Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site

Abstract

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. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. 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 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 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 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.

FIG 1

Generation of recombinant reassortant viruses. Three parent viruses were used to generate the reassortant viruses that were used in the present study. LPAI H7N3 A/chicken/BC/CN006/2004 and HPAI H7N3 A/chicken/BC/CN007/2004 can be considered isogenic with the exception of the HA gene, which in the case of A/chicken/BC/CN007/2004 contains a 21-nucleotide/7-amino-acid insert in its cleavage site that is derived from the matrix gene. The HA cleavage sites are indicated in bold letters, and the basic amino acid residues are highlighted.

FIG 2

Survival of White Leghorn chickens following intranasal inoculation with reassortant viruses. Four- to six-week-old chickens were inoculated intranasally with 0.2 ml of an inoculum containing various PFU of the respective reassortant viruses and mortality was monitored over a period of 20 days. (A) Five chickens each were inoculated with 10,000, 1,000, or 100 PFU of rH5N1. (B) Five chickens each were inoculated with 10,000, 1,000, or 100 PFU of rH7N3. (C) Five chickens each were inoculated with 400,000, 40,000, or 4,000 PFU of rH5N3.

FIG 3

Multicycle replication of reassortant viruses in MDCK and QT-35 cells. MDCK and QT-35 cell monolayers were inoculated with each virus at an MOI of 0.00001. After the inoculum had adsorbed for 1 h, the cell monolayers were washed three times with PBS, and infection medium was added. Culture supernatants were collected at the indicated time points and titrated on MDCK cells. (A) MDCK cells; (B) QT-35 cells. *, P < 0.05 based on one-way ANOVA. The Bonferroni adjusted probabilities were 0.002 for rH5N3 versus rH7N3 at 72 h postinoculation in MDCK cells and 0.006 for rH5N3 versus rH5N1 at 96 h postinoculation in MDCK cells. The Bonferroni adjusted probabilities were 0.004 for rH5N3 versus rH5N1 at 72 h postinoculation in QT-35 cells, 0.005 for rH5N3 versus rH7N3 at 72 h postinoculation in QT-35 cells, and 0.003 for rH5N1 versus rH7N3 at 96 h postinoculation in QT-35 cells.

FIG 4

Survival of White Leghorn chickens following intranasal inoculation with P0 to P6 rH5N3 virus. The percent survival of chickens following intranasal inoculation of each passaged virus was determined. Intravenous pathogenicity indices (IVPI) are indicated for P0, P2, P4, and P6 rH5N3 viruses.

FIG 5

Comparison of histopathology and immunohistochemistry findings in brains of P6 rH5N3 (A and B)- and P0 rH5N3 (C and D)-inoculated birds at 3 dpi. (A) Perivascular cuffing with mononuclear inflammatory cells (arrowhead) was observed throughout the brain. Note area of gliosis and necrosis (arrow). Bar, 50 μm; H&E stain. (B) Extensive positive immunostaining for influenza A virus antigen. Bar, 500 μm. (C) Perivascular cuffing was observed only in the periventricular area of one bird (arrow). Bar, 50 μm; H&E stain. (D) Detection of viral antigen was limited to the ependymal cells lining the ventricles. Bar, 500 μm.

FIG 6

Comparison of the neuraminidase activities of rH5N3 P0 versus rH5N3 P6. (A) Neuraminidase enzyme kinetic curves for rH5N3 P0 and rH5N3 P6 expressed in relative fluorescent units per second as a function of MUNANA concentration. (B) Elution of rH5N3 P0 versus rH5N3 P6 from chicken erythrocytes. rH5N3 P0 and rH5N3 P6 were adjusted to a HA titer of 1:128, and 2-fold serial dilutions were incubated with equal volumes of 0.5% (vol/vol) chicken erythrocyte suspensions in V-bottom microtiter plates at 4°C for 1 h. The microtiter plates were then incubated at 37°C, and the reduction in HA titers was recorded every hour over a period of 6 h.

FIG 7

Comparison of multicycle replication of rH5N3 P0 and rH5N3 P6 in MDCK and QT-35 cells. MDCK and QT-35 cell monolayers were inoculated with each virus at an MOI of 0.0001. After the inoculum had adsorbed for 1 h the cell monolayers were washed three times with PBS, and infection medium was added. Culture supernatants were collected at the indicated time points and titrated on MDCK cells. (A) QT-35 cells; (B) MDCK cells. (C) Representative viral plaques formed by rH5N3 P0 and rH5N3 P6 in QT-35 cells. *, P < 0.05 based on the Student t test.

FIG 8

Survival of White Leghorn chickens and transmission of virus to naive contact birds following intranasal inoculation with P0 rH5N3 versus P6 rH5N3. (A) Ten chickens, each in separate isolation cubicles, were intranasally inoculated with 40,000 PFU of either P0 rH5N3 or P6 rH5N3, and survival was monitored over a period of 20 days. (B and D) Oropharyngeal and cloacal swabs from P0 rH5N3- and P6 rH5N3-inoculated birds (B) or from P0 rH5N3 and P6 rH5N3 contact birds (D) were tested by qRT-PCR to assess virus shedding. (C) One day following inoculation, five naive contact birds were placed in each isolation cubicle and allowed to commingle with the infected birds. The survival of the contact birds was monitored for a period of 19 days.