Influenza C virus in humans and animals

Abstract

Influenza C virus (ICV) was discovered in 1947 and detected in humans, with natural infections occurring periodically. However, early studies on ICV were challenging in diagnosis because the virus is difficult to culture. As a result, the disease burden and pathogenicity of ICV have been underestimated. Recent studies using molecular biological techniques such as real-time polymerase chain reaction have provided further insights into prevalence, seasonality, genomic diversity, and evolution. In addition, the possibility of interspecies transmission was suggested based on the high similarity between the nucleotide sequence of ICV confirmed to infect animals and the sequence of ICV isolated from humans. In this review, we summarize current data on the epidemiology and clinical features, viral genome analysis, and animal studies of ICV.

INTRODUCTION

Influenza viruses are well known zoonotic pathogen. Influenza viruses belong to the Orthomyxoviridae family, are characterized by segmented, negative-sense RNA genomes, and are classified into four types: influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV) [1]. These influenza viruses are genetically and epidemiologically distinct (Fig. 1).

Fig. 1. Type and structure of Influenza virus. The genome of IAV and IBV is composed of 8 segmented gene, while the genome of ICV and IDV is composed of 7 segmented gene. HEF protein is a surface glycoprotein of ICV and IDV that functions as HA and NA of IAV and IBV. IAV has a wide host range, including humans, bird, and swine. IBV and ICV primarily infect humans, and IDV primarily infects cattle.

IAV, influenza A virus; IBV, influenza B virus; ICV, influenza C virus; IDV, and influenza D virus; HEF, hemagglutinin-esterase fusion; HA, hemagglutinin; NA, neuraminidase; NP, nucleoprotein; M, matrix; NS, nonstructural.

IAV and IBV have eight-segmented genomes, while ICV and IDV have seven-segmented genomes. Differences in the number of genome segments among influenza viruses can be differentiated based on the composition of their surface glycoproteins. IAV and IBV have 2 surface glycoprotein, hemagglutinin (HA) and neuraminidase (NA). In contrast, ICV and IDV have a single surface glycoprotein, the hemagglutinin-esterase fusion (HEF) protein, which combines the functions of both HA and NA [1,2]. Differences in surface glycoproteins also results in variations in receptor binding during host invasion: IAV and IBV bind to α2,3- or α2,6-linked sialic acid, while ICV and IDV utilize 9-O-acetylated sialic acid as binding receptor [1,2,3]. The other 6 segmented genes encode proteins of the polymerase complex (PB2, PB1, and P3/PA), as well as the nucleoprotein (NP), matrix, and nonstructural proteins.

IAV infects a wide range of hosts, including birds, pigs, and humans. IBV and ICV primarily cause respiratory infections in humans, whereas IDV is predominantly associated with respiratory infections in cattle. Additionally, IAV and IBV cause seasonal epidemics in humans annually [4]. Therefore, annual vaccination is recommended, and various studies have been conducted in the fields of molecular biology and immunology with high disease burden of influenza. In contrast, ICV generally causes mild illness or asymptomatic infection in humans and is not thought to cause seasonal epidemics. Therefore, ICV has been neglected, although it primarily cause infection in humans. However, with the development of molecular biological diagnostic technology such as real-time polymerase chain reaction (RT-PCR) and next-generation sequencing, some clinical epidemiological studies on ICV have been reported [5,6,7]. Recent studies have shown that ICV can cause lower respiratory tract infections in children, especially those under 2 years of age [5,6]. In addition, recent research on ICV has expanded our understanding of the clinical characteristics of ICV, as well as its seasonality and molecular epidemiology. IDV was discovered in swine relatively recently in Oklahoma in 2011 [8] and although it is known to infect other animals such as cattle and goats [9,10,11], it is not known to infect humans. In this article, the epidemiology, characteristics, and zoonotic potential of ICV were reviewed.

EPIDEMIOLOGY

ICV was discovered in 1947 and the strain C/Taylor/1233/1947 was first isolated in the USA [12]. Before the development of molecular diagnostics, egg-based or cell culture-based virus isolation method was the only way to detect ICV. However, because it was difficult to isolate ICV strain, early studies on ICV were mainly serological studies measuring hemagglutinin inhibition (HAI) antibody titers, and clinical characteristics and disease burden were poorly described. Recent epidemiological studies have provided additional insight into the ICV infection using highly diagnostic RT-PCR methods as a means of detecting ICV.

