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Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other mammalian hosts. By using traditional hemagglutination assay coupled with sialoglycan microarray (SGM) platform and functional assays, we demonstrated that IDV is more efficient in recognizing both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac 2 ) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) than influenza C virus (ICV), a ubiquitous human pathogen. ICV seems to strongly prefer Neu5,9Ac 2 over Neu5Gc9Ac. Since Neu5Gc9Ac is different from Neu5,9Ac 2 only by an additional oxygen in the group at the C5 position, our results reveal that the hydroxyl group in Neu5Gc9Ac plays a critical role in determining receptor binding specificity, which as a result may discriminate IDV from ICV in communicating with 9-O-acetylated SAs. These findings shall provide a framework for further investigation towards better understanding of how newly discovered multiple-species-infecting IDV exploits natural 9-O-acetylated SA variations to expand its host range., Competing Interests: Declaration of competing interest The authors have read the journal's policy and have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Influenza viruses type A, B, and C are human pathogens that share common structural and functional features, yet they do not form natural reassortants. To determine to what extent type-specific interactions of the polymerase complex with template RNA contribute to this lack of genotypic mixing, we investigated whether homotypic or heterotypic polymerase complexes support the expression and replication of model type A, B, or C RNA templates in vivo. A plasmid-based expression system, as initially described by Pleschka et al. [(1996) J. Virol. 70, 4188-4192] for influenza A virus, was developed for influenza viruses B/Harbin/7/94 and C/Johannesburg/1/66. The type A core proteins expressed heterotypic model RNAs with similar efficiencies as the homotypic RNA. The influenza B virus model RNA was efficiently expressed by all three types of polymerase complexes. Although no functional polymerase complex could be reconstituted with heterotypic P protein subunits, when the influenza A virus P proteins were expressed together with heterotypic nucleoproteins, significant, albeit limited, expression of RNA templates of all influenza virus types was detected. Taken together, our results suggest that less strict type-specific interactions are involved for the polymerase complex of influenza A compared with influenza B or C viruses., (Copyright 1999 Academic Press.)

The open reading frame (ORF) and the regulated synthesis of the influenza C viral NS1 protein were analyzed in view of viruses possessing different biological activities. We provide evidence for a 246-amino-acid NS1-ORF, encoded by five viral strains and variants. Prokaryotic expression of the prototype NS1-ORF resulted in a product of 27 kDa, confirming the predicted molecular weight. Using an antiserum raised against recombinant NS1 protein, nonstructural proteins of wild-type virus were detected in infected cells for a limited course of time, whereas a persistent virus variant was characterized by a long-term nonstructural gene expression. As examined by infection experiments, the intracellular distribution of nonstructural protein was nuclear and cytoplasmic, whereas in NS1 gene-transfected cells, the cytoplasmic localization occurred in a fine-grained structure, suggesting an analogy to influenza A viral NS1 protein. Concerning persistent infection, NS1 protein species differing in sizes and posttranslational modifications were observed for a persistent virus variant, as particularly illustrated by a high degree of NS1 phosphorylation. Virus reassortant analyses proved the importance of the NS-coding genomic segment: the minimal viral properties required for the establishment of persistence were transferred with this segment to a monoreassortant virus. Thus the influenza C viral NS1 protein is a 246-amino-acid nuclear-cytoplasmic phosphoprotein that can be subject to specific variations being functionally linked to a persistent virus phenotype., (Copyright 1999 Academic Press.)

The antigenic and genetic relationships among 15 strains of influenza C virus isolated between August, 1988, and May, 1990, in Yamagata City, Japan, were investigated. Based on the results of antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoproteins and oligonucleotide mapping of total vRNA, the isolates were divided into two distinct groups closely related to either C/Yamagata/26/81 or C/Aichi/1/81. Antigenic differences between two groups could be detected clearly with heterogeneous antiviral sera. Comparison of the HE gene sequences between the representative Yamagata isolates and the previous isolates from other countries showed that C/Yamagata/26/81-like and C/Aichi/1/81-like viruses had high degrees of nucleotide sequence homology with C/pig/Beijing/115/81 and C/Johannesburg/1/66, respectively. These observations suggest that two lineages of influenza C virus, markedly different from each other in both antigenic and genetic structures, were simultaneously present in Yamagata City during 1988-1990 and that viruses belonging to each of the two lineages may also be prevalent in other areas of Japan and even in other countries.

