Biology subjects

Rajao, D. S.

Publications and source records attributed to Rajao, D. S..

10 recordsLinked to original sources

bioRxiv2025

Vaccine-induced antigenic drift of a human-origin H3N2 Influenza A virus in swine alters glycan binding and sialic acid avidity

Interspecies transmission of human influenza A viruses (FLUAV) to swine occurs frequently, yet the molecular factors driving adaptation remain poorly understood. Here we investigated how vaccine-induced immunity shapes the evolution of a human-origin H3N2 virus in pigs using an in vivo sustained transmission model. Pigs (seeders) were vaccinated with a commercial inactivated swine vaccine and then infected with an antigenically distinct FLUAV containing human-origin HA/NA. Contact pigs were introduced two days later. After 3 days, seeder pigs were removed, and new contacts introduced. This was repeated for a total of 4 contacts. Sequencing of nasal swab samples showed the emergence of mutations clustered near the HA receptor binding site, enabling immune escape and abolishing binding to N-glycolylneuraminic acid. Mutant viruses recognized 2,6-sialosides with 3 N-acetyllactosamine repeats, which are rare in swine lungs, while the parental virus bound structures with a minimum of 2 repeats. Adaptative HA mutations enhanced avidity for 2,6-linked sialic acid, likely compensating for the low abundance of extended glycans. Notably, residues outside the canonical HA binding pocket contribute to glycan binding, suggesting a trade-off between receptor breadth and avidity. These findings show that non-neutralizing immunity promotes viral adaptation by fine-tuning receptor engagement and immune evasion. SIGNIFICANCEUnderstanding how vaccination shapes influenza A virus (FLUAV) evolution across species barriers is critical for predicting and preventing zoonotic and reverse-zoonotic events. Our study demonstrates that vaccine-induced immune pressure can drive antigenic drift in a human-origin H3N2 virus, altering HA receptor binding properties that could inadvertently facilitate adaptation to swine. These changes shifted glycan specificity toward extended poly-LacNAc structures and enhanced 2,6-linked sialoglycans binding while abolishing Neu5Gc recognition. By revealing how non-neutralizing immunity fine-tunes HA-glycan interactions by engaging antigenically relevant residues in glycan binding, this work highlights vaccination as an underappreciated driver of host adaptation and viral evolution.

microbiology↗

bioRxiv2025

Impact of Maternal Antibodies and Weaning Stress on the Replication and Transmission of Human H3N2 Influenza A in Piglets

Modern swine production facilitates indoor respiratory contact between human employees and pigs in their care, creating conditions for interspecies transmission of influenza A virus (IAV). Sow vaccination is routinely practiced in the U.S. to transfer maternal derived antibodies (MDA) to piglets. Weaning is a highly stressful period for piglets that requires increased human interaction. This study investigates the effect of maternal antibodies on the susceptibility of weaned piglets to a human-origin H3N2 IAV. Weaned piglets often possess mixed immunity from MDA, which may be antigenically matched or mismatched to circulating viruses. Given the repeated spillover of human seasonal H3N2 into swine, we specifically examined how matched and mismatched MDA, acquired from vaccinated sows, influenced piglet susceptibility. Additionally, we assessed the impact of weaning-related stress on the outcome of viral challenge. The H3N2 virus was generated by reverse genetics to mimic the 2010.1 H3N2 introduction from humans to swine. Challenged seeder piglets were divided by immune and weaning status. Two days post inoculation, naive direct contact pigs were placed with seeders. IAV qRT-PCR and virus titration were performed on nasal swabs and bronchoalveolar lavage fluid to evaluate shedding and transmission kinetics. Matched MDA were effective in reducing shedding in challenged pigs and minimizing transmission to contacts. There was an increase in shedding and transmission in weaned pigs compared to littermates that remained on the sow. These results identify critical control points in production where changing practices could mitigate human-to-swine and swine-to-swine transmission to prevent establishment of novel lineages in pig populations. ImportanceDefining the factors that increase the susceptibility of pigs to infection with human influenza A viruses (IAV) is critical to understand why those viruses transmit to the new host. IAV is frequently detected in nursing pigs, where it was shown that maternal derived antibodies (MDA) may reduce clinical signs but may not prevent infection and transmission. Infected weaned piglets can then move viruses from the sow farm to offsite nurseries, where they can cause outbreaks with clinical disease as MDA wanes. Determining management practices that can be modified to reduce interspecies transmission of viruses to pigs is economically beneficial to the swine industry and could help define measures to prevent new spillover events. Reducing spillover of human IAV into pig populations also benefits public health by reducing genomic and phenotypic diversity in swine and the subsequent potential for zoonotic transmission.