Serological studies on ICV infection have shown that ICV is widespread in the global population and natural infections occur periodically. A serological study of the U.S. population, including children and adults, has shown that the antibodies to ICV increase with age in childhood and remain high in adulthood [13]. In particular, the geometric mean titers of antibody against ICV remained high each year in sera from adults collected for non-respiratory diseases in the summer and fall of 1943-1951: 263 in 1943, 182 in 1944, 129 in 1945, 191 in 1946, 159 in 1947, 162 in 1948, 219 in 1949, 200 in 1950, 204 in 1951. The stability of HAI titers in adults from year to year suggests periodic circulation that maintains antibody levels. These results indicated that ICV has been widespread in the U.S. for many years.

Other studies on the age distribution of ICV antibodies have also shown that seropositivity increases with age, persists throughout adulthood, and declines after the age of 65 years [14,15,16]. The decline in seropositivity among the elderly (≥65 years) is thought to be due to waning immunity. The increase in seropositivity with age in children suggests that the primary exposure to ICV occurs during childhood. In particular, a Japanese study found that seropositivity in infants (<6 months) was mediated by maternal antibodies, that no antibodies to ICV were detected in children from 6 months to 1 year, and that seropositivity increased between 1 and 10 years of age, indicating that ICV infection mainly occurs during this period [16]. Similar results have been reported in various countries [17,18,19,20], and consequently, these data indicate that ICV infection is widespread globally and that most infections occur during childhood. Human challenge studies found that administration of ICV to volunteers resulted in increased antibody levels against ICV, indicating infection; however, most volunteers were asymptomatic or exhibited only common cold symptoms [21,22].

A recent study from Hong Kong reported that 56.5–71.7% of patients diagnosed with ICV were children under 5 years of age, 3.5%–13.0% were aged 6–15 years, 6.2%–14.9% were aged 16–45 years, 4.4%–9.2% were aged 46–65 years, and 5.7%–17.4% were over 65 years of age [23]. Although the rates varied depending on the outbreak period, ICV was most frequently diagnosed in children under 5 years of age. Pabbaraju et al. [24] also showed that 7 (2.32%) out of 474 patients were diagnosed with ICV, and all diagnosed patients were children aged 1 to 10 years (sample size=430), with no cases in individuals aged 10 to 100 years (sample size=44). Nesmith et al. [25] reported that among 4,200 adults who presented with acute respiratory symptoms, only 13 (0.3%) tested positive for ICV on nasal/throat swab samples. Consequently, ICV was rarely associated with healthcare visits in adults. As a result, clinical characterization studies have primarily focused on children.

CLINICAL CHARACTERISTICS

The most well-known symptoms associated with ICV infection are common cold symptoms such as fever, rhinorrhea, and cough, but recent reports have shown that it can also cause lower respiratory diseases such as pneumonia, bronchiolitis, and bronchitis in children. Matsuzaki et al. reported that among 170 patients diagnosed with ICV in children younger than 15 years, 29 were hospitalized, and 21 (72.4%) presented with lower respiratory disease such as pneumonia (n=15, 51.7%), bronchiolitis (n=3, 10.3%), and bronchitis (n=3, 10.3%) [26]. Additionally, this study found that the risk of complications due to lower respiratory disease was particularly high in children under 2 years of age. A Spanish study also observed bronchitis (n=4, 19%) and pneumonia (n=2, 10%) in patients diagnosed with ICV [27]. However, 81% of patients with ICV were co-infected with other pathogens.

Co-infections with other pathogens are common in patients with ICV infection, and children hospitalized with ICV infection often have underlying conditions such as prematurity and asthma [28]. The most common co-infections associated with ICV are rhinovirus/enterovirus and respiratory syncytial virus. Similar to influenza types A and B, fatal cases of ICV infection in adults associated with Staphylococcus aureus have also been reported in the UK [29]. The frequent co-infection with other pathogens in ICV-positive patients complicates the interpretation of the clinical significance and disease burden of ICV. Further studies on the role of ICV in bacteria-virus or virus-virus co-infections are needed to clearly understand the clinical features of ICV.

OUTBREAKS

The seasonality of ICV is unclear, unlike seasonal influenza. Serological studies suggest that ICV circulates periodically [13,16]. Additionally, various studies have shown that ICV is diagnosed or isolated between November and April, when influenza viruses are prevalent [23,28,30,31]. However, ICV infections have also been reported during the summer [32,33], suggested that ICV may exhibit less seasonal variation than IAV and IBV.

