Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to hostspecific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus—comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus—that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian–human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures. Although H5N1 viruses continue to cause outbreaks in poultry and there are cases of human infection in Indonesia, Vietnam, Egypt and elsewhere (http://www.who.int/influenza/human_animal_interface/H5N1_ cumulative_table_archives/en/index.html), they have not acquired the ability to cause human-to-human transmission. Investment in H5N1 vaccines has therefore been questioned. However, because humans lack immunity to influenzaviruses possessing anH5HA, the emergence of a transmissible H5-HA-possessing virus would probably cause a pandemic. To prepare better for such a scenario, it is critical that we understand themolecular changes thatmay renderH5-HA-possessing viruses transmissible in mammals. Such knowledge would allow us to monitor circulating or newly emerging variants for their pandemic potential, focus eradication efforts on viruses that already have acquired subsets of molecular changes critical for transmission in mammals, stockpile antiviral compounds in regionswhere suchviruses circulate, and initiate vaccine generation and large-scale production before a pandemic. Therefore, we studied the molecular features that would render H5-HA-possessing viruses transmissible in mammals. Previous studies suggested that HA has a major role in host-range restriction of influenzaA viruses. TheHAof human isolates preferentially recognizes sialic acid linked to galactose by a2,6-linkages (Siaa2,6Gal), whereas the HA of avian isolates preferentially recognizes sialic acid linked to galactose by a2,3-linkages (Siaa2,3Gal). A small number of avian H5N1 viruses isolated from humans show limited binding tohuman-type receptors, a property conferred by several amino acid changes in HA. None of the H5N1 viruses tested transmitted efficiently in a ferret model, although, while our paper was under review, one study reported that a virus with a mutant H5 HA and a neuraminidase (NA) of a human virus in the H5N1 virus background caused respiratory droplet transmission in one of two contact ferrets. To identify novel mutations in avian H5 HAs that confer humantype receptor-binding preference, we introduced random mutations into the globular head (amino acids 120–259 (H3 numbering), which includes the receptor-binding pocket) of A/Vietnam/1203/2004 (H5N1; VN1203) HA (Supplementary Fig. 1). Although this virus was isolated froma human, itsHA retains avian-type receptor-binding properties. We also replaced the multibasic HA cleavage sequence with a non-virulent-type cleavage sequence, allowing us to perform studies in biosafety level 2 containment (http://www.who.int/ csr/resources/publications/influenza/influenzaRMD2003_5.pdf). The mutated polymerase chain reaction (PCR) products were cloned into RNA polymerase I plasmids containing the VN1203 HA complementary DNA, which resulted in Escherichia coli libraries representing the randomly generated HA variants. Sequence analysis of 48 randomly selected clones indicated an average of 1.0 amino acid changes per HA globular head (data not shown). To generate an H5N1 virus library, plasmids for the synthesis of themutated HA gene and the unmodified NA gene of VN1203 were transfected into human embryonic kidney (293T) cells together with plasmids for the synthesis of the six remaining viral genes of A/Puerto Rico/8/34 (H1N1; PR8), a laboratory-adapted human influenza A virus. Turkey red blood cells (TRBCs; which possess both Siaa2,6Gal and Siaa2,3Gal on their surface (data not shown)) were treated with Salmonella enterica serovar Typhimurium LT2 sialidase, which preferentially removes a2,3-linked sialic acid (that is, avian-type receptors), creating TRBCs that predominantly possess Siaa2,6Gal on the cell surface (Siaa2,6-TRBCs; Supplementary Fig. 2). The virus library was then adsorbed to Siaa2,6-TRBCs at 4 uC and extensively washed to removenonspecifically orweakly boundviruses. Boundviruseswere eluted by incubation at 37 uC for 30min, and then diluted to approximately ,0.5 viruses per well (on the basis of a pilot experiment that