Characterization of a novel influenza virus in cattle and Swine: proposal for a new genus in the Orthomyxoviridae family

Abstract

We have recently reported the isolation of a novel virus, provisionally designated C/swine/Oklahoma/1334/2011 (C/OK), with 50% overall homology to human influenza C viruses (ICV), from a pig in Oklahoma. Deep RNA sequencing of C/OK virus found a matrix 1 (M1) protein expression strategy that differed from that of ICV. The novelty of C/OK virus prompted us to investigate whether C/OK virus could exist in a nonswine species. Significantly, we found that C/OK virus was widespread in U.S. bovine herds, as demonstrated by reverse transcription (RT)-PCR and serological assays. Genome sequencing of three bovine viruses isolated from two herds in different states further confirmed these findings. To determine whether swine/bovine C/OK viruses can undergo reassortment with human ICV, and to clarify the taxonomic status of C/OK, in vitro reassortment and serological typing by agar gel immunodiffusion (AGID) were conducted. In vitro reassortment using two human ICV and two swine and bovine C/OK viruses demonstrated that human ICV and C/OK viruses were unable to reassort and produce viable progeny. Antigenically, no cross-recognition of detergent split virions was observed in AGID between human and nonhuman viruses by using polyclonal antibodies that were reactive to cognate antigens. Taken together, these results demonstrate that C/OK virus is genetically and antigenically distinct from ICV. The classification of the new virus in a separate genus of the Orthomyxoviridae family is proposed. The finding of C/OK virus in swine and bovine indicates that this new virus may spread and establish infection in other mammals, including humans. IMPORTANCE Influenza C viruses (ICV) are common human pathogens, infecting most people during childhood and adolescence, and typically cause mild respiratory symptoms. While ICV have been isolated from both pigs and dogs, humans are thought to be the natural viral reservoir. Previously, we characterized an ICV-like virus isolated from pigs exhibiting symptoms of influenza virus-like illness. Here, we show molecular and serological data demonstrating widespread circulation of similar viruses in bovines. Deep RNA sequencing, phylogenetic analysis, and in vitro reassortment experiments demonstrate that animal ICV-like viruses are genetically distinct from human ICV. Antigenically, we show that ICV-like viruses are not recognized by ICV antibodies. En masse, these results suggest that bovine influenza virus warrants classification as a new genus of influenza virus. The finding of this novel virus that can infect multiple mammalian species warrants further research into its role in human health.

FIG 1

Transcriptome maps of C/OK virus segments. Numbers of normalized sequence reads (100 nucleotides each read) were mapped to each nucleotide position across the respective segments of C/OK virus genome. The x axis represents the length of each segment, while the y axis represents the number of sequencing reads mapping to each genomic segment. Arrow signs indicate intron regions present in NS and M segments that were identified by next-generation sequencing (NGS) analysis.

FIG 2

Splicing strategies of C/OK virus for the NS segment (A) and M segment (B). Panel A schematically illustrates a splicing strategy of the C/OK virus NS segment to produce NS2 protein in comparison to its counterpart in human influenza C viruses (ICV), while panel B describes a splicing strategy of the M segment to produce M1 protein in comparison to human ICV’s M1 protein synthesis.

FIG 3

Phylogenetic trees of the seven segments of bovine C/OK viruses. Maximum likelihood analysis in combination with 500 bootstrap replicates was used to derive trees based on the nucleotide sequences of the genomic segments. Bootstrap values are shown above and to the left of the major nodes.

FIG 4

Mean hemagglutination inhibition titers for eight bovine herds (11 to 27 animals per herd) to bovine C/660 and swine C/OK. Bovine sera from eight herds located in five different states were screened individually (11 to 27 animals per herd) in the HI assay using C/OK and C/660 as antigens. The x axis represents the sample size of each herd tested, while the y axis indicates the average HI titer and standard error from each herd.