Researchers test-drive seasonal flu reassortant possibilities

Jan 25, 2011 (CIDRAP News) – As the Northern Hemisphere's flu season unfolds, featuring a mix of virus strains, researchers from the Netherlands who tested different genetic combinations reported that a mixture of the 2009 H1N1 and H3N2 strains could transmit and cause more severe disease.

Public health officials have warned that flu is unpredictable, with reassortment between last season's pandemic virus and one of the circulating influenza A strains among the possibilities. Though the new study shows what can happen with reverse genetics and tests on ferrets, there's no evidence that any of this year's circulating strains have reassorted outside of the lab.

The research team, from Erasmus Medical Center in Rotterdam, includes Dr Albert Osterhaus and Dr Ron Fouchier. The findings appeared yesterday in an early online edition of Emerging Infectious Diseases.

They compared four reassortant viruses with the wild-type 2009 H1N1 virus to observe how they replicated, caused disease, and transmitted in ferrets, which have been found to be good animal models for influenza infection and spread.

The group made recombinant viruses using 2009 H1N1 and seasonal H1N1 and H3N2 viruses that were isolated from patients during flu outbreaks in the Netherlands. For testing, they settled on four with favorable in vitro replication properties.

The 2009 H1N1–seasonal H1N1 recombinants included the basic polymerase 2 (PB2) alone or with acidic polymerase (PA) of the seasonal H1N1 virus. The 2009 H1N1–seasonal H3N2 recombinants both included the neuraminidase (NA) of the seasonal H3N2 virus, with one also containing basic polymerase 1 (PB1).

After characterizing the reassortant viruses in vitro using Madin-Darby canine kidney cells and samples from inoculated ferrets, they conducted a series of tests to determine the pathogenicity of each of the four reassortants in ferrets. Five groups of six ferrets were infected with the 2009 H1N1 virus or one of four reassortants.

In the transmission part of the experiment, four ferrets for 2009 H1N1 and two for each reassortant virus were individually housed in transmission cages and infected. Researchers measured the animals' daily weights to detect signs of disease.

They observed immunohistochemical and pathological changes by examining lung tissue from infected ferrets.

Findings revealed that replication of the two H1N1 reassortants was similar to the wild-type 2009 H1N1 virus and that the two H3N2 reassortants showed slightly higher virus titers after inoculation.

Virus shedding from the nose was lower in the ferrets infected with one of the H1N1 reassortants and significantly higher in animals infected with one of the H3N2 reassortants.

The virological examination of respiratory tissues suggested attenuated replication in the two H1N1 reassortants. Shedding with one of the H3N2 reassortants from the nose and trachea was higher than the 2009 H1N1 virus alone. In contrast to the 2009 virus alone and one of the H1N1 reassortants, ferrets infected with one of the H3N2 reassortants showed moderate to abundant virus antigen expression in bronchial tissues that was associated with moderate to marked inflammation.

Transmission through aerosol or respiratory droplets was similar to all viruses tested, the group reported.

Though the differences in replication and shedding differences among the viruses were small, with a small number of animals in each group, the results still suggest that the 2009 H1N1-H3N2 reassortants were not attenuated in ferrets, they found.

Also, inoculation with the H3N2 reassortants was associated with more severe lesions throughout the lower respiratory tract compared with the 2009 H1N1 virus alone. "Increased severity of lesions may be related to higher virus replication in the lung, to strong host immune responses, or both," they wrote.

Though the results can't be extrapolated directly to humans, the group wrote that the ferret findings are useful, because they are susceptible to respiratory disease, lung pathology develops similar to that in humans, and flu virus attachment to respiratory cells resembles that in humans.

Findings that the H3N2 reassortants were viable, remained transmissible, and were more pathogenic than 2009 H1N1 alone underscores the importance of flu surveillance programs, they concluded.

They pointed out that the 1968 H3N2 pandemic virus continued reassorting for the next 3 years, eventually acquiring a different N2 gene. Previous flu seasons have yielded H1N1-H3N2 reassortants, and H1N2 viruses have been detected globally in pigs, they added.

Surveillance programs should closely monitor 2009 H1N1 reassortment, the group emphasized, especially when clinicians detect changes in disease severity and transmission.

Schrauwen EJ, Herfst S, Chutinimitkul S, et al. Possible increased pathogenicity of pandemic (H1N1) 2009 influenza virus upon reassortment. Emerg Infect Dis 2011 Feb;17(2):[Full text]

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