gene Expression ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; genetics ; metabolism ; virology ; Microglia ; metabolism ; virology ; Poultry Diseases ; genetics ; metabolism ; virology ; Toll-Like Receptor 1 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Transcription, Genetic

The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter (LP2EP2) while the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ were characterized by Western blot (F and gB proteins) and indirect immunofluorescence test (F, HN and gB proteins). The results demonstrated that all four foreign proteins, which were encoded within a 10 kb gene fragment, could be expressed authentically and efficiently. Compared to the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in chicken embryo fibroblasts (CEF) cell culture. Overall, our work suggests that FPV can be a useful live virus vector for the expression of multi- foreign genes against multiple avian pathogens.
Animals ; Cloning, Molecular ; Fibroblasts ; virology ; Fowlpox virus ; genetics ; Gene Expression ; Genetic Engineering ; methods ; HN Protein ; genetics ; Herpesvirus 1, Gallid ; genetics ; physiology ; Newcastle disease virus ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; Viral Envelope Proteins ; genetics ; Viral Fusion Proteins ; genetics