Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

The Sialyl Lewis X Glycan Receptor Facilitates Infection of Subtype H7 Avian Influenza A Viruses.

Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLeX]) in chickens and five common dabbling duck species and the association between SLeX and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLeX expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLeX facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.

Intrauterine Fetal Demise in the Third Trimester of Pregnancy Associated With Mild Infection With the SARS-CoV-2 Delta Variant Without Protection From Vaccination.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a higher infection rate in pregnant women than age-matched adults. With increased infectivity and transmissibility, the Delta variant is predominant worldwide. METHODS: In this study, we describe intrauterine fetal demise in unvaccinated women with mild symptoms of SARS-CoV-2 Delta variant infection. RESULTS: Histology and elevated proinflammatory responses of the placenta suggest that fetal demise was associated with placental malperfusion due to Delta variant infection. CONCLUSIONS: This study suggests that the Delta variant can cause severe morbidity and mortality to fetuses. Vaccination should continue to be advocated and will likely continue to reduce SARS-CoV-2 infection risks for pregnant women and their fetuses.

Tissue Tropisms of Avian Influenza A Viruses Affect Their Spillovers from Wild Birds to Pigs.

Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations.IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

Placental SARS-CoV-2 in a pregnant woman with mild COVID-19 disease.

The full impact of coronavirus disease 2019 (COVID-19) on pregnancy remains uncharacterized. Current literature suggests minimal maternal, fetal, and neonatal morbidity and mortality. COVID-19 manifestations appear similar between pregnant and nonpregnant women. We present a case of placental severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a woman with mild COVID-19 disease, then review the literature. Reverse transcriptase polymerase chain reaction was performed to detect SARS-CoV-2. Immunohistochemistry staining was performed with specific monoclonal antibodies to detect SARS-CoV-2 antigen or to identify trophoblasts. A 29-year-old multigravida presented at 40-4/7 weeks for labor induction. With myalgias 2 days prior, she tested positive for SARS-CoV-2. We demonstrate maternal vascular malperfusion, with no fetal vascular malperfusion, as well as SARS-CoV-2 virus in chorionic villi endothelial cells, and also rarely in trophoblasts. To our knowledge, this is the first report of placental SARS-CoV-2 despite mild COVID-19 disease (no symptoms of COVID-19 aside from myalgias); patient had no fever, cough, or shortness of breath, but only myalgias and sick contacts. Despite her mild COVID-19 disease in pregnancy, we demonstrate placental vasculopathy and presence of SARS-CoV-2 virus across the placenta. Evidence of placental COVID-19 raises concern for placental vasculopathy (potentially leading to fetal growth restriction and other pregnancy complications) and possible vertical transmission-especially for pregnant women who may be exposed to COVID-19 in early pregnancy. This raises important questions of whether future pregnancy guidance should include stricter pandemic precautions, such as screening for a wider array of COVID-19 symptoms, increased antenatal surveillance, and possibly routine COVID-19 testing throughout pregnancy.

Aerosol Transmission of Gull-Origin Iceland Subtype H10N7 Influenza A Virus in Ferrets.

