key: cord-296573-4c9gch5b authors: To, Kelvin KW; Chan, Jasper FW; Chen, Honglin; Li, Lanjuan; Yuen, Kwok-Yung title: The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1: a tale of two cities date: 2013-08-19 journal: Lancet Infect Dis DOI: 10.1016/s1473-3099(13)70167-1 sha: doc_id: 296573 cord_uid: 4c9gch5b Infection with either influenza A H5N1 virus in 1997 or avian influenza A H7N9 virus in 2013 caused severe pneumonia that did not respond to typical or atypical antimicrobial treatment, and resulted in high mortality. Both viruses are reassortants with internal genes derived from avian influenza A H9N2 viruses that circulate in Asian poultry. Both viruses have genetic markers of mammalian adaptation in their haemagglutinin and polymerase PB2 subunits, which enhanced binding to human-type receptors and improved replication in mammals, respectively. Hong Kong (affected by H5N1 in 1997) and Shanghai (affected by H7N9 in 2013) are two rapidly flourishing cosmopolitan megacities that were increasing in human population and poultry consumption before the outbreaks. Both cities are located along the avian migratory route at the Pearl River delta and Yangtze River delta. Whether the widespread use of the H5N1 vaccine in east Asia—with suboptimum biosecurity measures in live poultry markets and farms—predisposed to the emergence of H7N9 or other virus subtypes needs further investigation. Why H7N9 seems to be more readily transmitted from poultry to people than H5N1 is still unclear. The infl uenza viruses belong to the RNA virus family Orthomyxoviridae. On the basis of antigenic and genetic diff erences between the internal proteins, nucleoprotein and matrix protein, infl uenza viruses are classifi ed into A, B, and C. The infl uenza A virus has a negative-sense, single-stranded, and eight-segmented genome. On the basis of two surface proteins, 17 haemagglutinin subtypes and ten neuraminidase subtypes can be found (fi gure 2). 19 Except H17 and N10, which have been found in bats only, all haemagglutinin and neuraminidase subtypes have been found in wild waterfowl. 20 Unlike other neuraminidase subtypes, N10 does not have neuraminidase activity. 21 The 17 haemagglutinin subtypes are further classifi ed into group 1 and group 2 on the basis of the antigenic and phylogenetic characteristics of haem agglutinin. The HA1 subunit of the haemagglutinin attaches onto host cell sialic acid receptor. The haemagglutinin precursor protein HA0 is cleaved by proteases into HA1 and HA2 before HA2 can mediate virion-cell fusion. The internal proteins are encoded by six other gene segments encoding nucleoprotein NP, matrix protein M1, ion channel protein M2, the polymerase complex proteins PB1, PB2, PA, including PB1-F2, PA-X, PA-N155, PA-N182, and non-structural proteins including PB1-F2, PA-X, PA-N155, PA-N182, NS1, and NS2. 19, 22 The eight gene segments can reassort when two diff erent viruses infect an animal host cell. Phylogenetic analysis shows that the 1997 H5N1 virus infecting human beings was a reassortant with goose H5N1 haemagglutinin, teal H6N1 neuraminidase, and internal genes from quail H9N2 and teal H6N1. 23, 24 The 2003 H7N7 virus that infected human beings in the Netherlands was a reassortant between avian infl uenza A H7N3, infl uenza A H10N7, and other Eurasian avian infl uenza viruses (fi gure 3). 9 On the basis of available sequences in the public domain, the 2013 H7N9 virus is also a reassortant of avian infl uenza viruses consisting of haemagglutinin and neuraminidase most closely related to H7N3 viruses isolated from ducks in Zhejiang and H7N9 viruses from wild birds in Korea, respectively. The M, NP, and NS genes are most closely related to H9N2 viruses from chickens in eastern China, whereas Personal View the PA, PB1, and PB2 genes are most closely related to those of H9N2 viruses from brambling in Beijing. 