Biology subjects

Carnaccini, S.

Publications and source records attributed to Carnaccini, S..

12 recordsLinked to original sources

bioRxiv2026

Comparative Distribution of α2,3- and α2,6-Linked Sialic Acid Receptors and Influenza A(H5N1) Virus Binding in Sheep, Goats, and Alpacas

Highly pathogenic avian influenza virus (HPAIV) H5N1 continues to pose a significant threat to animal and human health. Since 2022, an expanded host range has been observed, as illustrated by the H5N1 outbreak in dairy cows. Other minor livestock species, such as sheep, goats, and alpacas, have also tested positive for H5N1 for the first time, raising concerns about its impact on previously overlooked livestock species. In this study, we first characterized the distribution of sialic acid (SA) receptors across respiratory and non-respiratory tissues of sheep, goats, and alpacas using Maackia amurensis (SA 2,3) and Sambucus nigra (SA 2,6) lectins. We found that both SA receptors are present in all tissues across species, with notable differences in their distribution and abundance. Next, we developed recombinant LPAIV H5N1 viruses over a laboratory-adapted backbone to evaluate tissue-specific virus binding profiles. The results show that a representative H5N1 virus from clade 2.3.4.4b binds effectively to respiratory and non-respiratory tissues across all species. The strongest virus binding was observed in sheep, followed by goats and, lastly, alpacas. Similar binding patterns were observed with an LPAIV version of clade 2.3.4.4b and a Gs/Gd lineage precursor prototype virus. Finally, we show that H1N1 and H3N2 viruses also bind to respiratory tissues of sheep and goats. These findings provide important insights into the potential susceptibility of minor livestock species to HPAIV H5N1 and other influenza subtypes. Our results contribute to a better understanding of influenza tissue tropism across livestock species and its potential implications for zoonotic transmission.

microbiology↗

bioRxiv2025

Viral lineage and mode of exposure modulate within host spatial dynamics of influenza A viruses

The upper and lower respiratory tracts (URT and LRT) present distinct environments for influenza A virus (IAV) replication. Their differential features have major implications for viral evolutionary dynamics, transmission potential, and pathogenesis. To investigate the implications of differential viral replication in the URT and LRT, we assessed dispersal of IAVs throughout the guinea pig respiratory system. Guinea pigs were inoculated intranasally with a 300 L volume to deliver inoculum to both the URT and LRT. Two strains were used to represent the circulating seasonal IAV lineages: influenza A/TX/50/2012 (H3N2) and influenza A/CA/07/2009 (H1N1) virus. The inclusion of a diverse genetic barcode enabled high-resolution tracing of viral dispersal for the H1N1 virus. While infectious virus was consistently detected in the URT, the H1N1 virus could be detected in LRT while the H3N2 virus could not. To determine whether replication of the H1N1 virus in the LRT extends to other modes of infection, virus distribution was evaluated following infection via aerosol exposure or transmission. Infectious virus in lung homogenates was observed in both cases, confirming the LRT tropism of the H1N1 virus. Sequencing genetic barcodes revealed that diversity was largely maintained in nasal samples and trachea but contracted upon dispersal to the lungs. This loss of diversity was associated with increased distance to and branching from the major airways, implicating long distance dispersal through the airways in imposing within-host population bottlenecks. These data underline the implications for within-host viral dynamics of the distinct environments of the upper and lower respiratory tracts. ImportanceThe upper (URT) and lower (LRT) respiratory tracts create different conditions for influenza A virus (IAV) spread and evolution. We studied how the virus moves through guinea pigs airways after infection with H3N2 or H1N1 strains of IAV. Whether delivered intranasally, by aerosol or by transmission, the H1N1 virus replicated in the nasal cavity, trachea, and lungs. By contrast, the H3N2 virus stayed mostly in the nasal cavity. Genetic barcodes were used to track how the H1N1 virus moved and changed. The populations replicating in the nasal cavity and trachea maintained high diversity but those sampled from the lungs showed low diversity. This bottlenecking effect was stronger for viral populations present deeper in the lungs. These findings show that the different environments of the URT and LRT strongly shape how influenza spreads and evolves inside a host.

