Host Range, Biology, and Species Specificity of Seven-Segmented Influenza Viruses—A Comparative Review on Influenza C and D

Other than genome structure, influenza C (ICV), and D (IDV) viruses with seven-segmented genomes are biologically different from the eight-segmented influenza A (IAV), and B (IBV) viruses concerning the presence of hemagglutinin–esterase fusion protein, which combines the function of hemagglutinin and neuraminidase responsible for receptor-binding, fusion, and receptor-destroying enzymatic activities, respectively. Whereas ICV with humans as primary hosts emerged nearly 74 years ago, IDV, a distant relative of ICV, was isolated in 2011, with bovines as the primary host. Despite its initial emergence in swine, IDV has turned out to be a transboundary bovine pathogen and a broader host range, similar to influenza A viruses (IAV). The receptor specificities of ICV and IDV determine the host range and the species specificity. The recent findings of the presence of the IDV genome in the human respiratory sample, and high traffic human environments indicate its public health significance. Conversely, the presence of ICV in pigs and cattle also raises the possibility of gene segment interactions/virus reassortment between ICV and IDV where these viruses co-exist. This review is a holistic approach to discuss the ecology of seven-segmented influenza viruses by focusing on what is known so far on the host range, seroepidemiology, biology, receptor, phylodynamics, species specificity, and cross-species transmission of the ICV and IDV

Gene-Specific Substitution Profiles Describe the Types and Frequencies of Amino Acid Changes during Antibody Somatic Hypermutation

Somatic hypermutation (SHM) plays a critical role in the maturation of antibodies, optimizing recognition initiated by recombination of V(D)J genes. Previous studies have shown that the propensity to mutate is modulated by the context of surrounding nucleotides and that SHM machinery generates biased substitutions. To investigate the intrinsic mutation frequency and substitution bias of SHMs at the amino acid level, we analyzed functional human antibody repertoires and developed mGSSP (method for gene-specific substitution profile), a method to construct amino acid substitution profiles from next-generation sequencing-determined B cell transcripts. We demonstrated that these gene-specific substitution profiles (GSSPs) are unique to each V gene and highly consistent between donors. We also showed that the GSSPs constructed from functional antibody repertoires are highly similar to those constructed from antibody sequences amplified from non-productively rearranged passenger alleles, which do not undergo functional selection. This suggests the types and frequencies, or mutational space, of a majority of amino acid changes sampled by the SHM machinery to be well captured by GSSPs. We further observed the rates of mutational exchange between some amino acids to be both asymmetric and context dependent and to correlate weakly with their biochemical properties. GSSPs provide an improved, position-dependent alternative to standard substitution matrices, and can be utilized to developing software for accurately modeling the SHM process. GSSPs can also be used for predicting the amino acid mutational space available for antigen-driven selection and for understanding factors modulating the maturation pathways of antibody lineages in a gene-specific context. The mGSSP method can be used to build, compare, and plot GSSPs1; we report the GSSPs constructed for 69 common human V genes (DOI: 10.6084/m9.figshare.3511083) and provide high-resolution logo plots for each (DOI: 10.6084/m9.figshare.3511085)

Migration of the Swine Influenza Virus -δ Cluster Hemagglutinin NLinked Glycosylation Site from N142 to N144 Results in Loss of Antibody Cross-Reactivity