immunology↗

bioRxiv2025

Passage of human-origin influenza A virus in swine tracheal epithelial cells selects for adaptive mutations in the hemagglutinin gene

AbstractFrequent spillover of influenza A viruses from humans to swine contributes to the increasing diversity of influenza viruses circulating in pigs. Although these events are common, little is known about the adaptation processes that take place when viruses jump between the two species. We examined the changes that occurred during serial passages of a reassortant H3N2 virus (VIC11pTRIG) containing human seasonal surface genes (Hemagglutinin and Neuraminidase) and a swine-adapted internal gene constellation in differentiated primary swine tracheal epithelial cells (pSTECs). The VIC11pTRIG reassortant virus was serially passaged 8 times in pSTECs and compared to a control swine-adapted strain (OH/04p) containing the same internal gene constellation. Viral RNA from passages 0 (inoculum), 1, 3, 4-8 were sequenced via next generation or Sanger sequencing. Hemagglutinin diversity was highest at passage 3. Two amino acid mutations in the Hemagglutinin protein (N165K and N216K) were fixed at passages 7 and 5, respectively. These changes were associated with increased fitness of the virus in pSTECs compared to the original parental strain. Our results suggest that the adaptation of human seasonal H3N2 to swine cells may lead to the selection of HA mutations located near the receptor binding site. These mutations may result in increased fitness of human-origin H3N2 strains to adapt in swine.

microbiology↗

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

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

Characterizing a century of genetic diversity and contemporary antigenic diversity of N1 neuraminidase in IAV from North American swine

Influenza A viruses (IAV) of the H1N1 classical swine lineage became endemic in North American swine following the 1918 pandemic. Additional human-to-swine transmission events after 1918, and a spillover of H1 viruses from wild birds in Europe, potentiated a rapid increase in genomic diversity via reassortment between introductions and the endemic classical swine lineage. To determine mechanisms affecting reassortment and evolution, we conducted a phylogenetic analysis of N1 and paired HA swine IAV genes in North America between 1930 and 2020. We described fourteen N1 clades within the N1 Eurasian avian lineage (including the N1 pandemic clade) and the N1 classical swine lineage. Seven N1 genetic clades had evidence for contemporary circulation. To assess antigenic drift associated with N1 genetic diversity, we generated a panel of representative swine N1 antisera and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assays and antigenic cartography. Within the N1 lineage, antigenic similarity was variable and reflected shared evolutionary history. Sustained circulation and evolution of N1 genes in swine had resulted in significant antigenic distance between the N1 pandemic clade and classical swine lineage. We also observed a significant increase in the rate of evolution in the N1 pandemic clade relative to the classical lineage. Between 2010 and 2020, N1 clades and N1-HA pairings fluctuated in detection frequency across North America, with hotspots of diversity generally appearing and disappearing within two years. We also identified frequent N1-HA reassortment events (n = 36), which were rarely sustained (n = 6) and sometimes also concomitant with the emergence of new N1 genetic clades (n = 3). These data form a baseline from which we can identify N1 clades that expand in range or genetic diversity that may impact viral phenotypes or vaccine immunity and subsequently the health of North American swine.