microbiology↗

bioRxiv2025

Development and Characterization of Chicken Lung Organoids for Future In Vitro Modeling of Avian Influenza Virus-Host Cell Interaction

High pathogenicity avian influenza viruses pose a growing threat to poultry, livestock, wildlife, and humans as they undergo accelerated expansion of host and geographical ranges. Since 2020, these viruses have driven a panzootic characterized by extensive viral diversification and spillover into species previously considered to be resistant to the disease. There is currently a lack of physiologically relevant in vitro models that can be used to screen the rapidly changing viral landscape. To address this need, we describe the first chicken lung organoids derived from adult stem cells of specific pathogen free White Leghorns. We analyze their gene expression with bulk RNA sequencing, confirm their cellular heterogeneity via single-nuclei RNA sequencing, and provide basic morphological characterization using hematoxylin and eosin staining, immunohistochemistry, immunofluorescence, and transmission electron microscopy. The results indicate that the organoids contained several cell types, including non-ciliated columnar, cuboidal, squamous, and mucin-producing cells, representative of different regions of the avian respiratory system. Furthermore, expression of genes relevant to influenza A virus infection and replication appeared to be conserved across different sample types. These organoids have the potential to effectively and efficiently model viral infection of the chicken lung, enabling the investigation of viral pathogenesis and evolutionary potential, virus-host interactions, and discovery of targets for antiviral treatments.

cell biology↗

bioRxiv2024

Air-Liquid Interface Model for Influenza Aerosol Exposure In Vitro

Airborne transmission is an essential mode of infection and spread of influenza viruses among humans. However, most studies use liquid inoculum for virus infection. To better replicate natural airborne infections in vitro, we generated a calm-aerosol settling chamber system designed to examine the aerosol infectivity of influenza viruses in different cell types. Aerosol inoculation was characterized for multiple influenza A virus (FLUAV) subtypes, including a pandemic 2009 H1N1, a seasonal swine H3N2, and an avian H9N2 using this exposure system. While each FLUAV strain displayed high infectivity within MDCK cells via liquid inoculation, differences in infectivity were observed during airborne inoculation. This was further observed in recently developed immortalized differentiated human airway epithelial cells (BCi-NS1.1) cultured in an air-liquid interface. The airborne infectious dose 50 for each virus was based on the exposure dose per well. Our findings indicate that this system has the potential to enhance our understanding of the factors influencing influenza transmission via the airborne route. This could be invaluable for conducting risk assessments, potentially reducing the reliance on extensive and costly in vivo animal studies. ImportanceThis study presents a significant advancement in influenza research by developing a novel in vitro system to assess aerosol infectivity, a crucial aspect of influenza transmission. The systems ability to differentiate between mammalian-adapted and avian-adapted influenza viruses based on their aerosol infectivity offers a valuable tool for pre-screening the pandemic potential of different strains. This could potentially streamline the risk assessment process and inform public health preparedness strategies. Moreover, the systems capacity to examine aerosol infectivity in human airway epithelial cells provides a more relevant model for studying virus-host interactions in natural airborne infections. Overall, this study provides an accessible platform for investigating aerosol infectivity, which could significantly contribute to our understanding of influenza transmission and pandemic preparedness.

microbiology↗

bioRxiv2024

Dispersal of influenza virus populations within the respiratory tract shapes their evolutionary potential

Viral infections are characterized by dispersal from an initial site to secondary locations within the host. How the resultant spatial heterogeneity shapes within-host genetic diversity and viral evolutionary pathways is poorly understood. Here we show that dispersal within and between the nasal cavity and trachea maintains diversity and is therefore conducive to adaptive evolution, whereas dispersal to the lungs is stochastic and gives rise to population heterogeneity. We infected ferrets either intranasally or by aerosol with a barcoded influenza A/California/07/2009 (H1N1) virus. At 1, 2 or 4 days post infection, dispersal was assessed by collecting 52 samples from throughout the respiratory tract of each animal. Irrespective of inoculation route, barcode compositions across the nasal turbinates and trachea were similar and highly diverse, revealing little constraint on the establishment of infection in the nasal cavity and descent through the trachea. By comparison, infection of the lungs produced genetically distinct viral populations. Lung populations were pauci-clonal, suggesting that each seeded location received relatively few viral genotypes. While aerosol inoculation gave distinct populations at every lung site sampled, within-host dispersal after intranasal inoculation produced larger patches, indicative of local expansion following seeding of the lungs. Throughout the respiratory tract, barcode diversity declined over time, but new diversity was generated through mutation. De novo variants were often unique to a given location, indicating that localized replication following dispersal resulted in population divergence. In summary, dispersal within the respiratory tract operates differently between regions and contributes to the potential for viral evolution to proceed independently in multiple within-host subpopulations.