11, 12, 13, 25, 26 Because the origin of all gene segments of the 2013 H7N9 virus are avian, gene reassortment probably took place in an avian host. Although reassortment events of avian infl uenza viruses have been recorded in pigs in Korea, 27 the 2013 H7N9 virus and the immediate precursors have not been found in pigs. 28 The haemagglutinin sequence of one of the 2013 H7N9 virus human isolates (A/Shanghai/1/2013) was phylogenetically distinct from other human and avian isolates, 12, 29 suggesting that this new reassortant might have been circulating for some time to achieve this degree of diversity. Because of the roughly 5% nucleotide diff erences in the H7 and N9 genes between the 2013 H7N9 virus and the closest avian virus genes, more extensive virological surveillance of wild or domestic avian and non-avian animal species, as has been done for the Severe acute respiratory syndrome coronavirus, 30 is needed to understand the evolutionary pathway of this H7N9 virus, which is the fi rst N9 subtype virus infecting human beings. Up to now, only one wild pigeon from Nanjing has tested positive for the 2013 H7N9 virus, 31 but the epidemiological and genomic details of this virus isolate are not yet available. One unique feature of the 2013 H7N9 human epidemic is the absence of preceding die-off s in poultry or wild birds, which makes epidemiological control diffi cult. The start of the H5N1 outbreak in 1997 was marked by three highly pathogenic avian infl uenza virus outbreaks on farms 1 month before the fi rst human case and about 32 Avian infl uenza virus can be divided into highly pathogenic avian infl uenza virus and low pathogenic avian infl uenza virus on the basis of pathogenicity in chickens. The World Organization for Animal Health has defi ned highly pathogenic avian infl uenza virus as any infl uenza virus that is lethal for six or more of eight 4-8-week-old susceptible chickens by 10 days after intravenous inoculation with 0·2 mL of a 1/10 dilution of a bacteriafree infective allantoic fl uid, or that has an intravenous pathogenicity index of greater than 1·2, which is calculated by scores on the severity of illness. 33 One key feature of highly pathogenic avian infl uenza virus is the presence of multibasic aminoacids at the haemagglutinin cleavage site, which render haemagglutinin susceptible to cleavage by many diff erent proteases. 34 Hence, H5 or H7 viruses that have low pathogenicity in chickens are still regarded as highly pathogenic avian infl uenza virus if their HA0 cleavage sequence is similar to that of other highly pathogenic avian infl uenza viruses. The haemagglutinin cleavage site from all reported 2013 H7N9 viruses do not possess multibasic aminoacids. 11, 12, 35 Data from preliminary pathogenicity testing suggest that chickens and quails infected with H7N9 do not show signs of illness. 36 A low pathogenic avian infl uenza virus can mutate to become highly pathogenic during an outbreak in domestic poultry. For the H7N3 virus outbreaks in 2002 and 2004, the diff erence in the cleavage site between low pathogenic avian infl uenza virus and highly pathogenic avian infl uenza virus was speculated to be a sequence insertion from the neuraminidase or M gene at the haemagglutinin cleavage site through intersegmental recombination. 37 Close monitoring of the evolution of the 2013 H7N9 is needed. Another possible explanation for the apparent absence of a preceding infection with H7N9 in poultry in the 2013 outbreak is that previous infection by a closely related low pathogenic avian infl uenza H7 virus elicited crossprotection for this H7N9 virus because H7N3 virus was recently reported in ducks in Zhejiang, China. 38 Furthermore, prevalent H9N2 infections in poultry or the extensive use of the H5N1 vaccine in poultry in China might induce cell-mediated immunity against highly conserved internal viral proteins without inducing any neutralising antibody. This process might be suffi cient to militate against poultry death, but not asymptomatic viral shedding. 39 The eff ect of previous infection by other avian infl uenza virus subtypes and previous H5N1 vaccine on the susceptibility to infection by H7N9 deserves further investigation. Interspecies jumping of an avian infl uenza virus into human beings is favoured by several factors: viral mutations that allow the virus to bind, replicate, and spread in human beings; a high inoculum of virus during the transmission between the poultry and human being; host susceptibility such as the presence of underlying diseases; extremes of age; and genetic predispositions. One key viral factor governing host adaptation and transmission is the relative binding affi nity between haemagglutinin and the sialic acid receptor on the host cell surface. 