Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses

Abstract

H9N2 avian influenza viruses circulate in poultry throughout much of Asia, the Middle East and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right as well as in the generation of novel zoonotic viruses, for example, H7N9. In recent years it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titres in various systems and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as rapidly expanding geographical range seen in these viruses. Importance Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here we take representative viruses and investigate the genetic basis for this ‘fitness'. A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes in viral replication as well as contribute in the virus accessory functions – however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.

PA-X is an avian virulence factor in H9N2 avian influenza virus

“…UDL-01 WT virus reduced cellular protein synthesis by over 50 % compared to uninfected cells while the FS mutant only caused <20 % host shutoff (). Furthermore, we have recently shown that UDL-01 WT virus is able to cause host cell shutoff in avian cells [37]. These data corroborate the plasmid-based methods previously used and showed that in the context of infectious virus, UDL-01 expresses a classically active PA-X protein.…”

Natural Selection of H5N1 Avian Influenza A Viruses with Increased PA-X and NS1 Shutoff Activity

“…We show that, among the eight amino acid differences between H5N1 PA-X OR and PA-X CIR , two of them, T118I and V127I, are responsible for the increased ability of H5N1 PA-X to inhibit host gene expression, whereas amino acid changes T20A and A85T have little or no effect on PA-X´s host shutoff activity ( Figure 3 and Figure 4 ). Notably, substitution I118T is located within the endonuclease active site in the PA-X N-terminal domain [ 59 ]. Similar to our results, using an H9N2 strain, it has been shown that amino acid change T118I increases the ability of PA-X to inhibit host gene expression, whereas the amino acid change T20A has no effect on PA-X-mediated inhibition of host gene expression [ 59 ].…”

“…Notably, substitution I118T is located within the endonuclease active site in the PA-X N-terminal domain [ 59 ]. Similar to our results, using an H9N2 strain, it has been shown that amino acid change T118I increases the ability of PA-X to inhibit host gene expression, whereas the amino acid change T20A has no effect on PA-X-mediated inhibition of host gene expression [ 59 ]. Interestingly, four of these amino acid changes (T20A, A85T, T118I, and I127V), especially two of them that mostly affect PA-X´s activity (T118I, I127V), also affect the protein sequence of PA; therefore, it is difficult to know whether these identified amino acid changes were selected due to a beneficial effect on PA-X, PA, or both.…”

Co-infection of chickens with H9N2 and H7N9 avian influenza viruses leads to emergence of reassortant H9N9 virus with increased fitness for poultry and enhanced zoonotic potential

“…For two influenza viruses to reassort most efficiently in a host, the viruses must establish a state of co-infection (Lowen, 2018), for which, productive infection of hosts is required (Fonville et al, 2015). In view of previous studies of experimental chicken infections where a high dose was indicated for H7N9 Anhui/13 (Slomka et al, 2018;Spackman et al, 2015), and a low dose was sufficient for H9N2 UDL/08 (Clements et al, 2020) to cause productive infections, we inoculated chickens with a high virus dose mix of Anhui/13 and UDL/08 which included a predominance of the former. The observed virus shedding from directly infected chickens indicated that a productive infection was established.…”