Biology subjects

Meliopoulos, V. A.

Publications and source records attributed to Meliopoulos, V. A..

12 recordsLinked to original sources

bioRxiv2025

Susceptibility of bovine respiratory and mammary epithelial cells to avian and mammalian derived clade 2.3.4.4b H5N1 highly pathogenic avian influenza viruses

Zoonotic transmission of avian influenza viruses into mammals is relatively rare due to anatomical differences in the respiratory tract between species. Recently, clade 2.3.4.4b highly pathogenic H5N1 avian influenza viruses were detected circulating in North American cattle. Sporadic transmission between cattle, humans, and other animals proximal to cattle or after consuming products from infected cattle has occurred, but thus far there is no evidence of human-to-human transmission. However, the virus has the potential to adapt to the mammalian respiratory tract with every transmission event that occurs, making it crucial to understand cellular and species tropism of the H5N1 2.3.4.4b viruses. We compared viral kinetics of clade 2.3.4.4b viruses isolated from birds and mammals in respiratory epithelial cells derived from cattle, human, swine, and ferret. We found that avian derived viruses could replicate in swine cells only, yet mammalian derived strains could replicate efficiently in all tracheal and nasal epithelial cells tested. Interestingly, only bovine mammary epithelial cells (MEC) and swine respiratory epithelial cells were permissive to both avian and mammalian derived strains, possibly due to increased sialic acid expression on bovine MEC compared to bovine tracheal epithelial cells (TEC). However, sialic acid expression differed between dairy and beef cows: TEC derived from a dairy cow had increased expression of 2,3 sialic acid receptors compared to TEC from a beef-dairy cow cross. This study highlights the ability of clade 2.3.4.4b H5N1 viruses derived from mammals but not wild birds to infect the respiratory epithelium of other mammalian hosts.

microbiology↗

bioRxiv2024

Intraductal infection with H5N1 clade 2.3.4.4b influenza virus

The ongoing outbreak of highly pathogenic avian influenza (HPAI) H5N1 of the clade 2.3.4.4b in dairy cows has led to numerous questions including how the virus transmits amongst cattle given the limited respiratory infection. One hypothesis is that the virus is spreading through fomites from udder to udder. In these studies, we demonstrate that intraductal inoculation of H5N1 but not H1N1 influenza virus results in infection in mice. This model will be useful to our understanding of the impact of influenza virus on the mammary gland, the potential as a new route of transmission, and can be used to assess if antiviral treatments prevent infections in the mammary gland.

microbiology↗

bioRxiv2024

Immune History Modifies Disease Severity to HPAI H5N1 Clade 2.3.4.4b Viral Challenge

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity--either from LAIV vaccination and/or from Cal09 infection--showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

microbiology↗

bioRxiv2024

Effects of Oral Exposure to HPAI H5N1 Pasteurized in Milk on Immune Response and Mortality in Mice

In March 2024, there was the first reported outbreak of a highly pathogenic avian H5N1 influenza (HPAI) clade 2.3.4.4b virus in dairy cows in the United States. Since then, there have been several spillover events to cats, poultry, and humans. Multiple reports have discovered infectious virus in raw milk from infected dairy cows. Infectious virus can also last over a period on milking machine surfaces as a potential route of spread in cattle and contamination in raw milk. While the U.S. Food and Drug Administration has cleared commercial pasteurized milk as safe for consumption given the lack of infectious virus, there have been numerous reports that up to 30 percent of commercial milk tested were positive for HPAI H5N1 influenza virus genome copies. This is not necessarily unique to the HPAI H5N1 virus, as retrospective studies have identified H1N1 and H3N2 seropositivity in cows linked to decreased milk production. However, it is unknown how repeat exposure to the remaining viral proteins and genomic material in pasteurized milk modulates immune responses once ingested. We developed a successful in-house pasteurization protocol that inactivated high viral loads of the pandemic H1N1 strain A/California/04/2009 (Cal09) or bovine-derived HPAI H5N1 (A/bovine/Ohio.B24OSU-439/2024) viruses in raw milk. Mice were administered this milk daily for five days and rechallenged with each respective virus. We found that repeated oral exposure to inactivated virus was not sufficient to prevent or accelerate mortality from lethal challenge of HPAI H5N1, though it did result in a [~]0.5 log10 reduction viral titers in the brain and delayed clinical signs. In contrast, oral gavage of mice with pre-existing immunity to H1N1 influenza virus with virus pasteurized in milk were protected from morbidity and mortality upon bovine H5N1 viral challenge. These findings suggest that ingestion of inactivated HPAI H5N1 has limited potential health risk and does not prevent protective immune history-mediated responses to lethal infection.

microbiology↗

bioRxiv2023

Maternal immunization with distinct influenza vaccine platforms elicits unique antibody profiles that impact the protection of offspring

Pregnant women and infants are considered high-risk groups for increased influenza disease severity. While influenza virus vaccines are recommended during pregnancy, infants cannot be vaccinated until at least six months of age. Passive transfer of maternal antibodies (matAbs) becomes vital for the infants protection. Here, we employed an ultrasound-based timed-pregnancy murine model and examined matAb responses to distinct influenza vaccine platforms and influenza A virus (IAV) infection in dams and their offspring. We demonstrate vaccinating dams with a live-attenuated influenza virus (LAIV) vaccine or recombinant hemagglutinin (rHA) proteins administered with adjuvant resulted in enhanced and long-lasting immunity and protection from influenza in offspring. In contrast, a trivalent split-inactivated vaccine (TIV) afforded limited protection in our model. By cross-fostering pups, we show the timing of antibody transfer from vaccinated dams to their offspring (prenatal versus postnatal) can shape the antibody profile depending on the vaccine platform. Our studies provide information on how distinct influenza vaccines lead to immunogenicity and efficacy during pregnancy, impact the protection of their offspring, and detail roles for IgG1 and IgG2c in the development of vaccine administration during pregnancy that stimulate and measure expression of both antibody subclasses.