Subtype H10 influenza A viruses (IAVs) have been recovered from domestic poultry and various aquatic bird species, and sporadic transmission of these IAVs from avian species to mammals (i.e., human, seal, and mink) are well documented. In 2015, we isolated four H10N7 viruses from gulls in Iceland. Genomic analyses showed four gene segments in the viruses were genetically associated with H10 IAVs that caused influenza outbreaks and deaths among European seals in 2014. Antigenic characterization suggested minimal antigenic variation among these H10N7 isolates and other archived H10 viruses recovered from human, seal, mink, and various avian species in Asia, Europe, and North America. Glycan binding preference analyses suggested that, similar to other avian-origin H10 IAVs, these gull-origin H10N7 IAVs bound to both avian-like alpha 2,3-linked sialic acids and human-like alpha 2,6-linked sialic acids. However, when the gull-origin viruses were compared with another Eurasian avian-origin H10N8 IAV, which caused human infections, the gull-origin virus showed significantly higher binding affinity to human-like glycan receptors. Results from a ferret experiment demonstrated that a gull-origin H10N7 IAV replicated well in turbinate, trachea, and lung, but replication was most efficient in turbinate and trachea. This gull-origin H10N7 virus can be transmitted between ferrets through the direct contact and aerosol routes, without prior adaptation. Gulls share their habitat with other birds and mammals and have frequent contact with humans; therefore, gull-origin H10N7 IAVs could pose a risk to public health. Surveillance and monitoring of these IAVs at the wild bird-human interface should be continued.IMPORTANCE Subtype H10 avian influenza A viruses (IAVs) have caused sporadic human infections and enzootic outbreaks among seals. In the fall of 2015, H10N7 viruses were recovered from gulls in Iceland, and genomic analyses showed that the viruses were genetically related with IAVs that caused outbreaks among seals in Europe a year earlier. These gull-origin viruses showed high binding affinity to human-like glycan receptors. Transmission studies in ferrets demonstrated that the gull-origin IAV could infect ferrets, and that the virus could be transmitted between ferrets through direct contact and aerosol droplets. This study demonstrated that avian H10 IAV can infect mammals and be transmitted among them without adaptation. Thus, avian H10 IAV is a candidate for influenza pandemic preparedness and should be monitored in wildlife and at the animal-human interface.

Mutation W222L at the Receptor Binding Site of Hemagglutinin Could Facilitate Viral Adaption from Equine Influenza A(H3N8) Virus to Dogs.

An outbreak of respiratory disease caused by the equine-origin influenza A(H3N8) virus was first detected in dogs in 2004 and since then has been enzootic among dogs. Currently, the molecular mechanisms underlying host adaption of this virus from horses to dogs is unknown. Here, we have applied quantitative binding, growth kinetics, and immunofluorescence analyses to elucidate these mechanisms. Our findings suggest that a substitution of W222L in the hemagglutinin of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to receptor glycans with N-glycolylneuraminic acid (Neu5Gc) and sialyl Lewis X (SLeX) motifs. We have demonstrated these motifs are abundantly located in the submucosal glands of dog trachea. Our findings also suggest that in addition to the type of glycosidic linkage (e.g., α2,3-linkage or α2,6-linkage), the type of sialic acid (Neu5Gc or 5-N-acetyl neuraminic acid) and the glycan substructure (e.g., SLeX) also play an important role in host tropism of influenza A viruses.IMPORTANCE Influenza A viruses (IAVs) cause a significant burden on human and animal health, and mechanisms for interspecies transmission of IAVs are far from being understood. Findings from this study suggest that an equine-origin A(H3N8) IAV with mutation W222L at its hemagglutinin increased binding to canine-specific receptors with sialyl Lewis X and Neu5Gc motifs and, thereby, may have facilitated viral adaption from horses to dogs. These findings suggest that in addition to the glycosidic linkage (e.g., α2,3-linked and α2,6-linked), the substructure in the receptor saccharides (e.g., sialyl Lewis X and Neu5Gc) could present an interspecies transmission barrier for IAVs and drive viral mutations to overcome such barriers.

