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Title:   Author  item
                                            Yu, Qingzhongitem
                                            Estevez, Carlositem
                                            King, Danielitem
                                            Kapczynski, Darrellitem
                                            Zsak, Laszlo
  Submitted to: Symposium on Avian Pneumovirus
                                    Publication Type: Proceedings
                                    Publication Acceptance Date: 9/7/2007
                                    Publication  Date: 9/27/2007
                                    Citation: Yu, Q., Estevez, C., King, D.J., Kapczynski, D.R., Zsak, L. 2007. Rescue of recombinant avian metapneumovirus subgroup C viruses from cloned DNAs for vaccine development and pathogenesis studies.  In: Proceedings of 2007 Turkey Research Review and Avian Pneumovirus Symposium, September 26-27, 2007 Willmar, Minnesota. 2007 CDROM.Interpretive Summary: Avian metapneumovirus (aMPV) causes an acute upper respiratory tract disease of turkeys, and is associated with swollen head syndrome in chickens.  The disease is usually accompanied by secondary bacterial infections that can increase morbidity and mortality.  The United States was considered free of aMPV disease until 1996 when outbreaks of aMPV subtype C infection of turkeys occurred in Colorado, and subsequently in Minnesota where the disease has emerged as a major economic problem for turkey industry.  The virus has also spread to other states, such as North Dakota, South Dakota, Iowa, and Wisconsin.  Efforts have been made in the US to develop vaccines for control of the disease.  Some levels of protection in response to immunization with cell culture attenuated viruses were reported, whereas the killed vaccine was not effective.  There is a pressing need to develop highly efficacious and safe next generation aMPV vaccines to control and eradicate the disease.  In this study we developed a genetics system for recovery of infectious aMPV-C from cloned DNAs.  By using this system, we generated aMPV-C and Newcastle disease virus (NDV) chimeras.  Subsequently, these chimeras were tested in naive chickens and turkeys to see if they cause any diseases.  The results showed that the aMPV/NDV chimeras were attenuated and safe to be used as vaccine vectors in bids.  The successful development of the reverse genetic technology for aMPV-C provides a powerful tool for developing safe and efficacious multivalent vaccines to control and eradicate aMPV-C and other pathogen diseases.
                                Technical Abstract: Avian metapneumovirus subgroup C (aMPV-C) causes an upper respiratory disease in turkeys, resulting in significant economic losses for the US turkey industry.  To study the disease pathogenesis and to eventually develop a safe and effective vaccine against aMPV-C disease, we developed a reverse genetics system for rescue of recombinant aMPV-C viruses from cloned DNAs.  A full length cDNA clone of aMPV-C and plasmids expressing the N, P, M2-1, and L proteins of aMPV-C were constructed.  Co-transfection of the full length clone and the viral protein expression plasmids in vaccinia/T7 (MVA-T7) virus-infected HEp-2 cells resulted in rescue of infectious aMPV-C.  By using this system, we generated an aMPV-C and Newcastle disease virus (NDV) chimera, raMPV/NDV F-HN, in which the aMPV-C fusion (F) and attachment (G) protein genes were exchanged with the F and Hemagglutinin-Neuraminidase (HN) genes of the mesogenic NDV Anhinga strain.  In addition, we generated a foreign gene expression recombinant virus, raMPV/GFP/NDV F-HN, by inserting a green fluorescence protein (GFP) gene into the aMPV/NDV F-HN chimera.  The pathogenicity of these aMPV recombinants was assessed by conducting the intracerebral pathogenicity index (ICPI) assay and inoculation of susceptible day-old SPF chickens and 2-week-old SPF turkeys via natural routes of infection.  The ICPI results indicated that the aMPV/NDV F-HN and aMPV/GFP/NDV F-HN chimeras were attenuated when compared with the NDV Anhinga strain, the donor of F and HN protein genes.  Neither day-old SPF chickens nor 2 week-old SPF turkeys produced any clinical signs of the diseases after the natural route inoculation of these chimeras.  The successful development of the reverse genetic technology for aMPV-C provides a powerful tool for developing bivalent or multivalent recombinant vaccines and identifying pathogenic determinants of the virus.


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