Structural and evolutionary characteristics of HA, NA, NS and M genes of clinical influenza A/H3N2 viruses passaged in human and canine cells.
BACKGROUND
Canine (MDCK) cells and chicken eggs are usually used for isolation of human influenza viruses. Viruses isolated by these procedures often differ from those present in the clinical specimens, since adaptive changes occur during virus transmission from the human host to cells of heterologous origin.


OBJECTIVE
To minimize these species-dependent changes, CACO-2 cells derived from human intestinal epithelium were used to isolate virus from influenza patients.


METHODS
Influenza A viruses of subtype H3N2 were primarily isolated in CACO-2 and then passaged in parallel in CACO-2 and MDCK cells. Structural properties of passaged virus variants were compared and analyzed for evolutionary relationships.


RESULTS
Influenza viruses were isolated in CACO-2 with higher efficiency than in MDCK and chicken eggs. The following observations were made: (i) recent isolates showed an about 2-fold increase in the number of glycosylation sites of HA and NA when compared to isolates from 1968 to 1970; (ii) during passages of clinical strains in CACO-2 and MDCK cells HA and NA mutated cooperatively with strain-specific variations implying that functioning of the HA-NA complex varied from strain to strain in one influenza outbreak; (iii) there were no amino acid exchanges in the HA receptor binding site although the viruses acquired the ability to agglutinate avian erythrocytes after passage in MDCK cells, suggesting that virus adsorption is regulated by several factors; (iv) quasispecies characterized by deletion of 66 nucleotides (22 amino acids) in the stalk region of the NA gene was dominant in naso-pharyngeal washes of all patients whereas during passaging in CACO-2 cells this deleted genotype in isolates from different patients was either stably retained as prevalent quasispecies or rapidly replaced for that one containing full length NA gene; (v) the M2 protein of clinical viruses was sensitive to amantadine; (vi) the NS segment of human viruses, unlike the most of avian ones, contained an additional positive-sense open reading frame encoding a hypothetical 25kD polypeptide (negative strand protein, NSP).


CONCLUSIONS
The data suggest that (i) clinical influenza viruses can be isolated from respiratory tract of humans more effectively in human than in canine cells; (ii) heterologous virus population circulates during one influenza outbreak; (iii) increasing numbers of glycosylation sites on HA and NA and stalk shortening of NA take place during virus evolution in humans.
Zhirnov
O P
OP
D.I. Ivanovsky Institute of Virology, Gamaleya 16, Moscow 123098, Russia. zhirnov@inbox.ru
Vorobjeva
I V
IV
Saphonova
O A
OA
Poyarkov
S V
SV
Ovcharenko
A V
AV
Anhlan
D
D
Malyshev
N A
NA
D000220:Adaptation, Biological; D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D004285:Dogs; D006031:Glycosylation; D006389:Hemagglutinins, Viral / Q000235:genetics*; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009439:Neuraminidase / Q000235:genetics*; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014763:Viral Matrix Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D006389:Hemagglutinins, Viral; C488936:M1 protein, Influenza A virus; C108309:NS protein, influenza virus; D012367:RNA, Viral; D014763:Viral Matrix Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C458881:hemagglutinin fusogenic peptide, influenza virus; C487630:NA protein, influenza A virus; D009439:Neuraminidase