evolutionary biology↗

bioRxiv2022

Independent evolution of influenza A virus H1N1 in pigs in Guatemala

Commercial swine farms provide unique systems for interspecies transmission of influenza A viruses (FLUAVs) at the animal-human interface. Bidirectional transmission of FLUAVs between pigs and humans plays a significant role in the generation of novel strains that become established in the new host population. Active FLUAV surveillance was conducted for two years on a commercial pig farm in Southern Guatemala with no history of FLUAV vaccination. Nasal swabs (n=2,094) from fattening pigs (6 to 24 weeks old) with respiratory signs were collected from May 2016 to February 2018. Swabs were screened for FLUAV by RRT-PCR and full virus genomes of FLUAV-positive swabs were sequenced by next-generation sequencing (NGS). FLUAV prevalence was 12.0% (95% CI: 10.6% - 13.4%) with two distinct periods of high infection. All samples were identified as FLUAVs of the H1N1 subtype within the H1 swine clade 1A.3.3.2 and whose ancestors are the human origin 2009 H1N1 influenza pandemic virus (H1N1 pdm09). Compared to the prototypic reference segment sequence, 10 amino acid signatures were observed on relevant antigenic sites on the hemagglutinin. We also found that Guatemalan swine-origin FLUAVs show independent evolution from other H1N1 pdm09 FLUAVs circulating in Central America. The zoonotic risk of these viruses remains unknown, but strongly calls for continued FLUAV surveillance in pigs in Guatemala. ImportanceDespite increased surveillance efforts, the epidemiology of FLUAVs circulating in swine in Latin America remains understudied. For instance, the 2009 H1N1 influenza pandemic strain (H1N1 pdm09) emerged in Mexico, but its circulation remained undetected in pigs. In Central America, Guatemala is the country with the largest swine industry. We found a unique group of H1N1 pdm09 sequences that suggests independent evolution from similar viruses circulating in Central America. These viruses may represent the establishment of a novel genetic lineage with the potential to reassort with other co-circulating viruses, and whose zoonotic risk remains to be determined.

evolutionary biology↗

bioRxiv2022

Transmission of human influenza A virus in pigs selects for adaptive mutations on the HA gene

Influenza A viruses (IAV) cause respiratory diseases in many host species, including humans and pigs. The spillover of IAV between swine and humans has been a concern for both public health and the swine industry. With the emergence of the triple reassortant internal gene (TRIG) constellation, establishment of human-origin IAVs in pigs has become more common, leading to increased viral diversity. However, little is known about the adaptation processes that are needed for a human-origin IAV to transmit and become established in pigs. We generated a reassortant IAV containing surface gene segments from a human IAV strain and internal gene segments from the 2009 pandemic and TRIG IAV lineages and demonstrated that it can replicate and transmit in pigs. Sequencing and variant calling analysis identified a mutant that emerged during replication in pigs, which was mapped to a region near the receptor binding site of the hemagglutinin (HA). The variant was present in all contact pigs and replicated more efficiently in differentiated swine tracheal cells compared to the virus containing the wildtype human-origin HA. These results show that variants are selected quickly after replication of human-origin HA in pigs, leading to improved fitness in the swine host, likely contributing to transmission. ImportanceInfluenza A viruses (IAV) cause respiratory disease in several species, including humans and pigs. The bidirectional transmission of IAV between humans and pigs plays a significant role in the generation of novel viral strains, greatly impacting viral epidemiology. However, little is known about the evolutionary processes that allow human IAV to become established in pigs. In this study, we generated reassortant viruses containing human seasonal HA and NA on different constellations of internal genes and tested their ability to replicate and transmit in pigs. We demonstrated that a virus containing a common internal gene constellation currently found in U.S. swine was able to transmit efficiently via the respiratory route. We identified a specific amino acid mutation that was fixed in the respiratory contact pigs that was associated with improved replication in primary swine tracheal epithelial cells, suggesting it was crucial for the transmissibility of the human virus in pigs.

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↗