Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity AT7519 HCl rH5N3 increased its virulence transmission to naive contact birds was inefficient suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE To date the contribution that hemagglutinin-neuraminidase balance can have on the expression of a Rabbit Polyclonal to MAP9. highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment which can result in new hemagglutinin-neuraminidase combinations may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of AT7519 HCl the neuraminidase in complementing a polybasic HA0 cleavage site. Furthermore it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance. INTRODUCTION Avian influenza viruses (AIV) belong to the genus A in the family characterization of viruses. AT7519 HCl Multistep growth kinetics of each virus was determined in MDCK and quail fibrosarcoma QT-35 cells. Cells grown in six-well culture dishes were inoculated with each recombinant reassortant virus at a multiplicity of infection (MOI) of 0.0001 or 0.00001. The virus inoculum was allowed to adsorb for 1 h; the residual inoculum was then removed AT7519 HCl the cell monolayers were washed three times with PBS and 3 ml of fresh infection medium without trypsin was added. Culture supernatants were sampled at selected time points postinoculation for plaque titration. characterization of viruses. The pathogenic phenotypes of the viruses generated were assessed in 4- to 6-week-old White Leghorn chickens ((17). AT7519 HCl The intravenous pathogenicity index (IVPI) is the mean score per bird per observation over the 10-day period. A maximum score of 3.00 means that all birds died by 24 h postinoculation. Viruses with an IVPI of >1.2 are considered highly pathogenic. In an attempt to determine the 50% lethal dose (LD50) groups of five White Leghorn chickens were inoculated intranasally with 0.2 ml of 10-fold serial dilutions of virus. Serial passaging of recombinant reassortant H5N3 virus in chickens. Serial passaging of rH5N3 began with a cloacal swab specimen that was taken at 10 days postinoculation (dpi) from a single bird (Ck.