Interestingly, surveillance of ICV in Hong Kong between 2014 and 2020 revealed three ICV outbreaks (2015–2016, 2017–2018, 2019–2020) occurring in 2-year cycles [23]. In addition, a Japanese study conducted from 2006 to 2020 similarly confirmed ICV outbreaks occurring in 2-year cycles [34]. Consequently, ICV appears to circulate in a 2-year cycle, with its seasonality considered less evident than other human influenza viruses.

These studies also reported antigenic change and reassortment of ICV and suggested that these factors are involved in the recent outbreak of ICV [23,34]. In particular, antigenic changes in the HEF protein may reduce the binding affinity of antibodies, including neutralizing antibodies, to the viral glycoproteins, thereby affecting immune evasion. Consequently, antigenic changes and reassortment may alter the properties of ICV and influence its prevalence of ICV in the population.

GENETIC DIVERSITY AND EVOLUTION

Based on the HEF gene, ICV is classified into 6 lineages: Taylor lineage, Aichi lineage, Mississippi lineage, Yamagata lineage, Kanagawa lineage, and Sao Paulo lineage (Fig. 2). Recent studies have shown that Kanagawa and Sao Paulo lineages are the most widespread, while the other lineages have not been detected in recent surveillance [23,34,35,36]. The dominant ICV strains in the population may be influenced by environmental pressures as well as genetic factors, including antigenic variation and reassortment.

Fig. 2. Phylogenetic tree for the HEF gene of ICV. A phylogenetic tree was constructed using MEGA software. Bootstrap values (%) are shown above the branches, with values below 60% omitted. Branches are collapsed to highlight the major lineages of the ICV.

HEF, hemagglutinin-esterase fusion; ICV, influenza C virus.

Matsuzaki and colleagues have conducted various studies on antigenic change of HEF protein and evolution of ICV [26,33,34,37]. Matsuzaki et al. identified the antigenic site (A1-A4, B1-B4, and Y1) of ICV using various monoclonal antibodies (mAbs) and confirmed mutations occurring at the antigenic site in virus strains of each antigenic lineage [37]. They also confirmed that most of the epitopes recognized by mAbs capable of neutralizing ICV were located in the receptor binding domain (RBD, 151-310 a.a), and that mutations occurring at RBD in natural isolates lead to differences in the HAI potency of each mAb.

In addition, deletion mutations at position 192 to 195 or position 198 were identified in the escape mutants selected by mAbs. A deletion mutation at position 194 was also identified in viruses belonging to the Mississippi lineage in natural isolates. Consequently, it has been reported that the loop regions (165-172 a.a, 190-195 a.a) in the globular head portion of HEF protein play an important role in antigenic recognition [37]. Zhang et al. [38] identified three sites (176, 194, 198 a.a) located in the loop regions in the head portion of HEF that involved in positive selection by using a comparative approach integrating genetics, molecular selection analysis, and structural biology. These results indicate that mutations in the loop regions of the HEF head region may be associated with escape from the antibody response of host.

However, analysis of antibody reactivity using polyclonal antibodies to C/Ann Arbor/1/50 showed that reactivity was reduced, but not completely lost, for recently circulating strains with substitutions in the RBD compared to strains included in the Taylor lineage [37]. Despite the 68-year period, the low reactivity of the current circulating virus to polyclonal antibodies against C/Ann Arbor/1/50 suggests cross-reactivity within the ICV lineages. The reason for cross-reactivity is probably the low variability of ICV genome.

In an in vitro study, Matsuzaki et al. used mAbs to investigate the frequency of escape mutants resistant to each mAb. They found that the frequency of mutant selection in ICV (10^−4.62 to 10^−7.58 for C/Ann Arbor/1/50, 10^−7.11 to 10^−9.25 for C/Yamagata/15/2004) was lower than that observed for the A(H1N1)pdm virus (10^−2.97 to 10^−4.84) [37]. Moreover, analysis of genetic variation in HEF of ICV collected between 1947 and 2014 revealed that the evolution rate was significantly lower than hemagglutinin gene of IAV and IBV. Therefore, the HEF protein of ICV is generally stable and antigenic change rarely occurs. Additionally, ICV has only HEF glycoprotein, so antigenic shift cannot occur, limiting evolution through antigenic variants. Therefore, reassortment, which can increase genetic diversity by changing the internal genetic composition and acquire genetic compositions favorable for viral propagation and spread, may play an important role in the evolution of ICV.

