Neuraminidase-negative avian influenza viruses of subtype H5 and H7: Variation of virulence in the chicken model

Background A neuraminidase (NA)-negative virus originating from highly pathogenic avian influenza virus of subtype H5 (EscEgg50A; H5NAneg) was recently characterized as replication competent in vitro and highly attenuated in adult chicken (Kalthoff et al., 2013. J Virol. 87(24):13556-68). The suitability of the H5NAneg virus variant as a model for the induction of a very early onset of protection was demonstrated within the adult chicken, mouse and ferret model (Röhrs et al., 2014. Vaccine. 32(22):2631-6). Material and Methods The H5 of the EscEgg50 strain was exchanged by reverse genetics with H7 including a polybasic cleavage site resulting in strain H7NAneg. Both viruses were further evaluated in the chicken model using day-old chicks (H5NAneg) or 6 week-old adult chickens (H7NAneg). Results In contrast to the outcome with older chickens, day-old-chicks exhibited typical disease symptoms of highly pathogenic avian influenza infection after intramuscular as well as oronasal infection with H5NAneg (Fig 1A). The animals succumbed to the disease or had to be euthanized within 4 days (i.m. application) or 12 days (oronasal application). In-contact animals were also infected in both groups. Viral RNA loads from different tissues (e.g. myocard, lung, brain, skeletal muscle) confirmed a systemic infection. However, in one-week-old chickens inoculation with H5NAneg again did not result in clinical signs. Furthermore, adult chickens, intramuscularly inoculated with H7NAneg showed the typical clinical picture (Fig. 1B) of highly pathogenic avian influenza with 100% mortality until 4 days post inoculation. Transmission to sentinel chicken could be demonstrated in 2 out of 5 animals. Again, different organ samples scored positive for viral RNA, demonstrating severe systemic infection. Conclusions We evaluated the virulence of replication-competent NA-negative viruses in chickens of different ages. Interestingly, the virulence of H5NAneg is very much age-dependent. Day-old-chicks without maternally derived antibodies succumb to a systemic infection, while chickens one week of age do not show any clinical signs at all. Therefore, the day-old-chick organism is most likely not able to restrict replication of the virus as older chickens do. In addition to this host-derived component of virulence, our results from the H7NAneg inoculation experiment clearly demonstrated a hemagglutinin subtype-dependent variation in virulence despite using an identical genetic backbone. Inoculation with H7NAneg exhibited a highly pathogenic phenotype even in adult chickens, providing evidence, that the virulence of the H7 subtype might be less affected by the neuraminidase protein than that of the H5 virus. Our work with NA-negative avian influenza viruses provides a unique tool to study the role of the neuraminidase protein in replication cycle and pathogenesis of highly pathogenic avian influenza viruses.