40 Avian infl uenza viruses generally prefer α-2,3 sialic acid receptors abundantly found in avian alimentary tract and human infl uenza viruses generally prefer α-2,6 sialic acid receptors abundantly found in the human respiratory tract. H5N1 viruses can invade the lower respiratory tract of human beings because the viruses retain strong affi nity for the α-2,3-linked receptors present in the lower respiratory tract of human beings. 41 An increased affi nity for the α-2,6-linked receptors, which are abundant in the upper respiratory tract of human beings, is believed to be responsible for transmission between birds and human beings. In Egypt, which has had the highest number of H5N1 infections since 2009, 99% of virus isolates had a Thr160Ala (H3 numbering) mutation, which is associated with loss of glycosylation at position 158-160, and increased transmissibility in ferrets and guinea pigs. 42, 43 Thr160Ala was also present in all reported strains of 2013 H7N9 (table 2). The haemagglutinin mutations Gly186Val and Gln226Leu, which increase the binding of H5N1 and H7 viruses to α-2,6-linked receptor and H5N1 virus transmission between ferrets, are present in most strains of the human and avian 2013 H7N9, but none of the 1997 H5N1 strains (table 2). 11,12,35,45-47 However, a recent structural modelling and binding study with human tracheal epithelium and alveolar tissue sections suggested that 2013 H7N9 has weak binding to both α-2,3-linked and α-2,6-linked receptors. 48 Further studies should confi rm whether various strains of H7N9 diff er in binding affi nities. Neuraminidase enables release of virus particles from the host cell surface. Deletion in the stalk region of neuraminidase, which was found in the human H5N1 viruses and previous H7 viruses, is associated with increased virulence and replication in the respiratory tracts of chickens. 49 2013 H7N9 also has a fi ve aminoacid deletion in the stalk region, which might improve its adaptation to poultry. 11, 12, 29, 35 Comparative studies in wild waterfowl and domestic chickens will ascertain the degree of adaptation by this virus in these avian species. Mutations in the polymerase complex aff ect viral replication. PB2 Glu627Lys mutation is associated with increased viral replication at 33°C, the nasal body temperature in human beings, which is markedly lower than that of avian species. 50 Furthermore, this mutation is important in aerosol transmission of avian H1N1 virus between ferrets. 51 Glu627Lys was found only in human strains, but not the avian strains, of 1997 H5N1 or 2013 H7N9 (table 2) . However, Glu627Lys was specifi cally found in the Qinghai outbreak of H5N1 in 2005. 52 PB2 Asp701Asn is another important mutation associated with mammalian adaptation and transmission. 42, 53 Notably, most virus isolates from individuals infected with 2013 H7N9 had either Glu627Lys or Asp701Asn, but not both. Asp701Asn can compensate for the lack of Glu627Lys for transmission between guinea pigs. 53 Other mutations aff ect viral replication, transmission, and virulence of infl uenza viruses. NS1 protein is the key virulence factor counteracting innate host immunity. NS1 protein contains a four-aminoacid motif at the C terminal that disrupts cellular signalling by binding to the host PDZ domain-containing proteins. Recombinant infl uenza virus with PDZ-binding motif has increased pathogenicity in mice. 54 NS1 protein from human seasonal infl uenza viruses cannot bind to human proteins containing PDZ domain. 55 The NS1 of all 2013 H7N9 strains has a deletion of the PDZ motif. The structural M1 protein is needed for viral assembly and budding. Two mutations in M1, Asn30Asp and Thr215Ala, associated with increased virulence in a mice model, are found in all strains of the 2013 H7N9 virus. 56 Before the 2013 outbreak, the H7N9 virus had never been reported to cause human infection, although other H7 viruses had been associated with sporadic infections since 1979 (table 3) . 