Women's experience of polycystic ovary syndrome management: A systematic review and meta-synthesis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common chronic condition in women of child-bearing age. There is currently no effective treatment, so early and long-term management is essential. However, there are many problems in the practice of disease management in women with PCOS that make it difficult to achieve good outcomes. OBJECTIVE: To explore women's experience of PCOS management and identify the relevant facilitators and barriers to management. SEARCH STRATEGY: A structured search was undertaken in five bibliographic databases (MEDLINE, Web of Science, CINAHL, Embase, PubMed, and Cochrane) from the date of establishment of the database up to December 2022. SELECTION CRITERIA: All qualitative and mixed-methods studies available in English describing the experience of PCOS management from the patients' perspective were included. DATA COLLECTION AND ANALYSIS: The Joanna Briggs Institute Qualitative Assessment and Review Instrument was used to appraise study quality. The evidence was synthesized using a pragmatic meta-aggregative approach guided by the capability, opportunity, and motivation model of behavior (COM-B). MAIN RESULTS: A total of 13 studies were included with 85 equivocal findings and 12 credible findings. The findings were meta-aggregated into three themes: (1) capability of women with PCOS, including patients' attitudes toward disease and management, knowledge, and skills of the disease; (2) opportunities in PCOS management, including information about PCOS, diagnostic delay, disease characteristics, disease management plan, and logistical and environmental problems; and (3) motivation in PCOS management, including impact of symptoms, perceived needs, support and feedback, and unpleasant medical experience. CONCLUSIONS: This study identifies facilitators and barriers to PCOS management from the patient perspective, which can guide the design and implementation of PCOS management programs for patients. This study also provides information for future research into how the COM-B theory can be incorporated into specific management plans to promote patient action.

Healthcare professionals' perspectives on the challenges with managing polycystic ovary syndrome: A systematic review and meta-synthesis.

OBJECTIVE: To provide an overview of healthcare professionals' experience of PCOS management and identify the relevant facilitators and barriers. METHODS: A systematic search was conducted in MEDLINE, EMBASE, CINAHL, Web of Science, and Cochrane CENTRAL database from the earliest available date to April 2023. Qualitative and mixed methods studies that described healthcare professionals' experiences of PCOS management were included. RESULTS: A total of 74 findings were extracted from the 8 included studies, which were categorized into facilitators and barriers. The barriers were meta-aggregated into four themes: the weakness of clinical evidence; women's low adherence to PCOS management; various obstacles that healthcare professionals face, and the influence of social environment and culture. The facilitators were meta-aggregated into three themes: chronic disease healthcare plan, communication techniques and healthcare professionals' ability and awareness. CONCLUSION: The findings of this study have the potential to improve the care provided to women with PCOS. However, it is important for national health professionals and policy markers to consider the cultural context of their own country when implementing these findings. PRACTICAL IMPLICATIONS: This study illustrated several challenges in managing the heterogeneous condition of PCOS and provide insights for the development of medical policies and future research directions.

The Analysis, Description, and Examination of the Maize LAC Gene Family's Reaction to Abiotic and Biotic Stress.

Laccase (LAC) is a diverse group of genes found throughout the plant genome essential for plant growth and the response to stress by converting monolignin into intricate lignin formations. However, a comprehensive investigation of maize laccase has not yet been documented. A bioinformatics approach was utilized in this research to conduct a thorough examination of maize (Zea mays L.), resulting in the identification and categorization of 22 laccase genes (ZmLAC) into six subfamilies. The gene structure and motifs of each subgroup were largely consistent. The distribution of the 22 LAC genes was uneven among the maize chromosomes, with the exception of chromosome 9. The differentiation of the genes was based on fragment replication, and the differentiation time was about 33.37 million years ago. ZmLAC proteins are primarily acidic proteins. There are 18 cis-acting elements in the promoter sequences of the maize LAC gene family associated with growth and development, stress, hormones, light response, and stress response. The analysis of tissue-specific expression revealed a high expression of the maize LAC gene family prior to the V9 stage, with minimal expression at post-V9. Upon reviewing the RNA-seq information from the publicly available transcriptome, it was discovered that ZmLAC5, ZmLAC10, and ZmLAC17 exhibited significant expression levels when exposed to various biotic and abiotic stress factors, suggesting their crucial involvement in stress responses and potential value for further research. This study offers an understanding of the functions of the LAC genes in maize's response to biotic and abiotic stress, along with a theoretical basis for comprehending the molecular processes at play.

MAIVeSS: streamlined selection of antigenically matched, high-yield viruses for seasonal influenza vaccine production.

