Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to human influenza C viruses

Abstract

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

Figure 1. Ultrastructural analysis of C/OK virus isolate in cell culture as observed by negative stain (A) and thin-section (B) electron microscopy.

(A) Negative stain shows features of an orthomyxovirus particle (Bar = 200 nm) (B) Infected cells visualized with uranyl acetate and lead contrast. Note assembly and budding of virions at the apical pole. Free spherical virions 70–90 nm in diameter present surface spikes and internal electron-dense dots. Bar = 500 nm.

Figure 2. Phylogenetic trees of the coding regions of the seven segments of C/OK virus.

Maximum-likelihood analysis in combination with 1000 bootstrap replicates was used to derive trees based on the nucleotide sequences encoding respective proteins. A scale representing the number of nucleotide changes is shown in each panel. The trees are shown for PB2, PB1, P3, HE, NP, P42, and NS segments. Bootstrap values are shown above branches to the left of major nodes.

Figure 3. Replication of C/OK virus in ferrets challenged intranasally (donors) or exposed by direct or aerosol contact.

Three ferrets (donor ferrets) were inoculated intranasally with 10⁶ TCID₅₀ of C/OK virus in 1 ml of sterile PBS. Two inoculated ferrets were also housed separately for virus titration and histopathology in organs. At 23 h p.i., each of the three remaining donor ferrets was housed in a cage with one naïve direct-contact ferret (n = 3). An additional ferret (n = 3) was placed in an adjacent cage separated from the donor's cage by a two layers of wire mesh (∼5 cm apart) that prevented physical contact but allowed the passage of respiratory droplets. To monitor virus shedding, nasal washes were collected from ferrets 3, 5, 7, and 10 days p.i. Virus was titrated in ST cells as log₁₀ TCID₅₀/ml and values shown are the mean ± standard error.

Figure 4. Structural comparisons of C/OK and human C HE receptor-binding pocket and substrate-binding site.

(A) Superposition of the esterase active site of C/OK (magenta sticks) and human C/Johannesburg/1/66 (gray sticks). The potential hydrogen bonds shown were from a previous study and were indicated as dashed line . The analog of 9-O-sialic acid, 9-acetamindo-sialic acid α-methylglycoside, was colored cyan. (B) Superposition of the sialic acid binding site of the receptor binding domain of C/OK (magenta sticks) and human C/Johannesburg/1/66 (gray sticks). Hydrogen bonds between residues of C/Johannesburg/1/66 and sialic acid were shown as dashed yellow line. The structure of C/Johannesburg/1/66 HEF was transparently shown in cartoon mode (gray). (C) Electrostatic surface of human C/Johannesburg/1/66 HE. Blue colored region has strong positive potential while red colored region has negative potential. (D) Electrostatic surface of C/OK HE. For both (C) and (D), the nine carbons of sialic acid are numbered.