The influenza C virus is predominantly found in humans, and infection in humans can cause respiratory and febrile symptoms that are similar to those caused by influenza A and B viruses. Influenza C virus infections are generally mild and self-limited but can also cause more severe lower respiratory tract illness, such as bronchitis and pneumonia [1
]. The infection is considered common based on high seroprevalence [3
]. The influenza C virus has also been found in pigs, and there are some reports about interspecies transmission of the virus between humans and pigs [6
]. However, pigs are not considered to play a significant role in the transmission cycle of the viruses in humans [7
The influenza C virus is a member of the Orthomyxoviridae
family, enveloped and segmented negative-sense RNA virus with seven segments. The fourth segment of the viral genome encodes hemagglutinin-esterase (HE) glycoprotein [9
], which determines the major antigenicity of the virus and has a variety of functions in the viral replication cycle. At least nine antigenic sites (A-1 to A-5 and B-1 to B-4) of the HE glycoproteins are proposed; of them, amino acid positions responsible for four antigenic sites have been identified [10
]. In addition, glycosylation of the proteins also affects the virus’ antigenicity [10
]. The influenza C virus utilizes the HE glycoprotein as an attachment protein to a cellular receptor of the virus, 9-O
-acetylneuraminic acid [13
]. The HE glycoprotein also catalyzes fusion of the viral envelope with endocytic vesicles [15
]. Proteolytic cleavage of the protein into two subunits, HE1 and HE2, is an essential prerequisite for the membrane fusion activity [9
]. In addition, the HE glycoprotein has esterase activity that functions as the receptor-destroying enzyme to release the progeny of viral particles from infected cells [14
The influenza C virus was first isolated in 1947 [16
]. Since then, it has been reported that antigenically and genetically-distinct lineages of the virus are co-circulating [17
]. In the influenza A virus, there are two subtypes currently circulating among human populations; A/H1N1 and A/H3N2. In contrast to the influenza C virus, antigenically and genetically-similar strains of seasonal influenza A virus are circulating worldwide at a given time; and new antigenic variants, which are descendants of formerly circulating viruses, replace the previous viruses and become a predominant strain [19
]. The influenza B virus does not have subtypes, but currently circulating influenza B viruses are divided into two phylogenetically and antigenically distinct lineages: B/Victoria/2/87-like (B/Victoria) lineage and B/Yamagata/16/88-like (B/Yamagata) lineage [22
The seasonal influenza A virus accumulates mutations in the hemagglutinin (HA) gene, which encodes a major surface antigenic protein of the virus [21
]. The virus causes annual epidemics by continuous antigenic change (antigenic drift), which allows viruses to evade herd immunity [25
]. Therefore, positive selection that results in cumulative mutations in antigenic sites of the HA gene through evolutionary history has been observed [26
]. The HA protein of the influenza A virus can also gain or lose glycosylation sites, which can alter the antigenicity of the virus [30
]. There is a selection pressure on antigenic sites in the HA gene of influenza B viruses as well, although it is considered weaker than that of the influenza A viruses [32
]. Buonagurio et al. and Muraki et al. reported little or no accumulation of mutations in the HE gene of the influenza C virus in the 1980s and 1990s, respectively; although they analyzed limited numbers of sequences available then (<20 strains) [34
Differences in ecological, epidemiological, and evolutionary characteristics between influenza A, B, and C viruses are of great interest. Apart from antigenic analyses using viral isolates and sera or monoclonal antibodies against the viruses [17
], phylogenetic analyses using genetic sequence data of the HE gene could provide important information about the evolution of the influenza C virus. However, because isolation of the influenza C virus using cell cultures, such as MDCK and LLC-MK2
cells requires technical proficiency and intensive observation of inoculated cells, isolation of the virus is rarely performed. This is one of the reasons why sequence data of the virus are still limited.