Vaccines are the main pharmaceutical intervention used against the global public health threat posed by influenza viruses. Timely selection of optimal seed viruses with matched antigenicity between vaccine antigen and circulating viruses and with high yield underscore vaccine efficacy and supply, respectively. Current methods for selecting influenza seed vaccines are labor intensive and time-consuming. Here, we report the Machine-learning Assisted Influenza VaccinE Strain Selection framework, MAIVeSS, that enables streamlined selection of naturally circulating, antigenically matched, and high-yield influenza vaccine strains directly from clinical samples by using molecular signatures of antigenicity and yield to support optimal candidate vaccine virus selection. We apply our framework on publicly available sequences to select A(H1N1)pdm09 vaccine candidates and experimentally confirm that these candidates have optimal antigenicity and growth in cells and eggs. Our framework can potentially reduce the optimal vaccine candidate selection time from months to days and thus facilitate timely supply of seasonal vaccines.

Neu5Gc binding loss of subtype H7 influenza A virus facilitates adaptation to gallinaceous poultry following transmission from waterbirds but restricts spillback.

Migratory waterfowl, gulls, and shorebirds serve as natural reservoirs for influenza A viruses, with potential spillovers to domestic poultry and humans. The intricacies of interspecies adaptation among avian species, particularly from wild birds to domestic poultry, are not fully elucidated. In this study, we investigated the molecular mechanisms underlying avian species barriers in H7 transmission, particularly the factors responsible for the disproportionate distribution of poultry infected with A/Anhui/1/2013 (AH/13)-lineage H7N9 viruses. We hypothesized that the differential expression of N-glycolylneuraminic acid (Neu5Gc) among avian species exerts selective pressure on H7 viruses, shaping their evolution and enabling them to replicate and transmit efficiently among gallinaceous poultry, particularly chickens. Our glycan microarray and biolayer interferometry experiments showed that AH/13-lineage H7N9 viruses exclusively bind to Neu5Ac, in contrast to wild waterbird H7 viruses that bind both Neu5Ac and Neu5Gc. Significantly, reverting the V179 amino acid in AH/13-lineage back to the I179, predominantly found in wild waterbirds, expanded the binding affinity of AH/13-lineage H7 viruses from exclusively Neu5Ac to both Neu5Ac and Neu5Gc. When cultivating H7 viruses in cell lines with varied Neu5Gc levels, we observed that Neu5Gc expression impairs the replication of Neu5Ac-specific H7 viruses and facilitates adaptive mutations. Conversely, Neu5Gc deficiency triggers adaptive changes in H7 viruses capable of binding to both Neu5Ac and Neu5Gc. Additionally, we assessed Neu5Gc expression in the respiratory and gastrointestinal tissues of seven avian species, including chickens, Canada geese, and various dabbling ducks. Neu5Gc was absent in chicken and Canada goose, but its expression varied in the duck species. In summary, our findings reveal the crucial role of Neu5Gc in shaping the host range and interspecies transmission of H7 viruses. This understanding of virus-host interactions is crucial for developing strategies to manage and prevent influenza virus outbreaks in diverse avian populations.

SARS-CoV-2 Exposure in Norway Rats (Rattus norvegicus) from New York City.