5, 7, 9, [57] [58] [59] [60] [61] [62] [63] Serological evidence of H7 infection was present in 38% of a rural population in the Jiangsu Province near Shanghai. 64 However, a study including 1544 serum samples from poultry workers in eastern China in 2012 did not identify any seropositive samples by microneutralisation assay, suggesting that subclinical H7N9 infection was unlikely before 2013. 65 Infl uenza A subtypes H7N1, H7N2, H7N3, H7N7, and H7N8 were previously isolated from poultry and wild birds from China. 38, 66 Although studies of seroprevalence did not fi nd any evidence of H7 infection among sparrows or pigs in China, 67,68 H7N9 viruses were isolated from birds in nearby countries, including Mongolia and South Korea in 2008. 69, 70 Moreover, in the USA, H7N9 viruses have caused outbreaks in chickens. 71 Other H7N9 viruses were reported in wild birds or poultry from four continents. 10 Most of the laboratory-confi rmed cases of 2013 H7N9 were older adults living in the city, with a median age of 61 years. By contrast, in the 1997 H5N1 outbreak in Hong Kong, 61% of patients were younger than 18 years of age 1,74 and the mean age of patients with H5N1 in China between 2003 and 2008 was 28·1 years (table 4) . 78 The severe disease in older adults and the small number of cases in children in the 2013 H7N9 outbreak can be accounted for by several factors. First, H7N9 infection in children was mainly mild or asymptomatic (appendix); 14,79,80 therefore, infection in this age group might have been underdiagnosed because confi rmatory laboratory tests were usually reserved for severe cases. Two children were found to be infected with H7N9 during an enhanced surveillance that included 6333 children younger than 4 years of age with infl uenza-like illness. 79 Second, some adult patients were poultry workers; generally only adults go to live poultry markets and therefore adults might have had a greater dose or prolonged duration of exposure to H7N9 than did children. Additionally, adults might have an increased chance of exposure to pet birds. Finally, older adults might have impaired development of specifi c immune response to the H7N9 virus because of the immunological phenomenon of original antigenic sin. 81 Previous exposure to other infl uenza viruses might also lead to rapid increase in non-neutralising antibodies against these viruses, which can be associated with more severe H7N9 disease. 83, 84 Notably, most fatal or severe cases of 2013 H7N9 have comorbidities, and up to a quarter of adult patients were smokers. 15 The eff ect of genetic predisposition (eg, the IFITM3 and CD55 polymorphisms) on H7N9 infection warrants further investigation. 85, 86 A higher percentage of elderly patients aged 65 years or older and a higher proportion of males were infected during the 2013 H7N9 than were in the 1997 H5N1 outbreak. One explanation for this fi nding could be that most grandmothers in Shanghai take care of their grandchildren at home while the retired grandfathers go to live poultry markets for grocery shopping every day. 87 Another unique feature is that no poultry workers were infected with H5N1 in the Hong Kong epidemic. Seroepidemiology might ascertain the cause of this diff erence. Human beings probably acquired the H7N9 virus via direct contact with infected poultry or their surroundings, although aerosol transmission between ferrets was reported. 77 Ser variant associated with adaptation to pigs 9/10 All All All Increased binding to α-2,6-linked sialic acid receptor All All 3 of 5 6 of 10 Gly186Val Increased binding to α-2,6-linked sialic acid receptor 9 of 10 All ND ND Increased binding to α-2,6-linked sialic acid receptor 8 of 10 * 5 of 6 ND ND Cleavage by ubiquitous proteases ND ND All All Deletions in stalk region Increased virulence All All All All Neuraminidase resistance 2 of 11 ND ND ND Enhanced polymerase activity All All All All Improved viral replication at 33°C 7 of 9 ND 2 of 9 ND Mammalian adaptation 1 of 9 ND ND ND Enables droplet transmission in ferrets ND ND ND ND Enables droplet transmission in ferrets 5 of 7 All ND ND Full-length Full-length PB1-F2 needed for virulence in mice All 3 of 4 All All Increased virulence in a mice model ND ND ND ND Asn30Asp, Thr215Ala Increased virulence in a mice model All All All All Amantadine resistance All All ND ND Increased virulence in mice All All All All PDZ-binding motif Signalling of host proteins Deleted Deleted Avian type Avian type Personal View that 18% of transmission between farms was related to wind. 89 Up to May 22, 2013, 899 758 animal and environmental samples had been tested. 53 samples were positive for the 2013 H7N9 virus; 51 of these were collected from poultry or environmental samples in poultry markets, one sample was collected from a wild pigeon in Nanjing, and one sample was collected from a domestic racing pigeon at a household farm in Nantong. 28 2013 H7N9 viruses with very similar gene sequences have been found in pigeons, chickens, ducks, and environmental samples from poultry markets in aff ected areas. 11, 35, 36, 44 As in the H5N1 outbreak in Hong Kong, most confi rmed cases of H7N9 (55·9%) had history of contact with poultry. 15 Although some patients had contact with pigs, 2013 H7N9 has not been isolated from pigs. 14, 36 In the 2013 outbreak, the H7N9 virus might have originated from poultry farms and was amplifi ed in overcrowded live poultry markets before its transmission to human beings. Slaughtering and preparation of infected poultry constitute the highest risks of exposure to infected poultry secretions and excreta. Although transmission to human beings through contact with wild birds or consumption of inadequately cooked poultry is possible, documentation of such occurrences is scarce even though H7 viruses are able to survive and remain infective for a prolonged period. 90 No defi nitive evidence of continuing person-to-person transmission exists, although some fi rst-generation transmission might have occurred in four case clusters of which three were familial clusters. 14,91 However, exposure to a common avian or environmental source could not be excluded in these clusters. Few reports of person-to-person trans mission of H5N1 have been recorded. 92 The predilection of the 2013 H7N9 virus for the lower respiratory tract-shown by the higher virus burden in patients sputum than throat swab-might explain the low number of person-to-person transmissions as in the case of H5N1. 132 reported H7N9 infections in patients from March to May 2013 has far exceeded the 45 reported H5N1 infections in China within the past 10 years. 61 The crude case fatality of 2013 H7N9 infection is 30%, which is lower than that for H5N1 infection (60%), but much higher than the 0·1% of the 2009 pandemic or seasonal infl uenza. 3, 19 The true case fatality rate is uncertain because many of the patients are still receiving care in the intensive care units. Enhanced surveillance between March 7 and April 28, 2013, in which 20 739 patients with infl uenza-like illness were tested, identifi ed only two patients with H7N9 who were not hospitalised. 79 The fi rst human cases of H7N9 were detected in Shanghai, which is served by the farming and industrial establishments along the Yangtze River delta. Shanghai, a cosmopolitan megacity hardest hit by this novel virus, is similar to Hong Kong, a region that is served by the Pearl River delta and where the fi rst human cases of H5N1 were detected in 1997. 1,75,76 Both cities have well established health-care infrastructure with the necessary diagnostic methods for the detection and charac terisation of new viruses. Furthermore, both cities are located along the Asian-Australasian fl yway, where migratory birds stop at their wetlands and have surrounding poultry farms serving the densely populated areas that have many live poultry markets (table 4) . Between 1985 and 2011, consumption of poultry per person has increased 3·3 times from 3·2 kg to 10·6 kg in urban areas, whereas overall consumption of poultry increased 8·8 times from 1·53 to 13·54 million tonnes in China between 1987 and 2012. [93] [94] [95] Additionally, of all municipal areas or provinces in China, Shanghai has the highest densities of both people and poultry. 