Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets
Masaki Imai1, Tokiko Watanabe1’2, Masato Hatta1, Subash C. Das1, Makoto Ozawa1,3, Kyoko Shinya4, Gongxun Zhong1, Anthony Hanson1, Hiroaki Katsura5, Shinji Watanabe1,2, Chengjun Li1, Eiryo Kawakami2, Shinya Yamada5, Maki Kiso5, Yasuo Suzuki6, Eileen A. Maher1, Gabriele Neumann1 & Yoshihiro Kawaoka1,2,3,5
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 host-specific cellular receptors1-3. 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 H5 HA, 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.mt/mfluenza/human_animd_mterface/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 influenza viruses possessing an H5 HA, 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 the molecular changes that may render H5-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 regions where such viruses 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 influenza A viruses1-3. The HA of human isolates preferentially recognizes sialic acid linked to galactose by α2,6-linkages (Siaα2,6Gal), whereas the HA of avian isolates preferentially recognizes sialic acid linked to galactose by α2,3–linkages (Siaα2,3Gal)3. A small number of avian H5N1 viruses isolated from humans show limited binding to human-type receptors, a property conferred by several amino acid changes in HA4-9. None of the H5N1 viruses tested transmitted efficiently in a ferret model10-13, although, while our paper was under review, one study14 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 human-type 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 from a human, its HA retains avian-type receptor-binding properties6,15. 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 plasmids16 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 the mutated 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 Siaα2,6Gal and Siaα2,3Gal on their surface (data not shown)) were treated with Salmonella enterica serovar Typhimurium LT2 sialidase, which preferentially removes α2,3–linked sialic acid (that is, avian-type receptors), creating TRBCs that predominantly possess Siaα2,6Gal on the cell surface (Siaα2,6-TRBCs; Supplementary Fig. 2). The virus library was then adsorbed to Siaα2,6-TRBCs at 4°C and extensively washed to remove nonspecifically or weakly bound viruses. Bound viruses were eluted by incubation at 37°C for 30 min, and then diluted to approximately ~0.5 viruses per well (on the basis of a pilot experiment that
1Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA. 2ERATO infection-induced Host Responses Project, Saitama 332-0012, Japan. 3Department of Special Pathogens, international Research Centerfor infectious Diseases, institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 4Department of Microbiology and infectious Diseases, Kobe University, Hyogo 650-0017, Japan. 5Division of Virology, Department of Microbiology and immunology, institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 6Health Science Hills, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan.