New approach to delist highly pathogenic avian influenza viruses from BSL3+ Select Agents to BSL2 non-select status for diagnostics and vaccines.
Highly pathogenic avian influenza viruses (AIVs) are Select Agents in the United States and are required to be handled in bio-containment level-3 enhanced (BSL3+) facilities. Using a reverse genetics system, we attenuated a highly pathogenic virus, with the goal of making it low pathogenic and having it delisted as a Select Agent so that it could be handled in a bio-containment level-2 facility for diagnostic or vaccine production applications. We utilized two approaches to attenuate the target AIV by mutating the highly pathogenic hemagglutinin (HA) cleavage site to be low pathogenic and by replacing the full-length NS gene segment with a naturally truncated 124-amino acid NS1 coding gene from A/turkey/Oregon/73 (H7N3) virus (tkOR71 trNS1). To delist an AIV so that it can be handled in a BSL2 facility, the amino acid sequence of the HA cleavage site of the rescued virus must be confirmed to be compatible with a low-pathogenic AIV; it should not plaque in cell culture without supplementation of exogenous trypsin; and intravenous pathotyping in 4-6-wk-old specific-pathogen-free chickens must confirm that the virus is low pathogenic. The candidate A/duck/Vietnam/Baclieu/09/07 (rH5N1/PR8/trNS1) virus with five PR8 internal genes, tkOR71 trNS1 gene, and A/chicken/Indonesia/7/03 N1 neuraminidase gene was constructed. The virus was shown to not plaque in cell culture without addition of trypsin. The virus was low pathogenic in the standard intravenous pathotyping test (IVPI = 0) and also caused no disease in a separate intranasal inoculation test in 4-wk-old specific-pathogen-free chickens, thus demonstrating that the virus is suitable for deselection.
Jadhao
Samadhan J
SJ
Southeast Poultry Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 934 College Station Road, Athens, GA 30605, USA.
Suarez
David L
DL
D000818:Animals; D002460:Cell Line; D002645:Chickens; D004285:Dogs; D053124:Influenza A Virus, H5N1 Subtype; D009980:Influenza A virus / Q000145:classification* / Q000472:pathogenicity*; D007252:Influenza Vaccines / Q000276:immunology*; D005585:Influenza in Birds / Q000517:prevention & control* / Q000821:virology*; D016297:Mutagenesis, Site-Directed; D016865:Reassortant Viruses; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology; D014774:Virulence; D014779:Virus Replication
C085820:INS1 protein, influenza virus; D007252:Influenza Vaccines; D017361:Viral Nonstructural Proteins