94 This is similar to the situation in Hong Kong, where poultry consumption increased greatly before the 1997 H5N1 outbreak (fi gure 4). 75, 93, 96 The emergence of the H7N9 virus in 2013 might also be related to the selective pressure induced by the widespread use of the H5N1 vaccine because H5 and H7 are the common subtypes that generally cause poultry outbreaks. China uses more than 90% of the H5N1 vaccine worldwide. 97 9 Netherlands 89 human infections with one fatality; an outbreak in chicken arose before the human cases In the H7N9 and H5N1 outbreaks, infected individuals presented with acute severe community-acquired pneumonia that did not respond to typical and atypical antimicrobial coverage. In an analysis of 111 of 132 reported cases of human H7N9 infections in 2013, more than 90% of patients had fever or cough, and 24% of the 111 patients had haemoptysis. 15 71% of the patients had acute respiratory distress syndrome. Other complications associated with H7N9 infection are rhabdomyolysis, acute kidney injury, encephalopathy, and multiorgan dysfunction. 11, 15 Unlike the 1997 H5N1 outbreak, in which nearly 40% of patients had only fever and upper respiratory tract symptoms, none of the 111 patients with 2013 H7N9 infection had sore throat, rhinorrhoea, or conjunctivitis, although mild rhinorrhoea was reported in a 3·5-year-old boy. 80 Gastrointestinal symptoms (13·5%) were less common in people infected with H7N9 than in those with the 1997 H5N1 infection (50%). 1,15 Reye's syndrome was reported in a child with 1997 H5N1 infection. 1 Gastro intestinal haemorrhage, reactive haemo phagocytosis, and haemorrhagic pleural eff usion, which were present in some cases of 1997 H5N1, have not been reported for H7N9 infection. Coinfection at presentation or during admission to hospital can occur, as reported for H5N1. 2, 15, 98 Laboratory abnormalities in patients with either infections were prominent lymphopenia, thrombocytopenia, coagulo pathy, and raised serum transaminase, creatine kinase, and C-reactive protein concentrations. Radio logical features were similar in both infections: pulmonary consolidation (which progressed to involve bilateral lung fi elds), diff use ground glass opacities, pleural eff usions, and mediastinal emphysema. None of these clinical, laboratory, and radiological manifestations are pathognomonic for H7N9 infections. The defi nitive diagnosis depends on viral culture or real-time-PCR (RT-PCR) for the H7 and N9 gene targets from respiratory tract secretions. Because of the predilection of this virus for the lower respiratory tract, if available, sputum, endotracheal aspirate or bronchoalveolar lavage might give a better sensitivity than throat or nasopharyngeal specimens. However, point-of-care rapid antigen tests and multiplex RT-PCR assays have low sensitivity. 99 Acute and convalescent serum antibody testing by haemagglutination inhibition or neutralisation can be useful for epidemiology or retrospective diagnosis. Any febrile patients with history of travel to aff ected areas, or contact with poultry or infected patients can be tested for H7N9 as a screening strategy for the early identifi cation of imported cases. 2013 H7N9 is probably resistant to adamantanes because of the presence of an M2 Ser31Asn mutation (table 2). 12 Both genetic analysis and in-vitro testing showed that most strains of 2013 H7N9 are susceptible to neuraminidase inhibitors. 100 However, two reported strains carry the neuraminidase Arg292Lys (N2 numbering) mutation, which is associated with resistance to oseltamivir and zanamivir (increase in half maximal inhibitory concentration [IC 50 ] more than 9000-fold for oseltamivir and between four-fold and 25-fold for zanamivir). 101 Personal View therapy of more than 3 days after symptom onset was not an independent risk factor for acute respiratory distress syndrome in 2013 H7N9 infections, 15 previous experience from H5N1, 2009 pandemic, and seasonal infl uenza infections suggest that early oseltamivir treatment started less than 2 days after symptom onset can reduce morbidity and mortality. 19, 102 Inhalational neuraminidase inhibitors such as zanamivir and laninamivir are unlikely to be helpful in patients with respiratory failure. 