Near-Complete Genome Sequencing of Influenza C Virus in the Philippines between 2014 and 2019

We report 19 nearly complete genome sequences of influenza C virus isolated from clinical samples recovered from children in the Philippines between 2014 and 2019.

Temporal and gene reassortment analysis of influenza C outbreaks in Hong Kong SAR, China

From 2014 to week 07/2020 the Centre for Health Protection in Hong Kong conducted screening for influenza C virus (ICV). A retrospective analysis of ICV detections to week 26/2019 revealed persistent low-level circulation with outbreaks occurring biennially in the winters of 2015-16 and 2017-18 (1). Here we report on an outbreak occurring in 2019-20, reinforcing the observation of biennial seasonality in Hong Kong. All three outbreaks occurred in similar time-frames, were subsequently dwarfed by seasonal epidemics of influenza types A and B, and were caused by similar proportions of C/Kanagawa/1/76 (K-)lineage and C/São Paulo/378/82 S1- and S2-sublineage viruses. Ongoing genetic drift was observed in all genes, with some evidence of amino acid substitution in the HEF glycoprotein possibly associated with antigenic drift. A total of 61 ICV genomes covering the three outbreaks were analysed for reassortment and nine different reassortant constellations were identified, one K-lineage, four S1-sublineage and four S2-sublineage, with six of these being identified first in the 2019-20 outbreak (two S2-lineage and four S1-lineage). The roles that virus interference/enhancement, ICV persistent infection, genome evolution and reassortment might play in the observed seasonality of ICV in Hong Kong are discussed. Importance Influenza C virus (ICV) infection of humans is common with the great majority of people being infected during childhood, though re-infection can occur throughout life. While infection normally results in ‘cold-like’ symptoms, severe disease cases have been reported in recent years. However, knowledge of ICV is limited due to poor systematic surveillance and an inability to propagate the virus in large amounts in the laboratory. Following recent systematic surveillance in Hong Kong SAR, China and direct ICV gene sequencing from clinical specimens a two-year cycle of disease outbreaks (epidemics) has been identified with gene mixing playing a significant role in ICV evolution. Studies like those reported here are key to developing an understanding of the impact of influenza C virus infection in humans, notably where comorbidities exist and severe respiratory disease can develop.

TMPRSS2 activates hemagglutinin-esterase glycoprotein of influenza C virus

Influenza C virus (ICV) has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein. HE functions similarly to hemagglutinin (HA) and neuraminidase of the influenza A and B viruses (IAV/IBV). It has a monobasic site, which is cleaved by some host enzyme(s). The cleavage is essential to activating the virus, but the enzyme(s) in the respiratory tract has not been identified. This study investigated whether the host serine proteases, transmembrane protease serine S1, members 2 (TMPRSS2), and human airway trypsin-like protease (HAT), which reportedly cleave HA of IAV/IBV, are involved in HE cleavage. We established TMPRSS2- and HAT-expressing MDCK (MDCK-TMPRSS2, MDCK-HAT) cells. ICV showed multicycle replication with HE cleavage without trypsin in MDCK-TMPRSS2 cells as well as IAV did. The HE cleavage and multicycle replication did not appear in MDCK-HAT cells infected with ICV without trypsin, while HA cleavage and multi-step growth of IAV appeared in the cells. Amino acid sequences of the HE cleavage site in 352 ICV strains were completely preserved. Camostat and nafamostat suppressed the growth of ICV and IAV in human nasal surface epithelial (HNE) cells. Therefore, this study revealed that, at least, TMPRSS2 is involved in HE cleavage and suggested that nafamostat could be a candidate for therapeutic drugs of ICV infection. Importance Influenza C virus (ICV) is a pathogen that causes acute respiratory illness, mostly in children, but there are no anti-ICV drugs. ICV has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein on the virion surface, that possesses receptor binding, receptor destroying, and membrane fusion activities. The HE cleavage is essential for the virus to be activated, but the enzyme(s) in the respiratory tract has not been identified. This study revealed that transmembrane protease serine S1, members 2 (TMPRSS2), and not human airway trypsin-like protease (HAT), is involved in HE cleavage. This is a novel study on the host enzymes involved in HE cleavage, and the result suggests that the host enzymes, such as TMPRSS2, may be a target for therapeutic drugs of the ICV infection.

Prevalence of BRD-Related Viral Pathogens in the Upper Respiratory Tract of Swiss Veal Calves

The prevention of bovine respiratory disease is important, as it may lead to impaired welfare, economic losses, and considerable antimicrobial use, which can be associated with antimicrobial resistance. The aim of this study was to describe the prevalence of respiratory viruses and to identify risk factors for their occurrence. A convenience sample of 764 deep nasopharyngeal swab samples from veal calves was screened by PCR for bovine respiratory syncytial virus (BRSV), bovine parainfluenza-3 virus (BPI3V), bovine coronavirus (BCoV), influenza D virus (IDV), and influenza C virus (ICV). The following prevalence rates were observed: BRSV, 2.1%; BPI3V, 3.3%; BCoV, 53.5%; IDV, 4.1%; ICV, 0%. Logistic mixed regression models were built for BCoV to explore associations with calf management and housing. Positive swab samples were more frequent in younger calves than older calves (>100 days; p < 0.001). The probability of detecting BCoV increased with increasing group size in young calves. Findings from this study suggested that young calves should be fattened in small groups to limit the risk of occurrence of BCoV, although an extended spectrum of risk factors for viral associated respiratory disorders such as nutritional aspects should be considered in future studies.

