Limits...
Does pandemic A/H1N1 virus have the potential to become more pathogenic?

Ilyushina NA, Ducatez MF, Rehg JE, Marathe BM, Marjuki H, Bovin NV, Webster RG, Webby RJ - MBio (2010)

Bottom Line: When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected.These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains.Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

ABSTRACT
Epidemiologic observations that have been made in the context of the current pandemic influenza virus include a stable virulence phenotype and a lack of propensity to reassort with seasonal strains. In an attempt to determine whether either of these observations could change in the future, we coinfected differentiated human airway cells with seasonal oseltamivir-resistant A/New Jersey/15/07 and pandemic A/Tennessee/1-560/09 (H1N1) viruses in three ratios (10:90, 50:50, and 90:10) and examined the resulting progeny viruses after 10 sequential passages. When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected. These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains. The polymerase acidic protein mutation increased polymerase activity at 37°C, and the hemagglutinin change affected binding of the virus to α2,6-sialyl receptors. When the seasonal A/H1N1 virus was initially present in excess, the dominant progeny virus was a reassortant containing the hemagglutinin gene from the seasonal strain and the remaining genes from the pandemic virus. Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

Show MeSH

Related in: MedlinePlus

Replication of NJ/15, TN/560, and two recovered H1N1 genotypes, G1 and G2, in NHBE cells and ferrets. (A and B) NHBE cultures were infected via the apical side with each virus at an MOI of 0.1 at either 37°C (A) or 33°C (B). The progeny viruses released from the apical surfaces of infected cultures were collected at the indicated time points and titrated in MDCK cells by performing a plaque assay. Representative results expressed as log10 numbers of PFU/ml from three independent experiments are shown. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (C) Four ferrets were intranasally inoculated with 106 PFU of each virus, and 3 days later, tissues were collected and virus titers were determined. Virus titers are expressed as log10 TCID50/gram tissue for one ferret. (D) The images shown are hematoxylin-and-eosin-stained sections of lung tissue from donor ferrets inoculated with TN/560, G1, or G2 (H1N1) influenza viruses obtained on day 3 postinoculation. The TN/560 lung infection was primary restricted to the bronchioles and consisted of epithelial necrosis and regeneration associated with intraluminal sloughed epithelial cells and mononuclear inflammatory cells. The G1 infection involved the bronchioles and alveoli and consisted of bronchiolar epithelial necrosis and regeneration associated with alveolar pneumocytes hyperplasia and mononuclear inflammatory cell infiltrates. The pathologic changes in the G2-infected lungs were restricted to the alveolar interstitium only. The alveolar spaces were free of inflammatory cells, whereas the alveolar interstitial walls were hypercellular with mononuclear cells. Magnification, ×20.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2993010&req=5

f2: Replication of NJ/15, TN/560, and two recovered H1N1 genotypes, G1 and G2, in NHBE cells and ferrets. (A and B) NHBE cultures were infected via the apical side with each virus at an MOI of 0.1 at either 37°C (A) or 33°C (B). The progeny viruses released from the apical surfaces of infected cultures were collected at the indicated time points and titrated in MDCK cells by performing a plaque assay. Representative results expressed as log10 numbers of PFU/ml from three independent experiments are shown. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (C) Four ferrets were intranasally inoculated with 106 PFU of each virus, and 3 days later, tissues were collected and virus titers were determined. Virus titers are expressed as log10 TCID50/gram tissue for one ferret. (D) The images shown are hematoxylin-and-eosin-stained sections of lung tissue from donor ferrets inoculated with TN/560, G1, or G2 (H1N1) influenza viruses obtained on day 3 postinoculation. The TN/560 lung infection was primary restricted to the bronchioles and consisted of epithelial necrosis and regeneration associated with intraluminal sloughed epithelial cells and mononuclear inflammatory cells. The G1 infection involved the bronchioles and alveoli and consisted of bronchiolar epithelial necrosis and regeneration associated with alveolar pneumocytes hyperplasia and mononuclear inflammatory cell infiltrates. The pathologic changes in the G2-infected lungs were restricted to the alveolar interstitium only. The alveolar spaces were free of inflammatory cells, whereas the alveolar interstitial walls were hypercellular with mononuclear cells. Magnification, ×20.

Mentions: We first studied the growth of G1 and G2 viruses both in eggs and in MDCK cells (see Table S1 in the supplemental material). The yields of both viruses in cell culture were significantly higher (~1.5 logs) than those of their parental TN/560 and NJ/15 strains (P < 0.01). G1 and G2 formed larger plaques (diameter, 1.5 to 2.2 mm) than the parental counterparts (diameter, 0.6 to 1.1 mm) (Table S1). To assess the relative replication efficiencies of the G1 and G2 viruses, the multiple-cycle replication kinetics of these viruses in comparison with the levels for TN/560 and NJ/15 in NHBE cells were examined. The replication levels of G1 and G2 were significantly higher than those of both parents 24, 48, and 72 h after infection at 37°C (>0.9 to 1.8 logs; P < 0.05) (Fig. 2A). Higher titers of G1 and G2 than of TN/560 were also observed 48 and 72 hours after infection at 33°C (>0.6 to 1.3 logs; P < 0.05) (Fig. 2B).