We observed previously that cord-like structures which had lengths up to 500 microns or greater were protruding from the surface of HMV-II cells infected with influenza C/Yamagata/1/88 virus (Nishimura et al., 1990, Virology 179, 179-188). Comparison of the cord-forming ability among a number of influenza C isolates revealed that the C/Taylor/1233/47 strain was unique in lacking the ability. It was also found that the major structural proteins, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix (M), could each be distinguished between C/Yamagata/1/88 and C/Taylor/1233/47 viruses by SDS-polyacrylamide gel electrophoresis. To determine the genes involved in the cord formation, a series of reassortant viruses were prepared between C/Yamagata/1/88 and C/Taylor/1233/47, and parental derivation of genes coding for HE, NP, and M was determined by gel electrophoresis. All reassortants which derived M gene from C/Yamagata/1/88, irrespective of derivation of genes coding for HE and NP, had the ability to generate cords, whereas none of reassortants which derived M gene from C/Taylor/1233/47 were capable of producing cords. With respect to several representative reassortants, the origins of genes encoding the polymerase proteins (PB2, PB1, P3) and the nonstructural proteins (NS1, NS2) were determined by T1-oligonucleotide fingerprinting of the isolated RNA segments. The results suggested that none of genes coding for these proteins are exclusively associated with the cord formation. Thus, it is likely that the M gene is the key determinant of the cord-forming ability of influenza C viruses. Nucleotide sequence analysis of the M genes revealed that compared to C/Yamagata/1/88, C/Taylor/1233/47 had two amino acid substitutions in the M molecule at positions 24 (Ala-->Thr) and 133 (Asp-->Asn).

Neutralization-resistant variants of influenza C/Ann Arbor/1/50 virus were selected with monoclonal antibodies against four different antigenic sites on the hemagglutinin-esterase (HE) glycoprotein, and their HE genes were sequenced to identify amino acid residues important for the integrity of each site. Twelve different amino acid substitutions in a total of 18 antigenic variants were all located on the HE1 subunit. Although variants for antigenic site A-2 had a change at position 367, all substitutions in the variants for sites A-1, A-3, and A-4 occurred in the central region of the HE1 spanning amino acid positions 178 to 283. Furthermore, it was found that many of the substitutions in the variants selected with antibodies to sites A-1 and A-3 were clustered within or near one of the three variable regions revealed previously by comparing amino acid sequences of the HEs among various influenza C isolates (Buonagurio, D. A., Nakada, S., Fitch, W. M., and Palese, P., Virology 146, 221-232, 1985). The antigenic variants were also examined for their ability to agglutinate chicken and human erythrocytes in order to obtain information concerning the receptor-binding site on the HE molecule. The results suggested that the amino acid changes at residues 178, 186, 187, 190, 206, 212, and 226 decreased the hemagglutinating activity whereas those at residues 245, 266, and 283 produced an opposite effect.

From strain JHB/1/66 of influenza C virus a mutant was derived with a change in the cell tropism. The mutant was able to grow in a subline of Madin-Darby canine kidney cells (MDCK II) which is resistant to infection by the parent virus due to a lack of receptors. Inactivation of cellular receptors by either neuraminidase or acetylesterase and generation of receptors by resialylation of cells with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicated that 9-O-acetylated sialic acid is a receptor determinant for both parent and mutant virus. However, the mutant required less Neu5,9Ac2 on the cell surface for virus attachment than the parent virus. The increased binding efficiency enabled the mutant to infect cells with a low content of 9-O-acetylated sialic acid which were resistant to the parent virus. By comparing the nucleotide sequences of the glycoprotein (HEF) genes of the parent and the mutant virus only a single point mutation could be identified on the mutant gene. This mutation at nucleotide position 872 causes an amino acid exchange from threonine to isoleucine at position 284 on the amino acid sequence. Sequence similarity with a stretch of amino acids involved in the receptor-binding pocket of the influenza A hemagglutinin suggests that the mutation site on the influenza C glycoprotein (HEF) is part of the receptor-binding site.

