The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells

Abstract

Background: Two new subclades of influenza A(H3N2) viruses became prominent during the 2014-2015 Northern Hemisphere influenza season. The HA glycoproteins of these viruses showed sequence changes previously associated with alterations in receptor-binding properties. To address how these changes influence virus propagation, viruses were isolated and propagated in conventional MDCK cells and MDCK-SIAT1 cells, cells with enhanced expression of the human receptor for the virus, and analysed at each passage.

Methods: Gene sequence analysis was undertaken as virus was passaged in conventional MDCK cells and MDCK-SIAT1 cells. Alterations in receptor recognition associated with passage of virus were examined by haemagglutination assays using red blood cells from guinea pigs, turkeys and humans. Microneutralisation assays were performed to determine how passage-acquired amino acid substitutions and polymorphisms affected virus antigenicity.

Results: Viruses were able to infect MDCK-SIAT1 cells more efficiently than conventional MDCK cells. Viruses of both the 3C.2a and 3C.3a subclades showed greater sequence change on passage in conventional MDCK cells than in MDCK-SIAT1 cells, with amino acid substitutions being seen in both HA and NA glycoproteins. However, virus passage in MDCK-SIAT1 cells at low inoculum dilutions showed reducing infectivity on continued passage.

Conclusions: Current H3N2 viruses should be cultured in the MDCK-SIAT1 cell line to maintain faithful replication of the virus, and at an appropriate multiplicity of infection to retain infectivity.

Keywords: MDCK cells; MDCK-SIAT1 cells; antigenicity; influenza; receptor binding.

Figure 1

Comparison of plaque formation by 3C.2a and 3C.3a viruses on MDCK‐SIAT1 and MDCK‐parent cells. Plaque titrations were performed in parallel using MDCK‐SIAT1 (S) and MDCK‐parent (P) cells in duplicate with 10‐fold dilutions. Panel A: 3C.2a viruses: 1, A/Asturias/1951/2014 and 2, A/Buenos Aires/6367/2014 both retained the glycosylation site at HA1 158‐160, viruses with no polymorphism in NA 148/151; 3, A/Israel/O‐7414/2014, with no glycosylation site due to a T160K substitution in HA1 and no polymorphism at NA 148/151; 4, A/Hong Kong/7347/2014 with a partially lost glycosylation site due to polymorphism at HA1 160 (K/T) and no polymorphism at NA 148/151; 5, A/Hong Kong/6013/2014 retained glycosylation site at HA 158‐160 but with polymorphism at NA 148/151. Panel B: 3C.3a viruses: 1, A/Switzerland/9715293/2013 and 2, A/Norway/466/2014 have no polymorphism in NA 148/151; 3, A/Hong Kong/6315/2014 with polymorphism in NA 151. Viruses with polymorphisms were from passage 3 using conventional MDCK cells, and the extents of polymorphisms are as shown in Table 1

Figure 2

Propagation of recent influenza A (H3N2) viruses in MDCK‐parent and MDCK‐SIAT cells using undiluted culture supernatants serially passed from passage number 1 to 2 and from passage 2 to 3. ( MDCK‐SIAT1 cells, MDCK‐Parent cells). A, A/Hong Kong/6086/2014 and B, A/Hong Kong/6315/2014, both 3C.3a viruses; and C, A/Hong Kong/7127/2014 and D, A/Hong Kong/7364/2014, both 3C.2a viruses. cs=clinical sample, p=passage

Figure 3

Comparison of virus yield with different dilutions of inoculum for recent H3N2 viruses in MDCK‐SIAT1 cells. A, A/Hong Kong/6086/2014 and B, A/Hong Kong/6315/2014, both 3C.3a viruses; C, A/Hong Kong/7127/2014 and D, A/Hong Kong/7364/2014, both 3C.2a viruses. Virus passed using undiluted culture supernatants, Virus passed at 10⁻⁶ dilution of the inoculum from passage 1 to passage 2, and at 10⁻⁵ dilution from passage 2 to passage 3. cs=clinical sample, p=passage