Millions of Norway rats (Rattus norvegicus) inhabit New York City (NYC), presenting the potential for transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to rats. We evaluated SARS-CoV-2 exposure among 79 rats captured from NYC during the fall of 2021. Our results showed that 13 of the 79 rats (16.5%) tested IgG- or IgM-positive, and partial SARS-CoV-2 genomes were recovered from all 4 rats that were qRT-PCR (reverse transcription-quantitative PCR)-positive. Genomic analyses suggest these viruses were associated with genetic lineage B, which was predominant in NYC in the spring of 2020 during the early pandemic period. To further investigate rat susceptibility to SARS-CoV-2 variants, we conducted a virus challenge study and showed that Alpha, Delta, and Omicron variants can cause infections in wild-type Sprague Dawley (SD) rats, including high replication levels in the upper and lower respiratory tracts and induction of both innate and adaptive immune responses. Additionally, the Delta variant resulted in the highest infectivity. In summary, our results indicate that rats are susceptible to infection with Alpha, Delta, and Omicron variants, and wild Norway rats in the NYC municipal sewer systems have been exposed to SARS-CoV-2. Our findings highlight the need for further monitoring of SARS-CoV-2 in urban rat populations and for evaluating the potential risk of secondary zoonotic transmission from these rat populations back to humans. IMPORTANCE The host tropism expansion of SARS-CoV-2 raises concern for the potential risk of reverse-zoonotic transmission of emerging variants into rodent species, including wild rat species. In this study, we present both genetic and serological evidence for SARS-CoV-2 exposure to the New York City wild rat population, and these viruses may be linked to the viruses that were circulating during the early stages of the pandemic. We also demonstrated that rats are susceptible to additional variants (i.e., Alpha, Delta, and Omicron) that have been predominant in humans and that susceptibility to infection varies by variant. Our findings highlight the reverse zoonosis of SARS-CoV-2 to urban rats and the need for further monitoring of SARS-CoV-2 in rat populations for potential secondary zoonotic transmission to humans.

Exposure of white-tailed deer in North America to influenza D virus.

Influenza D virus (IDV) infections have been identified worldwide in cattle, swine, camelid, and small ruminants, mostly in domestic livestock. Here we report that the wild white-tailed deer in North America were exposed to IDVs, suggesting IDVs infect a wide range of hosts including wild animal populations.

Time-Dependent Proinflammatory Responses Shape Virus Interference during Coinfections of Influenza A Virus and Influenza D Virus.

Both influenza A virus (IAV) and influenza D virus (IDV) are enzootic in pigs. IAV causes approximately 100% morbidity with low mortality, whereas IDV leads to only mild respiratory diseases in pigs. In this study, we performed a series of coinfection experiments in vitro and in vivo to understand how IAV and IDV interact and cause pathogenesis during coinfection. The results showed that IAV inhibited IDV replication when infecting swine tracheal epithelial cells (STECs) with IAV 24 or 48 h prior to IDV inoculation and that IDV suppressed IAV replication when IDV preceded IAV inoculation by 48 h. Virus interference was not identified during simultaneous IAV/IDV infections or with 6 h between the two viral infections, regardless of their order. The interference pattern at 24 and 48 h correlated with proinflammatory responses induced by the first infection, which, for IDV, was slower than for IAV by about 24 h. The viruses did not interfere with each other if both infected the cells before proinflammatory responses were induced. Coinfection in pigs further demonstrated that IAV interfered with both viral shedding and virus replication of IDV, especially in the upper respiratory tract. Clinically, coinfection of IDV and IAV did not show significant enhancement of disease pathogenesis, compared with the pigs infected with IAV alone. In summary, this study suggests that interference during coinfection of IAV and IDV is primarily due to the proinflammatory response; therefore, it is dependent on the time between infections and the order of infection. This study facilitates our understanding of virus epidemiology and pathogenesis associated with IAV and IDV coinfection.

SARS-CoV-2 exposure in Norway rats (Rattus norvegicus) from New York City.

Millions of Norway rats (Rattus norvegicus) inhabit New York City (NYC), presenting the potential for transmission of SARS-CoV-2 from humans to rats and other wildlife. We evaluated SARS-CoV-2 exposure among 79 rats captured from NYC during the fall of 2021. Results showed that 13 of 79 rats (16.5%) tested IgG or IgM positive, and partial genomes of SARS-CoV-2 were recovered from four rats that were qRT-PCR positive. Using a virus challenge study, we also showed that Alpha, Delta, and Omicron variants can cause robust infections in wild-type Sprague Dawley (SD) rats, including high level replications in the upper and lower respiratory tracts and induction of both innate and adaptive immune responses. Additionally, the Delta variant resulted in the highest infectivity. In summary, our results indicated that rats are susceptible to infection with Alpha, Delta, and Omicron variants, and rats in the NYC municipal sewer systems have been exposed to SARS-CoV-2. Our findings highlight the potential risk of secondary zoonotic transmission from urban rats and the need for further monitoring of SARS-CoV-2 in those populations.

