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United States Department of Agriculture

Agricultural Research Service

Title: Electron Microscopy, Replication Characteristics in Cell Culture and Comparative Geonomic Sequence Analysis of the U.S. Avian Pneumovirus Isolate with European Subtypes

Authors
item Jacobs,, Janet - UNIV OF GA-ATHENS/USDA
item Seal, Bruce

Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 19, 2002
Publication Date: April 1, 2003
Citation: JACOBS,, J.A., SEAL, B.S. ELECTRON MICROSCOPY, REPLICATION CHARACTERISTICS IN CELL CULTURE AND COMPARATIVE GEONOMIC SEQUENCE ANALYSIS OF THE U.S. AVIAN PNEUMOVIRUS ISOLATE WITH EUROPEAN SUBTYPES. VETERINARY MICROBIOLOGY. 2003.

Interpretive Summary: Up until 1997 avian pneumoviruses, now known as avian metapneumovirus, were considered exotic to North America. However, the virus has become endemic to commercial turkeys in the north-central United States since that time. The virus causes a respiratory disease in turkeys that can be complicated by secondary infections with bacteria. Replication of the virus was examined by infecting cells in culture and the viral replication time was the same as that observed in turkeys that have become infected. The U.S. virus was examined utilizing the electron microscope and found to be structurally similar to avian metapneumoviruses found in other parts of the world. Visualizing the virus proteins in gels by electrophoresis further confirmed the structural similarity to other pneumovirus types. Gene sequences from the United States virus were compared to European viruses that have been categorized as types A and B. The gene comparisons correlate ewith antibody reactivities and further confirm that the United States viru should be categorized as type C avian metapneumovirus.

Technical Abstract: Basic replication characteristics of the U.S. isolate of avian metapneumovirus (AMPV/C) were investigated by electron microscopy of purified virus, generation of a replication curve in cell culture and northern blot hybridization of RNA from infected cells. Virus was detected in cell culture media by 24 hours post-infection (pi), replicating to a maximum titer approaching 107 by six days pi. Utilizing nucleoprotein (N) and matrix (M) protein gene probes transcription was detected by 24 hours pi and continued to five days pi. Virus purified by centrifugation through sucrose gradients was examined by electron microscopy and SDS-polyacrylamide gel electrophoresis. Pleomorphic particles with projections extending from the envelope typical of pneumovirus surface glycoproteins were observed. All potential metapneumovirus proteins were detected by Coomassie staining, while the fusion (F) and a possible attachment (G) protein were putatively identified by glycoprotein staining of duplicate gels. The second matrix (M2) protein, phosphoprotein (P) and N genes were sequenced for a European APV subtype B virus. Contiguous nucleotide coding sequences for the N, P, M, F and M2 genes were aligned from all three APV subtypes. Phylogenetic analysis further confirmed that the U.S. isolate is a separate C subtype compared to European AMPV strains.

Last Modified: 10/22/2014
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