A viral race for primacy: co-infection of a natural pair of low and highly pathogenic H7N7 avian influenza viruses in chickens and embryonated chicken eggs

Abstract

Highly pathogenic avian influenza virus (HPAIV) infection in poultry caused devastating mortality and economic losses. HPAIV of subtypes H5 and H7 emerge from precursor viruses of low pathogenicity (LP) by spontaneous mutation associated with a shift in the susceptibility of the endoproteolytic cleavage site of the viral hemagglutinin protein from trypsin- to furin-like proteases. A recently described natural pair of LP/HP H7N7 viruses derived from two spatio-temporally linked outbreaks in layer chickens was used to study how a minority of mutated HP virions after de novo generation in a single host might gain primacy. Co-infection experiments in embryonated eggs and in chickens were conducted to investigate amplification, spread and transmissionof HPAIV within a poultry population that experiences concurrent infection by an antigenically identical LP precursor virus. Simultaneous LPAIV co-infection (inoculum dose of 106 egg-infectious dose 50% endpoint (EID50)/0.5 mL) withincreasing titers of HPAIV from 101 to 105.7 EID50/0.5 mL) had a significant impeding impact on HP H7 replication, viral excretion kinetics, clinical signs and histopathological lesions (in vivo) and on embryo mortality (in ovo). LP/HP co-infected chickens required a hundredfold higher virus dose (HPAIV inoculum of 105 EID50) compared to HPAIV mono-infection (HPAIV inoculum of 103 EID50) to develop overt clinical signs, mortality and virus spread to uninfected sentinels. Escape and spread of HP phenotypes after de novo generation in an index host may therefore be highly precarious due to significant competition with co-circulating LP precursor virus.

The Emergence of H7N7 Highly Pathogenic Avian Influenza Virus from Low Pathogenicity Avian Influenza Virus Using an in ovo Embryo Culture Model

“…Influenza A viruses exist as a collection of closely related viral quasispecies, with reports that minority HP viral variants can be maintained in a predominantly LPAIV population [71,74]. A significant impeding impact on H7 HPAIV replication when co-infected with H7 LPAIV has been described in ovo and in vivo [75], suggesting that there may be a threshold at which a particular population is preferentially selected for (or outgrows) the other. However, results from the multi-cycle growth curve demonstrated that neither LPAIVs (H7N7 SB ; H7N7 DB ) nor HPAIV (H7N7 MB ) demonstrated an inherent replicative advantage in vitro when measuring nucleic acid over time (Figure 2).…”

Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites

“…embryos, indicative of enhanced pathogenicity, was observed with H9 viruses harboring a tribasic HACS (BD_11749 and IN_117, Table 2, Figure 3, Additional file 5). Endothelial and CNS infection, a hallmark of true HPAIV infection in the in ovo model [27], however, was not evident for any of the H9 isolates. Tissue tropism varied greatly with H9 viruses expressing different dibasic HACS motifs (EG_536, IN_118, BD_VP01, DU_121 and MO_166) and reflected a highly variable pathogenic potential of these viruses (Table 2; Additional file 5).…”

“…To visualize AIV-matrixprotein, immunohistochemistry (IHC) was performed using the avidin-biotin-peroxidase-complex (ABC) method essentially as described [27]. The distribution of AIV-matrixprotein was semiquantitatively assessed for each organ by scoring on a 0 to 3 scale: 0 = negative; 1 = focal or oligofocal, 2 = multifocal, 3 = coalescing to diffuse immunoreactive cells as previously described [27].…”

“…Based on previous data [27,42,43] embryonated chicken eggs (ECE) were chosen as an easily accessible and informative in vivo system to assess AIV pathogenicity. Expression of matrixprotein (M) in infected ECE was semiquantitatively assessed in the chorioallantoic membrane (CAM) and in various embryonic organs by immunohistochemistry (Additional file 5).…”

“…There is ample evidence that conversion by spontaneous mutation of a monobasic cleavage site into a polybasic one is at the basis of the pathogenicity shift from LP to HP phenotype in subtypes H5 and H7 [23]. Why and when such conversion events occur remains essentially unclear [27].…”