Terminal Epitope-Dependent Branch Preference of Siglecs Toward N-Glycans.

Siglecs are sialic acid-binding immunoglobulin-like lectins that play vital roles in immune cell signaling. Siglecs help the immune system distinguish between self and nonself through the recognition of glycan ligands. While the primary binding specificities of Siglecs are known to be divergent, their specificities for complex glycans remain unclear. Herein, we determined N-glycan binding profiles of a set of Siglecs by using a complex asymmetric N-glycan microarray. Our results showed that Siglecs had unique terminal epitope-dependent branch preference when recognizing asymmetric N-glycans. Specifically, human Siglec-3, -9, and -10 prefer the α1-3 branch when Siaα2-6Galß1-4GlcNAc terminal epitope serves as the binding ligand but prefer the opposite α1-6 branch when Siaα2-3Galß1-4GlcNAc epitope serves as the ligand. Interestingly, Siglec-10 exhibited dramatic binding divergence toward a pair of Neu5Ac-containing asymmetric N-glycan isomers, as well as their Neu5Gc-containing counterparts. This new information on complex glycan recognition by Siglecs provides insights into their biological roles and applications.

Cell-Adapted Mutations and Antigenic Diversity of Influenza B Viruses in Missouri, 2019-2020 Season.

Influenza B viruses (IBVs) are causing an increasing burden of morbidity and mortality, yet the prevalence of culture-adapted mutations in human seasonal IBVs are unclear. We collected 368 clinical samples from patients with influenza-like illness in Missouri during the 2019-2020 influenza season and recovered 146 influenza isolates including 38 IBV isolates. Of MDCK-CCL34, MDCK-Siat1, and humanized MDCK (hCK), hCK showed the highest virus recovery efficiency. All Missourian IBVs belonged to the Victoria V1A.3 lineage, all of which contained a three-amino acid deletion on the HA protein and were antigenically distant from the Victoria lineage IBV vaccine strain used during that season. By comparing genomic sequences of these IBVs in 31 paired samples, eight cell-adapted nonsynonymous mutations were identified, with the majority in the RNA polymerase. Analyses of IBV clinical sample-isolate pairs from public databases further showed that cell- and egg-adapted mutations occurred more widely in viral proteins, including the receptor and antibody binding sites on HA. Our study suggests that hCK is an effective platform for IBV isolation and that culture-adapted mutations may occur during IBV isolation. As culture-adapted mutations may affect subsequent virus studies and vaccine development, the knowledge from this study may help optimize strategies for influenza surveillance, vaccine strain selection, and vaccine development.

Pathogenicity and transmission of a swine influenza A(H6N6) virus.

Subtype H6 influenza A viruses (IAVs) are commonly detected in wild birds and domestic poultry and can infect humans. In 2010, a H6N6 virus emerged in southern China, and since then, it has caused sporadic infections among swine. We show that this virus binds to α2,6-linked and α2,3-linked sialic acids. Mutations at residues 222 (alanine to valine) and 228 (glycine to serine) of the virus hemagglutinin (HA) affected its receptor-binding properties. Experiments showed that the virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets. The internal genes of the influenza A(H1N1)pdm09 virus, which is prevalent in swine worldwide, increases the replication efficiency of H6N6 IAV in the lower respiratory tract of ferrets but not its transmissibility between ferrets. These findings suggest H6N6 swine IAV (SIV) currently poses a moderate risk to public health, but its evolution and spread should be closely monitored.