The complete nucleotide sequences of the hemagglutinin/esterase (HE) genes of human coronavirus (HCV) strain OC43 and bovine respiratory coronavirus (BRCV) strain G95 were determined from single-stranded cDNA fragments generated by reverse transcription of virus-specific mRNAs and amplified by polymerase chain reaction. An open reading frame of 1272 nucleotides was identified as the putative HE gene by homology to the bovine coronavirus HE gene. This open reading frame encodes a protein of 424 amino acids with an estimated molecular weight of 47.7 kDa. Ten potential N-linked glycosylation sites were predicted in the HE protein of HCV-OC43 while nine of them were present in BRCV-G95. Fourteen cysteine residues were conserved in the HE proteins of both viruses. Two hydrophobic sequences at the N-terminus and the C-terminus may serve as signal peptide and transmembrane anchoring domain, respectively. The predicted HE protein of HCV-OC43 was 95% identical to the HEs of BRCV-G95 and other bovine coronaviruses, and 60% identical to the HEs of mouse hepatitis viruses. Phylogenetic analysis suggests that the HE genes of coronaviruses and influenza C virus have a common ancestral origin, and that bovine coronaviruses and HCV-OC43 are closely related.

When HMV-II cells (a human malignant melanoma cell line) infected with a newly isolated influenza C strain (Yamagata/1/88) were examined by simple light microscopy, it was found that a large number of cord-like structures which had lengths up to about 500 microns or greater were emerging from the cell surface. The existence of viral glycoproteins (hemagglutinin-esterase, HE) on the surface of these huge structures was confirmed by hemadsorption experiments with erythrocytes from a variety of species as well as by immunofluorescent staining with anti-HE monoclonal antibody. Furthermore, electron microscopy revealed that numerous filamentous particles in the process of budding, each covered with a layer of surface projections approximately 13 nm in length, aggregated with their long axes parallel to form a cord-like structure visible under a light microscope. An electron-dense layer, which presumably consists of membrane protein (M), was seen in cross-sections of all filamentous virions whereas internal nucleocapsids were rarely seen. SDS-polyacrylamide gel electrophoresis of the purified cords also showed that they contained HE and M polypeptides but not nucleoprotein, confirming that long filamentous particles are mostly devoid of nucleocapsids. The emergence of cords on the cell surface was observed in various cell cultures infected with C/Yamagata/1/88 though their number and length varied markedly depending on cell type. The production of cord-like structures was also evident in HMV-II cells infected with any of several different influenza C strains, which suggests that the cord formation is a common feature of influenza C virus group.

Sequence analysis and comparison of hemagglutinin (HA) genes of different influenza C viruses isolated between 1947 and 1983 reveals that (1) the extent of difference among the HA genes is independent of the year in which these viruses were isolated and that (2) changes in the HA genes do not appear to accumulate with time. These results suggest that epidemiologically dominant variants of influenza C viruses do not emerge successively with time and that C virus variants derived from multiple evolutionary pathways cocirculate at any one time. Thus the epidemiology of influenza C viruses differs markedly from that of influenza A viruses, which is characterized by the emergence of successive variants. Based on the nucleotide sequence data, we propose different evolutionary models for influenza A and influenza C viruses.

The nucleotide sequences of nonstructural protein (NS) genes of human influenza C viruses isolated between 1947 and 1983 were determined and compared. Assuming constant evolutionary rates, the extent of nucleotide differences among NS genes does not correspond to the isolation years of the strains. This suggests that more than one gene lineage is present in the population. Furthermore, examination of the eight C virus NS gene sequences by the maximum parsimony method (W. M. Fitch, 1971, Syst. Zool. 20, 406-416) yielded phylogenetic trees that were grossly different from those obtained using the hemagglutinin genes for the same eight isolates. This result is compatible with the idea of reassortment of genes in nature across lineages of influenza C viruses. The sequence analysis also suggests that nucleotide substitutions occur at a lower rate in the C virus NS genes than in influenza A virus NS genes.