We recently reported the analysis of the full genome sequence of 102 strains of the influenza C virus and unveiled a history of frequent reassortment of the virus [18
]. Reports and sequence data of the virus from many parts of the world have also been increasing [37
]. In this study, we exploited sequence data available to date, many of which are from our recent study, with phylogenetic techniques to see the evolutionary pathway of the influenza C virus over a period of 68 years. We also compared the evolution of influenza C viruses with those of influenza A and B viruses.
In this study, we analyzed the evolutionary pathway of the influenza C virus using 218 viral sequences collected over 68 years. Slow evolution of the influenza C virus is characterized by weak and infrequent selective bottlenecks and a small number of mutations that possibly alter antigenicity. Most sequence data of the influenza C virus are from one country, Japan; while our datasets for influenza A and B viruses consist of sequence data from various parts of the world. There may be selection bias for the influenza C virus. Data collection from a restricted area, however, could lead to overestimation of the strength of selective bottlenecks. Even with the possibility of not underestimation, but overestimation, our analyses found that selective bottlenecks for the influenza C virus are weak and infrequent through its evolution pathway.
The unique evolutionary characteristics of the influenza C virus, have resulted in multiple lineages of the virus co-circulating in different countries. This is similar to the behavior of the influenza B virus whereby multiple lineages of the virus co-circulate, and antigenic variants appear to be slower than in influenza A viruses [33
]. In addition, our findings showed a much slower rate of evolution, less frequent selective bottlenecks, and weaker positive selection for the influenza C virus than the influenza B virus.
It is still unknown why the C/Taylor lineage, the C/Aichi lineage, the C/Mississippi lineage, and the C/Yamagata lineage disappeared, whereas the C/Kanagawa lineage and the C/Sao Paulo lineage persist. The existing two lineages experienced surge of genetic diversity (Figure 2
). As genetic diversity is associated with effective population size, occasional outbreak with a large number of infected people, which can lead to increase of genetic diversity and fitness of the virus, might be required to prevent it from extinction. It would be intriguing to see whether the high and constant genetic diversity of the influenza C virus would lead to the emergence of novel lineages that would co-circulate with existing lineages.
One limitation of this study is that we analyzed only one segment, the HE gene, of the influenza C virus. Analysis of the only one segment might lead to some loss of information. However, it is difficult to analyze evolutionary characteristics of the virus with the whole genome because of a complicated history of reassortment [18
] and an insufficient number of strains with whole-genome sequences. Published studies documented population dynamics of influenza viruses well by analysis with only one segment (the HA gene) for influenza A and B viruses [47
Further questions arise, such as what mechanisms underlie the evolutionary differences between influenza A, B, and C viruses. Selective bottlenecks of the HA/HE gene of influenza viruses at the population level must be mainly driven by the immune pressure of herd immunity [21
]. Multiple factors must be important to limit the genetic diversity of influenza viruses, including infection incidence rate, seroprevalence, protective immune response, duration of the immunity, and broadness of immunity against heterologous viruses [60
]. A rare selective bottleneck for the influenza C virus suggests unique characteristics of the virus and its infections. Seroprevalence and incidence of the influenza C virus do not seem as high as those of the influenza A virus [5
]. Further studies are necessary to understand the epidemiological and immunological aspects of influenza C viruses, such as whether multiple exposures throughout life are common, whether immunity against the virus can prevent reinfection, how long the immunity lasts, and whether the infection can induce strain non-specific immunity to constrain genetic diversity. We have limited knowledge of the epidemiological and immunological aspects of influenza C viruses, which must be responsible for its evolutionary characteristics.
Transmission dynamics, such as age at infection and global circulation patterns have also been suggested to affect the evolutionary pathway of influenza A and B viruses [47
]. There could be unique transmission dynamics of the influenza C virus affecting its evolution in addition to unique virological, immunological, and epidemiological characteristics of the virus. Further studies, combining experimental, epidemiological, and theoretical analyses, are needed for better understanding of the evolutionary and ecological aspects of the influenza C virus.