microbiology↗

bioRxiv2024

FLUAV RAM-IGIP: A modified live influenza virus vaccine that enhances humoral and mucosal responses against influenza

Current influenza A vaccines fall short, leaving both humans and animals vulnerable. To address this issue, we have developed attenuated modified live virus (MLV) vaccines against influenza using genome rearrangement techniques targeting the internal gene segments of FLUAV. The rearranged M2 (RAM) strategy involves cloning the M2 ORF downstream of the PB1 ORF in segment 2 and incorporating multiple early stop codons within the M2 ORF in segment 7. Additionally, the IgA-inducing protein (IGIP) coding region was inserted into the HA segment to further attenuate the virus and enhance protective mucosal responses. RAM-IGIP viruses exhibit similar growth rates to wild type (WT) viruses in vitro and remain stable during multiple passages in cells and embryonated eggs. The safety, immunogenicity, and protective efficacy of the RAM-IGIP MLV vaccine against the prototypical 2009 pandemic H1N1 strain A/California/04/2009 (H1N1) (Ca/04) were evaluated in Balb/c mice and compared to a prototypic cold-adapted live attenuated virus vaccine. The results demonstrate that the RAM-IGIP virus exhibits attenuated virulence in vivo. Mice vaccinated with RAM-IGIP and subsequently challenged with an aggressive lethal dose of the Ca/04 strain exhibited complete protection. Analysis of the humoral immune response revealed that the inclusion of IGIP enhanced the production of neutralizing antibodies and augmented the antibody-dependent cellular cytotoxicity response. Similarly, the RAM-IGIP potentiated the mucosal immune response against various FLUAV subtypes. Moreover, increased antibodies against NP and NA responses were observed. These findings support the development of MLVs utilizing genome rearrangement strategies in conjunction with the incorporation of immunomodulators. IMPORTANCECurrent influenza vaccines offer suboptimal protection, leaving both humans and animals vulnerable. Our novel attenuated MLV vaccine, built by rearranging FLUAV genome segments and incorporating the IgA-inducing protein, shows promising results. This RAM-IGIP vaccine exhibits safe attenuation, robust immune responses, and complete protection against lethal viral challenge in mice. Its ability to stimulate broad-spectrum humoral and mucosal immunity against diverse FLUAV subtypes makes it a highly promising candidate for improved influenza vaccines.

immunology↗

bioRxiv2023

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

Influenza A viruses (FLUAV) of the H9N2 subtype are zoonotic pathogens that cause significant economic damage to the poultry industry. Vaccination to prevent and control H9N2 infections in poultry is widely employed in the Middle East and Asia. We used phylogenetics and antigenic analysis to study the antigenic properties of the H9 hemagglutinin (HA) using sera produced in Japanese quail (Coturnix c. japonica). Consensus HA1 sequences were generated to capture antigenic diversity among isolates. We constructed chimeric H9N2 viruses containing the HA1 of each consensus sequence on a constant isogenic backbone. The resulting viruses were used to generate antisera from quail, a common and significant minor poultry species whose anti-HA response profiles remain poorly defined. Antigenic maps were generated by plotting the cross-hemagglutination inhibition (HI) data from the panel of quail sera against the chimeric constructs and 51 H9 field isolates. The chimeric antigens were divided into four different antigenic profiles (cyan, blue, orange, and red). Site-directed mutagenesis analysis showed 9 amino acid positions of antigenic relevance. Substitutions at amino acid positions 149, 150, and 180 (H9 HA numbering) had relatively significant impact on HI activity using quail sera. Substitutions E180A and R131K/E180A led to the most significant antigenic change transitions. This study provides insights into the antigenic profile of H9 FLUAVs, with important implications for understanding antigenic drift and improving vaccine development for use in minor poultry species. IMPORTANCEDetermining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 HA using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

microbiology↗

bioRxiv2022

Pathobiology and dysbiosis of the respiratory and intestinal microbiota in 14 months old Golden Syrian hamsters infected with SARS-CoV-2