103, 104 Because oseltamivir might not aff ect outcomes in patients with late and severe infl uenza pneumonia, intravenous zanamivir and peramivir should also be investigated in future treatment trials. 105 Other investigational antivirals include those acting on the haemagglutinin receptor (DAS181), nucleoprotein (nucleozin), poly merase (viramidine and T705), and protease (aprotinin). 106 Antivirals targeting the host machinery including nitazoxanide were also reported. [106] [107] [108] Although 2013 H7N9 is susceptible to neuraminidase inhibitors, the virus might develop resistance during treatment. 19 Mice infected with the infl uenza virus resistant to amantadine had improved survival when treated with the triple combination of amantadine, oseltamivir, and ribavirin. 109 Empirical antibiotics are initially needed to cover other common pathogens, such as Streptococcus pneumoniae and Staphylococcus aureus, but should be stopped when bacterial tests are negative. The role of immunomodulation remains controversial. Glucocorticoids have been used in many patients with 2013 H7N9, but the benefi t is unclear. 12, 15 Non-steroidal anti-infl ammatory drugs such as celecoxib improved survival of mice with H5N1 infection. 110 Confl icting outcomes of patients treated with statins and macrolides have been reported. 111 The plasma of convalescent patients, which contains specifi c neutralising antibodies, has been used in patients with the H5N1 virus, 1918 pandemic H1N1, and the 2009 pandemic H1N1. Hyperimmune globulin was also used in the 2009 pandemic. Both treatments have reduced morbidity and mortality in small studies. [112] [113] [114] [115] Supportive measures in intensive care are especially important for patients with respiratory or multiorgan failure. Extracorporeal membrane oxygenation could be benefi cial in patients with respiratory failure despite maximum mechanical ventilatory support. 116 In view of the many similarities between the Hong Kong and Shanghai outbreaks, many of the measures implemented in Hong Kong can be, and have already been, applied to the current outbreak in eastern China. In the 1997 Hong Kong outbreak, temporary closure of live poultry markets and cessation of poultry trading were instituted. Subsequently, diff erent poultry species were segregated to reduce the risk of further genetic Shanghai (registered population) Hong Kong 1 9 7 3 1 9 7 7 1 9 8 1 1 9 8 3 1 9 8 9 1 9 9 3 1 9 9 5 2 0 0 1 2 0 0 5 2 0 0 7 2 0 1 1 Year 1 9 7 5 1 9 7 9 1 9 8 5 1 9 8 7 1 9 9 1 1 9 9 7 1 9 9 9 2 0 0 3 2 0 0 9 reassortment. The regular cleansing of designated transport cages in live poultry markets was introduced to stop the virus traffi cking between farms and markets. To interrupt the amplifi cation of infl uenza virus, a monthly rest day with no live poultry allowed in the wet market was implemented in Hong Kong in July 2001. The isolation rate of H9N2 from poultry in live markets before the rest day could be as high as 10%, but reduced to less than 1% after the rest day. 117 To further reduce the spread of infl uenza virus in wet markets, overnight poultry storage was banned in July 2008. 118 A further 84% reduction in the isolation rate of H9N2 was reported. As in the H5N1 outbreak, poultry vaccination specifi cally against the H7N9 virus could probably reduce its transmission. A mathematical model showed that poultry vaccination with a matched H5N1 vaccine with coverage of only 30% of the poultry reduces the basic reproductive number to less than one. 117 The 1997 H5N1 human outbreak was stopped after a cull of all poultry in Hong Kong. 117 Subsequently, only registered farms with stringent biosecurity measures in China can import chickens to Hong Kong. If a highly pathogenic avian infl uenza virus outbreak arose, all live poultry and poultry products from the aff ected province would be suspended for up to 21 days. For unaff ected farms within 3 km of the index farm aff ected by the highly pathogenic avian infl uenza virus, there would be a 90 day suspension for live poultry and poultry products. For farms where 2013 H7N9 is detected by virological