Routine antigenic characterization of swine influenza virus isolates in a high-throughput serum neutralization (HTSN) assay found that approximately 20% of isolates were not neutralized by a panel of reference antisera. Genetic analysis revealed that nearly all of the neutralization-resistant isolates possessed a seasonal human-lineage hemagglutinin (HA; δ cluster). Subsequent sequencing analysis of full-length HA identified a conserved N144 residue present only in neutralization-resistant strains. N144 lies in a predicted N-linked glycosylation consensus sequence, i.e., N-X-S/T (where X is any amino acid except for proline). Interestingly, neutralization-sensitive viruses all had predicted N-linked glycosylation sites at N137 or N142, with threonine (T) occupying position 144 of HA. Consistent with the HTSN assay, hemagglutination inhibition (HI) and serum neutralization (SN) assays demonstrated that migration of the potential N-linked glycosylation site from N137 or N142 to N144 resulted in a \u3e8-fold decrease in titers. These results were further confirmed in a reverse genetics system where syngeneic viruses varying only by predicted N-glycosylation sites at either N142 or N144 exhibited distinct antigenic characteristics like those observed in field isolates. Molecular modeling of the hemagglutinin protein containing N142 or N144 in complex with a neutralizing antibody suggested that N144-induced potential glycosylation may sterically hinder access of antibodies to the hemagglutinin head domain, allowing viruses to escape neutralization. Since N-linked glycosylation at these sites has been implicated in genetic and antigenic evolution of human influenza A viruses, we conclude that the relocation of the hemagglutinin N-linked glycosylation site from N142 to N144 renders swine influenza virus δ-cluster viruses resistant to antibody-mediated neutralization

Isolation of a Novel Swine Influenza Virus from Oklahoma in 2011 Which Is Distantly Related to Human Influenza C Viruses

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has nonhuman maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/ OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution

Characterization of a Novel Influenza Virus in Cattle and Swine: Proposal for a New Genus in the Orthomyxoviridae Family

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

Influenza C and D Viruses Demonstrated a Differential Respiratory Tissue Tropism in a Comparative Pathogenesis Study in Guinea Pigs

Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses

Targeted Isolation of Antibodies Directed against Major Sites of SIV Env Vulnerability

The simian immunodeficiency virus (SIV) challenge model of lentiviral infection is often used as a model to human immunodeficiency virus type 1 (HIV-1) for studying vaccine mediated and immune correlates of protection. However, knowledge of the structure of the SIV envelope (Env) glycoprotein is limited, as is knowledge of binding specificity, function and potential efficacy of SIV antibody responses. In this study we describe the use of a competitive probe binding sort strategy as well as scaffolded probes for targeted isolation of SIV Env-specific monoclonal antibodies (mAbs). We isolated nearly 70 SIV-specific mAbs directed against major sites of SIV Env vulnerability analogous to broadly neutralizing antibody (bnAb) targets of HIV-1, namely, the CD4 binding site (CD4bs), CD4-induced (CD4i)-site, peptide epitopes in variable loops 1, 2 and 3 (V1, V2, V3) and potentially glycan targets of SIV Env. The range of SIV mAbs isolated includes those exhibiting varying degrees of neutralization breadth and potency as well as others that demonstrated binding but not neutralization. Several SIV mAbs displayed broad and potent neutralization of a diverse panel of 20 SIV viral isolates with some also neutralizing HIV-27312A. This extensive panel of SIV mAbs will facilitate more effective use of the SIV non-human primate (NHP) model for understanding the variables in development of a HIV vaccine or immunotherapy

Structural survey of HIV-1-neutralizing antibodies targeting Env trimer delineates epitope categories and suggests vaccine templates

HIV-1 broadly neutralizing antibodies are desired for their therapeutic potential and as templates for vaccine design. Such antibodies target the HIV-1-envelope (Env) trimer, which is shielded from immune recognition by extraordinary glycosylation and sequence variability. Recognition by broadly neutralizing antibodies thus provides insight into how antibody can bypass these immune-evasion mechanisms. Remarkably, antibodies neutralizing >25% of HIV-1 strains have now been identified that recognize all major exposed surfaces of the prefusion-closed Env trimer. Here we analyzed all 206 broadly neutralizing antibody-HIV-1 Env complexes in the PDB with resolution suitable to define their interaction chemistries. These segregated into 20 antibody classes based on ontogeny and recognition, and into 6 epitope categories (V1V2, glycan-V3, CD4-binding site, silent face center, fusion peptide, and subunit interface) based on recognized Env residues. We measured antibody neutralization on a 208-isolate panel and analyzed features of paratope and B cell ontogeny. The number of protruding loops, CDR H3 length, and level of somatic hypermutation for broadly HIV-1 neutralizing antibodies were significantly higher than for a comparison set of non-HIV-1 antibodies. For epitope, the number of independent sequence segments was higher (P < 0.0001), as well as the glycan component surface area (P = 0.0005). Based on B cell ontogeny, paratope, and breadth, the CD4-binding site antibody IOMA appeared to be a promising candidate for lineage-based vaccine design. In terms of epitope-based vaccine design, antibody VRC34.01 had few epitope segments, low epitope-glycan content, and high epitope-conformational variability, which may explain why VRC34.01-based design is yielding promising vaccine results