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS2) affected the geriatric population. Among research models, Golden Syrian hamsters (GSH) are one of the most representative to study SARS2 pathogenesis and host responses. However, animal studies that recapitulate the effects of SARS2 in the human geriatric population are lacking. To address this gap, we inoculated 14 months old GSH (resembling people over 60 years old) with a prototypic ancestral strain of SARS2 and studied the effects on virus pathogenesis, virus shedding, and respiratory and gastrointestinal microbiome changes. SARS2 infection led to high vRNA loads in the nasal turbinates (NT), lungs, and trachea as well as higher pulmonary lesions scores later in infection. Dysbiosis throughout SARS2 disease progression was observed in the pulmonary microbial dynamics with the enrichment of opportunistic pathogens (Haemophilus, Fusobacterium, Streptococcus, Campylobacter, and Johnsonella) and microbes associated with inflammation (Prevotella). Changes in the gut microbial community also reflected an increase in multiple genera previously associated with intestinal inflammation and disease (Helicobacter, Mucispirillum, Streptococcus, unclassified Erysipelotrichaceae, and Spirochaetaceae). Influenza A virus (FLUAV) pre-exposure resulted in slightly more pronounced pathology in the NT and lungs early on (3 dpc), and more notable changes in lungs compared to the gut microbiome dynamics. Similarities among aged GSH and the microbiome in critically ill COVID-19 patients, particularly in the lower respiratory tract, suggest that GSHs are a representative model to investigate microbial changes during SARS2 infection. The relationship between the residential microbiome and other confounding factors, such as SARS2 infection, in a widely used animal model, contributes to a better understanding of the complexities associated with the host responses during viral infections. Author SummaryThe SARS-CoV-2 pandemic led to millions of human losses, notably affecting the geriatric population, who are at greater risk of developing acute respiratory distress infection leading to prolonged hospitalization and death. However, the mechanism of age-related pathogenicity is not fully understood. Here, we utilized an aged Syrian hamster model resembling ~60-year-old humans to analyze the pathobiology, host response, and effects of SARS2 on the respiratory and intestinal microbiome. We identified specific microbial markers observed in severe COVID-19 patients within the lungs of aged hamsters infected with SARS-CoV-2. Prior influenza A virus (H1N1) exposure amplified these changes. Similarities among aged GSH and critically ill COVID-19 patients suggest that GSHs are a valuable model for investigating microbial changes during SARS2 infection. The relationship between the age, residential microbiome and viral pathogens contributes to a better understanding of the complexities associated with the host responses during viral infection while limiting potential environmental factors that may contribute to inter-individual variation.

microbiology↗

bioRxiv2022

Robustness of the ferret model for influenza risk assessment studies: a cross-laboratory exercise

Ferrets represent the preferred animal model for assessing the transmission potential of newly emerged zoonotic influenza viruses. However, heterogeneity among established experimental protocols and facilities across different laboratories may lead to variable results, complicating interpretation of transmission experimental data. Between 2018-2020, a global exercise was conducted by 11 participating laboratories to assess the range of variation in ferret transmission experiments using two common stock H1N1 influenza viruses that possess different transmission characteristics in ferrets. Inoculation route, dose, and volume were standardized, and all participating laboratories followed the same experimental conditions for respiratory droplet transmission, including a strict 1:1 donor:contact ratio. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored throughout. Overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. To attain high confidence in identifying zoonotic influenza viruses with moderate-to-high or low transmissibility, our analyses support that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment.

microbiology↗

bioRxiv2022

Influenza A virus reassortment in mammals gives rise to genetically distinct within-host sub-populations