A mouse monoclonal antibody against influenza C virus attenuates acetaminophen-induced liver injury in mice

Molecular mimicry is one of the main processes for producing autoantibodies during infections. Although some autoantibodies are associated with autoimmune diseases, the functions of many autoantibodies remain unknown. Previously, we reported that S16, a mouse (BALB/c) monoclonal antibody against the hemagglutinin-esterase fusion glycoprotein of influenza C virus, recognizes host proteins in some species of animals, but we could not succeed in identifying the proteins. In the present study, we found that S16 cross-reacted with acetyl-CoA acyltransferase 2 (ACAA2), which is expressed in the livers of BALB/c mice. ACAA2 was released into the serum after acetaminophen (APAP) administration, and its serum level correlated with serum alanine aminotransferase (ALT) activity. Furthermore, we observed that S16 injected into mice with APAP-induced hepatic injury prompted the formation of an immune complex between S16 and ACAA2 in the serum. The levels of serum ALT (p < 0.01) and necrotic areas in the liver (p < 0.01) were reduced in the S16-injected mice. These results suggest that S16 may have a mitigation function in response to APAP-induced hepatotoxicity. This study shows the therapeutic function of an autoantibody and suggests that an antibody against extracellular ACAA2 might be a candidate for treating APAP-induced hepatic injury.

Peeping into the Emerging Threat of Novel Influenza D Virus: A Review

In 2011, a new virus was isolated from swine with influenza-like symptoms in Oklahoma, USA. Later in 2013, it was also evidenced in cattle, considered as its natural reservoir, elsewhere in the USA. This new virus has certain resemblance with Influenza C virus (ICV), predominately a human pathogen. Due to its inability to re-assort with ICV, it is now classified as Influenza D virus (IDV) and is a new candidate in the Orthomyxoviridae family. It causes mild respiratory disease in several animal species and replicates in both upper and lower respiratory tract. To date, serological evidence was demonstrated in various animal species and humans in all continents except Australia. It is transmitted through direct contact or through aerosol routes. Not much is known about its potential impact to animal and human health but it poses a potential risk as an emerging threat to cattle-workers. Currently, limited data is available on its global occurrence and distribution. Therefore, in current review, we summarize the global available data regarding epidemiology, pathology, zoonotic potential and future perspectives of this virus. In conclusion, continuous surveillance and risk assessment of this emerging virus is required.

In situ structure and organization of the influenza C virus surface glycoprotein

The lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF’s membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

Growth Kinetics of Influenza C Virus Antigenic Mutants that Escaped from Anti-Hemagglutinin Esterase Monoclonal Antibodies and Viral Antigenic Changes Found in Field Isolates

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability.

New Diagnostic Assays for Differential Diagnosis Between the Two Distinct Lineages of Bovine Influenza D Viruses and Human Influenza C Viruses

Influenza D virus (IDV), a novel orthomyxovirus, is currently emerging in cattle worldwide. It shares &gt;50% sequence similarity with the human influenza C virus (HICV). Two clades of IDV are currently co-circulating in cattle herds in the U.S. New assays specific for each lineage are needed for accurate surveillance. Also, differential diagnosis between zoonotic human influenza C virus and the two clades of IDV are important to assess the zoonotic potential of IDV. We developed an enzyme-linked immunosorbent assay (ELISA) based on two different epitopes HEF and NP and four peptides, and fluorescent focus neutralization assay to differentiate between IDV bovine and swine clades. Calf sera were obtained, and bovine samples underwent surveillance. Our results highlight the importance of position 215 with 212 in determining the heterogeneity between the two lineages. We needed IFA and FFN for tissue culture–based analysis and a BSL2 facility for analyzing virus interactions. Unfortunately, these are not available in many veterinary centers. Hence, our second aim was to develop an iELISA using specific epitopes to detect two lineages of IDVs simultaneously. Epitope-iELISA accurately detects neutralizing and non-neutralizing antibodies against the IDV in non-BSL2 laboratories and veterinary clinics and is cost-effective and sensitive. To differentiate between IDVs and HICVs, whole antigen blocking, polypeptides, and single-peptide ELISAs were developed. A panel of ferret sera against both viruses was used. Results suggested that both IDV and ICV had a common ancestor, and IDV poses a zoonotic risk to individuals with prior or current exposure to cattle. IDV peptides IANAGVK (286–292 aa), KTDSGR (423–428 aa), and RTLTPAT (448–455 aa) could differentiate between the two viruses, whereas peptide AESSVNPGAKPQV (203–215 aa) detected the presence of IDV in human sera but could not deny that it could be ICV, because the only two conserved influenza C peptides shared 52% sequence similarity with IDV and cross-reacted with IDV. However, blocking ELISAs differentiated between the two viruses. Diagnostic tools and assays to differentiate between ICV and IDV are required for serological and epidemiological analysis to clarify the complexity and evolution and eliminate misdiagnosis between ICV and IDV in human samples.