testing, similar depopulation and perimetric moratorium strategies of poultry control are necessary to control the source. The general public was educated to avoid contact with birds or poultry, to cook poultry thoroughly, and to comply with hand hygiene because consumption of raw poultry might be a route of transmission. Public education and administrative measures have also led to a slow change in eating habits from live chicken to chilled or frozen chicken. To reduce the contact between human beings and poultry in Hong Kong, the Hong Kong Government implemented a voluntary surrender scheme in 2004, and subsequently a buyout scheme in 2008 for poultry retailers, wholesalers, transporters, and farmers. In 2006, domestic households were prohibited to keep any live poultry. With these measures, between 1997 and 2013, the number of local poultry farms in Hong Kong decreased from about 800 to 30 and the number of retail poultry stalls decreased from about 800 to 131. Since 1997, the number of imported live chickens was reduced from about 100 000 per day to 7000 per day. Enhanced surveillance of H5N1 and H7N9 in both live and dead wild birds, farm, and market poultries might be important for geographical areas along the migratory route of wild birds. With these measures, no local cases of H5N1 in Hong Kong have been identifi ed since 2007, despite the failure to implement central slaughtering because of cultural resistance. The 2013 H7N9 outbreak in human beings resembles the 1997 H5N1 outbreak in many ways, including substantial mortality associated with severe pneumonia, multiorgan dysfunction, and cytokine dysregulation, appearance in cities along the route of migratory birds with high density of poultry, predominant poultry-to-human transmission, and the predilection for the lower respiratory tract. 11, 119 The high proportion of elderly men infected with H7N9 could be attributable to diff erent social circumstances leading to this age and sex bias. The rapid increase in human beings infected with severe H7N9 to more than 130 cases within 2 months from March to May, 2013, is unprecedented for avian infl uenza viruses and could be related to enhanced transmissibility from poultry to human or improved virological testing. However, H7N9 is diffi cult to control because fl ock die-off did not precede human cases. Stringent measures to control the poultry outbreak will stop the human epidemic and perhaps decrease the risk of H7N9 evolving to become a pandemic agent. Even though this dangerous scenario has not happened with H5N1, the virus has spread from Hong Kong to the whole of China, southeast Asia, Middle East, and Africa. 2 Although the number of human beings infected with H7N9 was greatly reduced in May 2013 (possibly related to the rising ambient temperature or the epidemiological control measures in live poultry markets), high vigilance is still needed in view of the interval of 6 months between the fi rst and second 1997 H5N1 human case. Biosecurity measures for live poultry markets, farms, and traffi cking should be commensurate with increasing demand for poultry. Transmission cycles within markets or between markets and farms could be interrupted by a no overnight poultry policy with daily poultry-stall cleansing and ultimately central slaughtering in cities. Precautionary research and development of poultry and human H7N9 vaccines are important. Control in rural areas with many backyard farms relies on poultry vaccination. Virological surveillance of wild birds and domestic poultry in farms and markets is important in understanding the emergence of the 2013 H7N9 and other avian infl uenza viruses. Over 30 years from 1981 to 2011, the consumption of poultry in Hong Kong has tripled to 60 kg per person per year. In 2012, the consumption of poultry in China reached 10 kg per person per year, and the mainland cities such as Shanghai are likely to catch up with the amount of consumption in Hong Kong in coming years (fi gure 4). 95 Increasing problems with avian infl uenza in poultry and human beings are anticipated. We declare that we have no confl icts of interest. 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