Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses

Abstract

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA 225G and NA 315N substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA 225G and NA 315N as potential virulence markers in mammals.

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

“…Although swine influenza viruses only occasionally infect humans and rarely cause pandemics (39), they appear to pose an increasing risk. TRS viruses with human-like HA and NA proteins are airborne-transmissible in ferrets (40,41). Moreover, diverse swine viruses to which humans lack immunity are emerging through reassortment with the pH1N1 viruses (42).…”

The Homologous Tripartite Viral RNA Polymerase of A/Swine/Korea/CT1204/2009(H1N2) Influenza Virus Synergistically Drives Efficient Replication and Promotes Respiratory Droplet Transmission in Ferrets

“…Substitution of the isogenic CT-Sw/1204 HA alone or in combination with NA into the closely related Sw/1130 H1N2 virus enhanced replication and pathogenicity in mice, confirming the role of the HA 225G and NA 315N mutations found in CT-Sw/1204 after a single passage in ferrets (10). However, the reciprocal expression of Sw/1130 HA or HA-NA in the background of CT-Sw/1204 could not render the recombinant reassortant virus completely attenuated, implying that the remaining viral segments are required for pathogenicity.…”

“…The 50% lethal doses (LD 50 ) of viruses that killed more than 50% of the mice were determined in additional groups of mice that were inoculated intranasally with 10-fold serial dilutions (10 2 to 10 6 TCID 50 /50 l) of virus. Groups of three 14-to 16-week-old female outbred ferrets (Mustela putorius furo; Wuxi Sangosho Pet Park Co., China) that were seronegative for influenza virus exposure/infection by hemagglutination inhibition (HI) and NP enzyme-linked immunosorbent assays (ELISAs) were inoculated intranasally (300 l/nostril) and intratracheally (400 l) with the indicated recombinant virus (10 5.5 TCID 50 /ml) as previously described (10). At 1 day p.i., each inoculated ferret was paired with a seronegative ferret (in a 1:1 setup) in barrier-separated transmission isolators (35 mm apart) that permitted only aerosol contact.…”

“…However, their transmissibility by respiratory droplets (RD) in ferret models varies considerably depending on the HA and NA lineages (7). Recently, we have shown that the A/swine/Korea/1204/2009(H1N2) Korean swine virus, which is genetically related to North American strains and was similarly isolated before the 2009 pandemic, is virulent in ferrets and is efficiently transmitted to naive RD contacts (10). Viruses recovered from the RD contact ferrets (CT-Sw/1204) had acquired Asp-225-Gly (H3 numbering) and Ser-315-Asn (N2 numbering) mutations in their HA and NA proteins, respectively, contributing to the altered replicative ability, pathogenicity, and transmissibility.…”

Influenza A Virus Acquires Enhanced Pathogenicity and Transmissibility after Serial Passages in Swine

“…Most reported triple-reassortant swine H1 viruses caused mild disease, were inefficiently transmitted through the air in ferrets (34,35), and had restricted circulation in humans (36), even though they generally possess strong binding affinity for the ␣2,6-linked human-like receptor (37). Our previous (7) and present studies demonstrated that the parental rH1N1 virus had viral characteristics identical to those of these reported triple-reas- sortant viruses.…”