Does pandemic A/H1N1 virus have the potential to become more pathogenic?

Ilyushina NA, Ducatez MF, Rehg JE, Marathe BM, Marjuki H, Bovin NV, Webster RG, Webby RJ - MBio (2010)

Replication of NJ/15, TN/560, and two recovered H1N1 genotypes, G1 and G2, in NHBE cells and ferrets. (A and B) NHBE cultures were infected via the apical side with each virus at an MOI of 0.1 at either 37°C (A) or 33°C (B). The progeny viruses released from the apical surfaces of infected cultures were collected at the indicated time points and titrated in MDCK cells by performing a plaque assay. Representative results expressed as log10 numbers of PFU/ml from three independent experiments are shown. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (C) Four ferrets were intranasally inoculated with 106 PFU of each virus, and 3 days later, tissues were collected and virus titers were determined. Virus titers are expressed as log10 TCID50/gram tissue for one ferret. (D) The images shown are hematoxylin-and-eosin-stained sections of lung tissue from donor ferrets inoculated with TN/560, G1, or G2 (H1N1) influenza viruses obtained on day 3 postinoculation. The TN/560 lung infection was primary restricted to the bronchioles and consisted of epithelial necrosis and regeneration associated with intraluminal sloughed epithelial cells and mononuclear inflammatory cells. The G1 infection involved the bronchioles and alveoli and consisted of bronchiolar epithelial necrosis and regeneration associated with alveolar pneumocytes hyperplasia and mononuclear inflammatory cell infiltrates. The pathologic changes in the G2-infected lungs were restricted to the alveolar interstitium only. The alveolar spaces were free of inflammatory cells, whereas the alveolar interstitial walls were hypercellular with mononuclear cells. Magnification, ×20.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2993010&req=5

f2: Replication of NJ/15, TN/560, and two recovered H1N1 genotypes, G1 and G2, in NHBE cells and ferrets. (A and B) NHBE cultures were infected via the apical side with each virus at an MOI of 0.1 at either 37°C (A) or 33°C (B). The progeny viruses released from the apical surfaces of infected cultures were collected at the indicated time points and titrated in MDCK cells by performing a plaque assay. Representative results expressed as log10 numbers of PFU/ml from three independent experiments are shown. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (C) Four ferrets were intranasally inoculated with 106 PFU of each virus, and 3 days later, tissues were collected and virus titers were determined. Virus titers are expressed as log10 TCID50/gram tissue for one ferret. (D) The images shown are hematoxylin-and-eosin-stained sections of lung tissue from donor ferrets inoculated with TN/560, G1, or G2 (H1N1) influenza viruses obtained on day 3 postinoculation. The TN/560 lung infection was primary restricted to the bronchioles and consisted of epithelial necrosis and regeneration associated with intraluminal sloughed epithelial cells and mononuclear inflammatory cells. The G1 infection involved the bronchioles and alveoli and consisted of bronchiolar epithelial necrosis and regeneration associated with alveolar pneumocytes hyperplasia and mononuclear inflammatory cell infiltrates. The pathologic changes in the G2-infected lungs were restricted to the alveolar interstitium only. The alveolar spaces were free of inflammatory cells, whereas the alveolar interstitial walls were hypercellular with mononuclear cells. Magnification, ×20.
Mentions: We first studied the growth of G1 and G2 viruses both in eggs and in MDCK cells (see Table S1 in the supplemental material). The yields of both viruses in cell culture were significantly higher (~1.5 logs) than those of their parental TN/560 and NJ/15 strains (P < 0.01). G1 and G2 formed larger plaques (diameter, 1.5 to 2.2 mm) than the parental counterparts (diameter, 0.6 to 1.1 mm) (Table S1). To assess the relative replication efficiencies of the G1 and G2 viruses, the multiple-cycle replication kinetics of these viruses in comparison with the levels for TN/560 and NJ/15 in NHBE cells were examined. The replication levels of G1 and G2 were significantly higher than those of both parents 24, 48, and 72 h after infection at 37°C (>0.9 to 1.8 logs; P < 0.05) (Fig. 2A). Higher titers of G1 and G2 than of TN/560 were also observed 48 and 72 hours after infection at 33°C (>0.6 to 1.3 logs; P < 0.05) (Fig. 2B).

Bottom Line: When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected.These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains.Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

ABSTRACT
Epidemiologic observations that have been made in the context of the current pandemic influenza virus include a stable virulence phenotype and a lack of propensity to reassort with seasonal strains. In an attempt to determine whether either of these observations could change in the future, we coinfected differentiated human airway cells with seasonal oseltamivir-resistant A/New Jersey/15/07 and pandemic A/Tennessee/1-560/09 (H1N1) viruses in three ratios (10:90, 50:50, and 90:10) and examined the resulting progeny viruses after 10 sequential passages. When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected. These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains. The polymerase acidic protein mutation increased polymerase activity at 37°C, and the hemagglutinin change affected binding of the virus to α2,6-sialyl receptors. When the seasonal A/H1N1 virus was initially present in excess, the dominant progeny virus was a reassortant containing the hemagglutinin gene from the seasonal strain and the remaining genes from the pandemic virus. Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

Show MeSH
Related in: MedlinePlus