We have identified a novel reassortant influenza A (H7N9) virus that is associated with severe human infection. Currently, only 25 H7N9 viruses are available in GenBank. The H7N9 viruses we identified in the three patients were of avian origin, but only the NA gene was closely related to that from another H7N9 virus (KO14). The HA gene was similar to that of an H7N3 virus (ZJ12) from a nearby region (Zhejiang Province) in China. All the internal gene segments were closely related to those from avian H9N2 viruses, particularly a virus isolated from a brambling in Beijing (BJ16) (Figure 1, and Fig. S1 in the Supplementary Appendix). Thus, the human H7N9 viruses are the product of reassortment of viruses that are of avian-origin only. In addition, the phylogenetic trees showed that A/Shanghai/1/2013 is phylogenetically distinct from A/Anhui/1/2013 and A/Shanghai/2/2013 across all gene segments, which suggests that there have been at least two introductions into humans (Figure 1, and Fig. S1 in the Supplementary Appendix). Currently, there are no data to suggest that this reassortment occurred in a mammalian host, and the similarity of the human viruses to avian viruses may be stronger support for direct avian transmission of this virus. However, influenza surveillance of birds, swine, and humans is limited in China and nearby countries, making it difficult to resolve this question.

Although human infections with avian-origin H7 avian influenza viruses have been observed before,1,2,5,10,11 infection of humans with an N9 subtype influenza virus has not been reported previously. Human H7 influenza infections are generally mild, causing conjunctivitis or modest respiratory symptoms, although a fatal case was reported before this H7N9 outbreak.5 All three cases of H7N9 infection reported here were virulent, with the patients' conditions deteriorating rapidly with the development of severe pneumonia and ARDS, and ultimately resulted in death. All the patients had preexisting medical conditions, and two had a history of direct contact with poultry. Two patients presented with rhabdomyolysis, which has rarely been reported in patients infected with H1N1 or H5N112 influenza viruses. Encephalopathy, which is normally more common in pediatric patients with influenza,13 was observed in two patients.

The affinity of the influenza virus to different sialyl-sugar structures is an important determinant of range and pathogenicity in the viral host.14,15 Human influenza viruses preferentially bind to α2,6 sialyl glycan, whereas most avian viruses bind to α2,3 sialyl glycan.16,17 Q226L in the HA protein, which was first reported in H7 field viruses, as well as H5 subtypes, was expected to bind strongly to α-2,6 human-like receptors. A laboratory-produced Q226L mutation at the 210-loop of HA has been shown to change the receptor binding of avian origin to a human-type receptor binding and might increase the ability of the virus to be transmitted by air, as reported previously.18,19 Moreover, the lack of a glycosylation site on the 150-loop might decrease the affinity to α-2,3 avian-like receptors. The effects of these mutations require further study.

A deletion of five amino acids in the viral NA stalk has been observed in the novel reassortant H7N9 viruses. A similar deletion in the H5N1 avian virus has been shown to be responsible for the change in viral tropism to the respiratory tract20 or to enhance viral replication,21 and it has been suggested that this deletion may be associated with adaptation and transmission in domestic poultry.22,23 Since April 4, it has been reported that H7N9 viruses similar to those isolated from the three patients described here have been isolated from pigeons and chickens, indicating that the novel H7N9 viruses might currently be circulating in poultry. Moreover, the E627K substitution in the PB2 gene has been associated with increased virulence in mice and was reported to be associated with improved replication of avian influenza viruses in mammals.24,25 A combination of these substitutions may contribute to the human infection and severe disease. Other possible virulence molecular markers are shown in Table 2. The potential virulence mutations are described on the basis of previous studies in animals, but the pathogenesis in humans remains unknown.

The difference between the two Shanghai viruses and the similarity between the Shanghai/2 and Anhui/1 viruses argue against human-to-human transmission in these cases, and no close contacts of the patients have tested positive for these viruses. However, limited human-to-human transmission was observed in the H7 outbreak in the Netherlands in 200310; therefore, the pandemic potential of these novel avian-origin viruses should not be underestimated.

Currently there is no vaccine available for these novel viruses, and it is not known whether the current candidate H7 vaccine viruses, of which three are North American viruses and the other three are avian viruses from 2000 in the Netherlands, may be effective. The influenza H7N9 A/Anhui/1/2013 strain has been proposed to be one of the candidate vaccine strains since it grows to a very high titer in eggs. Heightened protective measures should be taken when dealing with these viruses, and increased surveillance and analyses of these viruses are needed.

Severe avian influenza A (H7N9) infections, characterized by high fever and severe respiratory symptoms, may pose a serious human health risk. We are concerned by the sudden emergence of these infections and the potential threat to the human population. An understanding of the source and mode of transmission of these infections, further surveillance, and appropriate counter measures are urgently required.