Structural survey of HIV-1-neutralizing antibodies targeting Env trimer delineates epitope categories and suggests vaccine templates

HIV-1 broadly neutralizing antibodies are desired for their therapeutic potential and as templates for vaccine design. Such antibodies target the HIV-1-envelope (Env) trimer, which is shielded from immune recognition by extraordinary glycosylation and sequence variability. Recognition by broadly neutralizing antibodies thus provides insight into how antibody can bypass these immune-evasion mechanisms. Remarkably, antibodies neutralizing >25% of HIV-1 strains have now been identified that recognize all major exposed surfaces of the prefusion-closed Env trimer. Here we analyzed all 206 broadly neutralizing antibody-HIV-1 Env complexes in the PDB with resolution suitable to define their interaction chemistries. These segregated into 20 antibody classes based on ontogeny and recognition, and into 6 epitope categories (V1V2, glycan-V3, CD4-binding site, silent face center, fusion peptide, and subunit interface) based on recognized Env residues. We measured antibody neutralization on a 208-isolate panel and analyzed features of paratope and B cell ontogeny. The number of protruding loops, CDR H3 length, and level of somatic hypermutation for broadly HIV-1 neutralizing antibodies were significantly higher than for a comparison set of non-HIV-1 antibodies. For epitope, the number of independent sequence segments was higher (P < 0.0001), as well as the glycan component surface area (P = 0.0005). Based on B cell ontogeny, paratope, and breadth, the CD4-binding site antibody IOMA appeared to be a promising candidate for lineage-based vaccine design. In terms of epitope-based vaccine design, antibody VRC34.01 had few epitope segments, low epitope-glycan content, and high epitope-conformational variability, which may explain why VRC34.01-based design is yielding promising vaccine results

GSSP (Gene-specific substitution profiles)

Somatic hypermutation (SHM) plays a critical role in the maturation of antibodies, optimizing recognition initiated by recombination of V(D)J genes. Previous studies have shown that the propensity to mutate is modulated by the context of surrounding nucleotides and that SHM machinery generates biased substitutions. To investigate the mutation frequency and substitution bias of SHMs, context-dependent substitution models have been developed at the nucleotide level. However, no context-dependent model has been developed to systematically characterize the intrinsic mutation frequency and substitution bias of SHMs at the amino acid level. In this study, we analyzed functional human antibody repertoires and developed mGSSP (method for Gene-Specific Substitution Profile), a method to construct amino acid substitution profiles from next-generation sequencing-acquired B cell transcripts. These gene-specific substitution profiles (GSSP) are unique to each V gene and highly consistent between donors. By implicitly incorporating context-dependence, GSSPs provide an alternative means for accurate modeling of the SHM process, and provide a means for characterizing features of SHM mutational space, predicting mutational propensities of SHMs, and for better understanding maturation pathways of antibody lineages in a gene-specific context. To date, extensive efforts have been invested to re-elicit naturally occurring broadly neutralizing antibodies against HIV-1 and other pathogens in humans and animal models. Incorporating GSSPs into the modeling of the SHM process may help predict the likelihood of re-eliciting key SHM patterns of interest. This dataset includes GSSPs for VH, VK, and VL genes with at least 300 lineages present in the pooled sequencing data from three normal donors