The three large RNA segments of influenza C virus C/JJ/50 were cloned and sequenced, and the deduced amino acid sequences were compared with those of the polymerase (P) proteins of influenza A and B viruses. The coding strategy of the C virus RNA segments is the same as that for the large A and B virus segments as one long open reading frame is present in each segment. RNA segment 1 of influenza C virus encodes the equivalent of the PB2 protein; it has an approximate 25% sequence identity with the corresponding (cap binding) influenza A and B virus PB2 proteins. The PB1 protein of influenza C virus, coded for by segment 2, has an approximate 40% sequence identity with the corresponding proteins of influenza A and B viruses including the Asp-Asp sequence motif found in many RNA polymerase molecules. The PB1 polymerase is thus the most highly conserved protein among the influenza A, B, and C viruses. Although the protein coded for by RNA 3 of influenza C virus shows an approximate 25% sequence identity with the acid polymerase (PA) proteins of the A and B viruses, its sequence does not display any acid charge features at neutral pH. This protein is thus referred to as the P3 (rather than the PA) protein of influenza C virus.

The nucleotide sequence of the unique region of coronavirus MHV-A59 mRNA 2 has been determined. Two open reading frames (ORF) are predicted: ORF1 potentially encodes a protein of 261 amino acids; its amino acid sequence contains elements which indicate nucleotide binding properties. ORF2 predicts a 413 amino acids protein; it lacks a translation initiation codon and is therefore probably a pseudogene. The amino acid sequence of ORF2 shares 30% homology with the HA1 hemagglutinin sequence of influenza C virus. A short stretch of nucleotides immediately upstream of ORF2 shares 83% homology with the MHC class I nucleotide sequences. We discuss the possibility that both similarities are the result of recombinations and present a model for the acquisition and the subsequent inactivation of ORF2; the model applies also to MHV-A59-related coronaviruses in which we expect ORF2 to be still functional.

A cDNA copy of RNA segment 4 of influenza C/Cal/78 virus was cloned into an SV40 vector and expressed in CV-1 cells. The gene product expressed from the SV40 recombinant virus was immunoprecipitated by monoclonal antibodies directed against the influenza C virus glycoprotein. Cells infected with the recombinant virus also exhibited C virus-specific hemagglutinin and O-acetylesterase activity. This suggests that the same C virus protein is associated with receptor-binding as well as receptor-destroying activity. The latter viral activity was measured using as substrates bovine submaxillary mucin or a low molecular weight compound p-nitrophenylacetate. In analogy to the parainfluenza virus HN protein, the influenza C virus glycoprotein was termed HE, because it possesses hemagglutinin and esterase (receptor-destroying) activity.

Sequence analyses and comparison of the genes coding for the nonstructural (NS) and hemagglutinin (HA) proteins of different influenza B viruses isolated between 1940 and 1987 reveal that the number of substitutions is not always proportional to the time between isolates. Examination of 14 influenza B virus NS gene and 10 HA gene sequences by the maximum parsimony method suggested that--as with influenza C viruses--there are multiple evolutionary lineages which can coexist for considerable periods of time. Comparison of the sequence divergence among genes of viruses belonging to type A, B, and C virus suggests that, in man, influenza B viruses evolve slower than A viruses and faster than C viruses. We propose an evolutionary model for influenza B viruses that is intermediate between the pattern for human influenza A viruses and that for influenza C viruses.

Three strains of influenza C virus (C/Kyoto/41/82, C/Nara/82, C/Hyogo/1/83) have been isolated from humans in the Kinki district of Japan between February 1982 and December 1983. Oligonucleotide mapping of total vRNA and antigenic analysis with anti-HE monoclonal antibodies have shown previously that they are closely similar to one another but dissimilar to any of the strains isolated in Japan before 1982. In the present study, the nucleotide sequences of the hemagglutinin-esterase (HE) genes of these three strains were determined and compared with those of the previously published strains. The results revealed that the Kinki isolates had a high nucleotide sequence homology (98.4-98.5%) with the virus isolated in 1980 in the United States (C/Mississippi/1/80). Similarity of the Kinki strains to C/Mississippi/1/80 was also confirmed by oligonucleotide mapping of total vRNA and antigenic comparison using a panel of 11 anti-HE monoclonal antibodies. The isolates from Kinki and Mississippi could be distinguished from the previously isolated strains in serological tests with heterogeneous sera, suggesting that they may have possessed epidemiological advantage in Japan around 1982-1983. These observations raise the possibility that the rapid spread of influenza C variant closely related to C/Mississippi/1/80 has occurred in Kinki in 1982-1983 presumably because this imported virus was largely different in antigenicity from the previously prevalent ones.