In particular, the NP gene of ICV mainly has amino acid substitutions in the C-terminal region, and mutations in the nuclear localization signals of the C-terminal region can affect the nuclear import of NP and the formation of viral ribonucleoprotein complexes, ultimately affecting viral replication and fitness [39]. Additionally, the study using Bayesian analysis has reported that ICV gene reassortment plays an important role in the generation of viruses with specific gene arrangements, thereby increasing genetic diversity and playing an important role in evolution [23]. Interestingly, ICV antigenic change and weather conditions did not play a major role in the 2-year cycle of ICV outbreaks, suggesting that the level of herd immunity within the population plays an important role in ICV outbreaks [23]. Consequently, genetic reassortment and antigenic changes contribute to the genetic diversity of ICV, but the evolution of ICV is also influenced by various factors, including environment conditions and herd immunity.

ZOONOTIC POTENTIAL

ICV was first identified in humans in 1947, in swine in 1981, and in cattle in 2016 [12,40,41]. Before the ICV was isolated from swine, only 1 study in animals reported ICV infection in horse by serological analysis [42]. Since ICV was isolated from swine, ICV infection has been studied in domestic animals including swine, dogs, camels, horses, and cattle (Fig. 3). From March 1983 to October 1984, 112 serum specimens were collected from mongrel dogs in Japan and serologically analyzed for ICV using radioimmunoprecipitation, immunoblotting, and HAI methods [43]. Two specimens were positive for all three assays. In addition, HAI analysis using dog serum showed seropositivity of 56.3% (n=101) in Spain, 50.6% (n=150) in Germany, and 32% in France [44,45,46]. These results indicated that ICV causes natural infection in dogs, although the virus has not been isolated.

Fig. 3. Timeline of ICV infection based on virus isolation and serological evidence. Since the first discovery of ICV in humans in 1947, species with confirmed ICV infection are described on the year bar. The species from which the virus was isolated are shown in black. The species shown in white are those for which infection with ICV was confirmed by seroprevalence.

ICV, influenza C virus.

Additionally, seroprevalence data showed susceptibility to ICV and IDV in horses [47] and camels [48]. In 2016, ICV was identified in cattle with bovine respiratory disease in the United States [41]. In RT-PCR results, 64 (4.20%) of 1,525 samples tested positive for ICV, 38 samples had cycle threshold (Ct) <36 and 26 samples had Ct 36–39. The bovine ICV isolates showed high nucleotide identity (95%) with human ICV isolate C/Mississippi/80. The high genetic similarity (over 95%) between human and bovine ICV strains suggests the potential for cross-species transmission, indicating that human ICV can infect cattle and vice versa.

Studies in pigs have also raised concerns about the interspecies transmission of ICV [49,50]. In a study, pigs were experimentally infected with both human and swine ICV strains, confirming pig-to-pig transmission. All viruses were successfully re-isolated from the infected pigs [50]. Additionally, cross-reactivity between human and swine ICV strains was demonstrated through HAI analysis. These findings highlight the need for further studies on the interspecies transmissibility and the zoonotic potential of ICV. However, in animals infected with ICV, most cases present as mild upper respiratory infections, similar to those observed in humans, and the virus is generally considered to have low pathogenicity [46,49,50,51].

CONCLUSION

While numerous studies have reported ICV infections in humans and animals in Japan and China, relatively few investigations have focused on ICV infection in Korea. In Korea, while diagnosis and viral genome sequencing of ICV in humans have been reported, no positive cases of ICV have been detected in swine farms [35,52]. However, ICV is distributed worldwide, and outbreaks primarily occur in humans, although dogs, swine, and cattle can also serve as intermediate hosts and be infected with ICV. ICV infection typically presents as a mild upper respiratory tract infection, however, in some cases, ICV is associated with lower respiratory illnesses, such as pneumonia, bronchitis, and bronchiolitis, particularly in children under the age of 5.

Since its first report in 1947, ICV has been studied far less than other influenza viruses. ICVs have multiple lineages, can undergo reassortment and antigenic changes due to mutations in HEF, and may be capable of interspecies transmission. Therefore, continuous surveillance in humans and animals is needed to understand the epidemiology and viral characteristics of ICV, and molecular biological and immunological studies on the interactions between hosts and viruses, and viruses and other pathogens are needed.