Influenza A virus (IAV) genetic exchange through reassortment has the potential to accelerate viral evolution and has played a critical role in the generation of multiple pandemic strains. For reassortment to occur, distinct viruses must co-infect the same cell. The spatio-temporal dynamics of viral dissemination within an infected host therefore define opportunity for reassortment. Here, we used wild type and synonymously barcoded variant viruses of a pandemic H1N1 strain to examine the within-host viral dynamics that govern reassortment in guinea pigs, ferrets and swine. The first two species are well-established models of human influenza, while swine are a natural host and a frequent conduit for cross-species transmission and reassortment. Our results show reassortment to be pervasive in all three hosts but less frequent in swine than in ferrets and guinea pigs. In ferrets, tissue-specific differences in the opportunity for reassortment are also evident, with more reassortants detected in the nasal tract than the lower respiratory tract. While temporal trends in viral diversity are limited, spatial patterns are clear, with heterogeneity in the viral genotypes detected at distinct anatomical sites revealing extensive compartmentalization of reassortment and replication. Our data indicate that the dynamics of viral replication in mammals allow diversification through reassortment but that the spatial compartmentalization of variants likely shapes their evolution and onward transmission.

microbiology↗

bioRxiv2021

Mild and severe SARS-CoV-2 infection induces respiratory and intestinal microbiome changes in the K18-hACE2 transgenic mouse model

Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and declining economies around the world. K18-hACE2 mice develop disease resembling severe SARS-CoV-2 infection in a virus dose-dependent manner. The relationship between SARS-CoV-2 and the intestinal or respiratory microbiome is not fully understood. In this context, we characterized the cecal and lung microbiome of SARS-CoV-2 challenged K18-hACE2 transgenic mice in the presence or absence of treatment with the Mpro inhibitor GC376. Cecum microbiome showed decreased Shannon and Inv Simpson diversity index correlating with SARS-CoV-2 infection dosage and a difference of Bray-Curtis dissimilarity distances among control and infected mice. Bacterial phyla such as Firmicutes, particularly Lachnospiraceae and Oscillospiraceae, were significantly less abundant while Verrucomicrobiota, particularly the family Akkermansiaceae, were increasingly more prevalent during peak infection in mice challenged with a high virus dose. In contrast to the cecal microbiome, the lung microbiome showed similar microbial diversity among the control, low and high challenge virus groups, independent of antiviral treatment. Bacterial phyla in the lungs such as Bacteroidota decreased while Firmicutes and Proteobacteria were significantly enriched in mice challenged with a high dose of SARS-CoV-2. In summary, we identified changes in the cecal and lung microbiome of K18-hACE2 mice with severe clinical signs of SARS-CoV-2 infection. IMPORTANCEThe COVID-19 pandemic has resulted in millions of deaths. The hosts respiratory and intestinal microbiome can affect directly or indirectly the immune system during viral infections. We characterized the cecal and lung microbiome in a relevant mouse model challenged with a low and high dose of SARS-CoV-2 in the presence or absence of an antiviral Mpro inhibitor, GC376. Decreased microbial diversity and taxonomic abundances of the phyla Firmicutes, particularly Lachnospiraceae, correlating with infection dosage was observed in the cecum. In addition, microbes within the family Akkermansiaceae were increasingly more prevalent during peak infection, which is observed in other viral infections. The lung microbiome showed similar microbial diversity to the control, independent of antiviral treatment. Decreased Bacteroidota and increased Firmicutes and Proteobacteria were observed in the lungs in a virus dose-dependent manner. These studies add to a better understanding of the complexities associated with the intestinal microbiome during respiratory infections.

microbiology↗

bioRxiv2021

Efficacy of GC-376 against SARS-CoV-2 virus infection in the K18 hACE2 transgenic mouse model

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC-376 is a Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice and produced milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls, most notably in the brain in mice challenged with a low virus dose. Although GC-376 was not sufficient to improve neither clinical symptoms nor survival, it did show a positive effect against SARS-CoV-2 in vivo. This study supports the notion that the K18-hACE2 mouse model is suitable to study antiviral therapies against SARS-CoV-2, and GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection.

microbiology↗