Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin - PubMed (original) (raw)

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

Troy D Cline et al. J Virol. 2011 Dec.

Abstract

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.

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Figures

Fig. 1.

Fig. 1.

Replicative capacity of reassortant pH1N1-H5N1 viruses in vitro. MDCK (A) and A549 (B) cells were infected at an MOI of 0.01, cell culture supernatants were collected at 6, 24, 48, and 72 hpi, and viral titers were determined by TCID50 analysis. (C) NHBE cells were infected at an MOI of 0.03 for 2 h at 37°C. Following infection, the cells were maintained in culture at an air-liquid interface. At 6, 24, 48, and 72 hpi, medium was added to the apical surface for 30 min and collected, and viral titers were determined by TCID50 analysis in triplicate. Data are representative of at least two experiments. Error bars represent the standard errors of the means. Individual influenza virus genes indicate reassortant CA/09 virus expressing a single gene from HK/483 virus.

Fig. 2.

Fig. 2.

Pathogenicity of reassortant pH1N1-H5N1 viruses in vivo. Female 6- to 8-week-old BALB/c mice (n = 10) were intranasally infected with 103 TCID50 units of the indicated viruses, and weight loss (A) and survival (B) were monitored for 14 dpi. (C) At 3, 6, and 10 dpi, lungs were collected from three mice per group, and viral titers were determined in homogenates by TCID50 analysis. Data are representative of at least two experiments, and error bars represent the standard errors of the means. Individual influenza virus genes indicate reassortant CA/09 virus expressing a single gene from HK/483 virus.

Fig. 3.

Fig. 3.

Histological analysis of CA/09-483HA infection. Female 6- to 8-week-old BALB/c mice (n = 3) were inoculated with 103 TCID50 units of the indicated viruses. Lung tissue was collected at 3 or 6 dpi, fixed in 10% buffered formalin, processed, and embedded in paraffin. Four-micron-thick sections were stained with hematoxylin and eosin.

Fig. 4.

Fig. 4.

Replication of serially passaged viruses in vitro. (A) NHBE cells were inoculated with the indicated viruses at an MOI of 0.03 at 37°C for 2 h. At 24, 48, and 72 hpi medium was added to the apical surface for 30 min and collected, and viral titers were determined by TCID50 analysis in triplicate. This represents one passage (p1). Equal TCID50 doses of each virus from the sample of p1 at 72 h was used to infect another monolayer of NHBE cells at an MOI of 0.03. This procedure was repeated until p3 virus was collected. Error bars represent the standard errors of the means. (B) At 48 hpi, inserts from NHBE cells infected with the p2 viruses were fixed, stained for the viral NP (green) and nuclei by DAPI (blue), and visualized by confocal microscopy.

Fig. 5.

Fig. 5.

In vivo pathogenicity of serially passaged viruses. Female 6- to 8-week-old BALB/c mice (n = 10) were intranasally inoculated with 103 TCID50 units of the indicated viruses, and weight loss (A) and survival (B) were monitored for 12 dpi. (C) At 3 and 6 dpi, lungs were collected from three mice per group and viral titers were determined in homogenates by TCID50 analysis. Error bars represent the standard errors of the means.

Fig. 6.

Fig. 6.

Transmissibility of CA/09-483HA in ferrets. Male 9- to 15-week-old ferrets (n = 2) were intranasally inoculated with 104 TCID50 units of the indicated viruses. Twenty-four hpi, two naïve contact ferrets were introduced and housed together with the inoculated ferrets for the duration of the experiment. Nasal washes were collected, and viral titers were determined by TCID50 analysis at 1, 3, 5, and 7 dpi for the inoculated ferrets and at 3, 5, 7, and 10 days postexposure for the contact ferrets. Error bars represent standard errors of the means.

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