title: Role of trypsin in the replication of Avian metapneumovirus subtype C (strain MN-2a) and its entry into the Vero cells.
abstract: To understand the molecular mechanisms of Avian metapneumovirus (aMPV) and the requirements involved in the infection and fusion, trypsin treatment was done in the different stages of virus; before infection, during entry and after virus infection followed by aMPV infection. The growth kinetics of aMPV was compared in time dependent manner. The effect of trypsin was found in the later stage of aMPV infection increasing the numbers of infected cells with the significant higher titer of infectious virions to that of trypsin treated before infection, during entry and aMPV. A serine protease inhibitor reduced aMPV replication in a significant way, whereas cysteine peptidase (E-64), aspartic protease (pepstatin A), and metalloprotease (phosphoramidon) inhibitors had no effect on aMPV replication. Inoculation of aMPV on Vero cells expressing the membrane-associated protease TMPRSS2 resulted in higher virus titers than that inoculated on normal Vero cells and is statistically significant (p < 0.05). Also, an inhibitor of clathrin/caveolae-mediated endocytosis had no effect on virus progeny, indicating that aMPV does not use the endocytic pathway for entry but undergoes direct fusion. The effect of lysosomotropic agents was not significant, suggesting that aMPV does not require low-pH environment in endosomes to fuse its envelope with the plasma membrane.
authors.affiliation: Laboratory of Infectious Diseases, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea.
authors.forename: Sarita
authors.initials: S
authors.lastname: Paudel
authors.affiliation: Laboratory of Infectious Diseases, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea; Research Institute of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea. Electronic address: shin0089@cnu.ac.kr.
authors.forename: Hyun-Jin
authors.initials: HJ
authors.lastname: Shin
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D008561:Membrane Fusion / Q000187:drug effects; D029121:Metapneumovirus / Q000145:classification / Q000187:drug effects / Q000502:physiology*; D018184:Paramyxoviridae Infections / Q000662:veterinary / Q000821:virology; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology*; D014709:Vero Cells; D019562:Viral Load / Q000187:drug effects; D053586:Virus Internalization / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D012697:Serine Endopeptidases; C421305:TMPRSS2 protein, human; D014357:Trypsin
keywords: Avian metapneumovirus; Direct fusion; Endocytosis; Lysosomotropic agents; Replication; Trypsin