Low growth ability of recent influenza clinical isolates in MDCK cells is due to their low receptor binding affinities.
Madin Darby canine kidney (MDCK) cells have generally been used to isolate influenza viruses from patients. However, in recent years, most fresh isolates of the H3N2 subtype have shown poor growth in MDCK cell cultures. Such low-growth viruses were often converted to high-growth viruses after several passages through MDCK cell cultures. In the present study, viruses were found to lose a potential glycosylation site near the receptor-binding pocket of hemagglutinin (HA), at the same time as they acquired the high-growth property. The growth curves of viruses in MDCK cell cultures revealed that multi-cycle replication did not function well in the low-growth viruses. However, the production of progeny viruses within a single cycle of growth did not differ much between the low- and high-growth viruses. The high-growth viruses showed higher infection efficiency in MDCK cell cultures than the low-growth viruses. The HA genes of both low- and high-growth viruses were separately cloned into the SV40 vector to compare their receptor binding affinities. The HA of high-growth viruses showed a much higher receptor binding affinity than that of low-growth viruses, when assayed by hemadsorption and the release kinetics of erythrocytes with bacterial neuraminidase. Reverse genetics studies demonstrated that HA was a crucial determinant for multi-cycle replication in MDCK cell cultures. Taken together, these results demonstrate that inefficient multi-cycle growth of fresh isolates is due to their low receptor binding affinities.
Asaoka
Naoko
N
Department of Microbiology, Kawasaki Medical School, Kurashiki 710-0192, Japan.
Tanaka
Yasuko
Y
Sakai
Tatsuya
T
Fujii
Yutaka
Y
Ohuchi
Reiko
R
Ohuchi
Masanobu
M
D000818:Animals; D002460:Cell Line; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000254:growth & development* / Q000302:isolation & purification / Q000378:metabolism* / Q000472:pathogenicity; D007251:Influenza, Human / Q000821:virology; D009439:Neuraminidase / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism*; D012692:Serial Passage
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011991:Receptors, Virus; D009439:Neuraminidase