We have previously shown that 11 patients became naturally coinfected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during coinfection of host animals and in vitro; however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two coinfecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally coinfected patients.
IMPORTANCEReassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural coinfection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen. Few investigations have addressed influenza coinfection in humans: in Japan coinfections with A/H3N2 or A/H1N1 and influenza B viruses were detected in 2004/2005 (9, 10), and in Corsica, France, in 2007 (11). In September 2009, six patients in Beijing, China, became coinfected with pdm/H1N1 and A/H3N2, but reassortment between the two viruses was not detected (12). In October 2009, two patients in Cambodia were also found to be coinfected with pdm/H1N1 and A/H3N2 (13). Patient material was plaque purified on MDCK cells, and six virus plaques were examined: no reassortment between the coinfecting viruses was detected (13). Recently, Rith et al. (14) reported reassortment between pdm/H1N1 and A/H3N2 in a naturally coinfected patient, and several groups have detected coinfection of avian H7N9 and human H1N1, H3N2, and influenza B viruses in human patients (14-17).In our study, we sought to (i) determine whether reassortant viruses could be recovered from clinical material from individuals naturally coinfected with different H1N1 viruses and (ii) assess the relative fitness of these reassortants compared to parental A/H1N1 and pdm/H1N1.
MATERIALS AND METHODSCell culture. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection and maintained in minimal essential medium (MEM; Gibco, Grand Island, NY) plus 5% fetal calf serum, antibiotic-antimycotic medium (Gibco), vitamins (Gibc...