microbiology↗

bioRxiv2023

Diet-induced obesity impacts influenza disease severity and transmission dynamics in ferrets

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a novel diet-induced obese ferret model and new tools to demonstrate that like humans, obesity resulted in significant changes to the lung microenvironment leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for longer making them more likely to transmit to contacts. These data suggest the obese ferret model may be crucial to understanding obesitys impact on influenza disease severity and community transmission, and a key tool for therapeutic and intervention development for this high-risk population. TeaserA new ferret model and tools to explore obesitys impact on respiratory virus infection, susceptibility, and community transmission.

microbiology↗

bioRxiv2023

The beta 6 Integrin Negatively Regulates TLR7-Mediated Epithelial Immunity via Autophagy During Influenza A Virus Infection

Integrins are essential surface receptors that sense extracellular changes to initiate various intracellular signaling cascades. The rapid activation of the epithelial-intrinsic {beta}6 integrin during influenza A virus (IAV) infection has been linked to innate immune impairments. Yet, how {beta}6 regulates epithelial immunity remains undefined. Here, we identify the role of {beta}6 in mediating the Toll-like receptor 7 (TLR7) through the regulation of intracellular trafficking. We demonstrate that deletion of the {beta}6 integrin in lung epithelial cells significantly enhances the TLR7-mediated activation of the type I interferon (IFN) response during homeostasis and respiratory infection. IAV-induced {beta}6 facilitates TLR7 trafficking to lysosome-associated membrane protein (LAMP2a) components, leading to a reduction in endosomal compartments and associated TLR7 signaling. Our findings reveal an unappreciated role of {beta}6-induced autophagy in influencing epithelial immune responses during influenza virus infection.

immunology↗

bioRxiv2023

Host obesity impacts genetic variation in influenza A viral populations

Obesity is a chronic health condition characterized by excess adiposity leading to a systemic increase in inflammation and dysregulation of metabolic hormones and immune cell populations. Obesity is well established as a risk factor for many noncommunicable diseases; however, its consequences for infectious disease are poorly understood. Influenza A virus (IAV) is a highly infectious pathogen responsible for seasonal and pandemic influenza. Host risk factors, including compromised immunity and pre-existing health conditions, can contribute to increased infection susceptibility and disease severity. During viral replication in a host, the negative sense single stranded RNA genome of IAV accumulates genetic diversity that may have important consequences for viral evolution and transmission. Here, we investigated the impact of host obesity on IAV genetic variation using a diet induced obesity ferret model. We infected obese and lean male ferrets with the A/Hong Kong/1073/1999 (H9N2) IAV strain. Using a co-caging study design, we investigated the maintenance, generation, and transmission of intrahost IAV genetic variation by sequencing viral genomic RNA obtained from nasal wash samples over multiple days of infection. We found evidence for an enhanced role of positive selection acting on de novo mutations in obese hosts that led to nonsynonymous changes that rose to high frequency. In addition, we identified numerous cases of recurrent low-frequency mutations throughout the genome that were specific to obese hosts. Despite these obese-specific variants, overall viral genetic diversity did not differ significantly between obese and lean hosts. This is likely due to the high supply rate of de novo variation and common evolutionary adaptations to the ferret host regardless of obesity status, which we show are mediated by variation in the hemagglutinin (HA) and polymerase genes (PB2 and PB1). As with single nucleotide variants, we identified a class of defective viral genomes (DVGs) that were found uniquely in either obese or lean hosts, but overall DVG diversity and dynamics did not differ between the two groups. Our study provides the first insight into the consequences of host obesity on viral genetic diversity and adaptation, suggesting that host factors associated with obesity alter the selective environment experienced by a viral population, thereby impacting the spectrum of genetic variation.

microbiology↗

bioRxiv2023

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

cell biology↗

bioRxiv2023

Astrovirus replication is dependent on induction of double membrane vesicles through a PI3K-dependent, LC3-independent pathway

Human astrovirus is a positive sense, single stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double membrane vesicles (DMVs). Here we show that astrovirus infection leads to an increase in DMV formation, and this process is replication-dependent. Our data suggest that astrovirus infection induces rearrangement of endoplasmic reticulum fragments, which may become the origin for DMV formation. Transcriptional data suggested that formation of DMVs during astrovirus infection requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Inhibition of the PI3K complex leads to significant reduction in viral replication and release from cells. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. ImportanceThese studies provide critical new evidence that astrovirus replication requires formation of double membrane vesicles, which utilize class III PI3K, but not LC3 conjugation autophagy machinery for biogenesis. These results are consistent with replication mechanisms for other positive sense RNA viruses. This suggests that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive sense RNA virus infections.

microbiology↗

bioRxiv2020

Respiratory bacteria stabilize and promote airborne transmission of influenza A virus

Influenza A virus (IAV) is a major pathogen of the human respiratory tract where the virus co-exists and interacts with bacterial populations comprising the respiratory microbiome. Synergies between IAV and respiratory bacterial pathogens promote enhanced inflammation and disease burden that exacerbate morbidity and mortality. We demonstrate that direct interactions between IAV and encapsulated bacteria commonly found in the respiratory tract promote environmental stability and infectivity of IAV. Antibiotic-mediated depletion of the respiratory bacterial flora abrogated IAV transmission in ferret models, indicating that these viral-bacterial interactions are operative for airborne transmission of IAV. Restoring IAV airborne transmission in antibiotic treated ferrets by co-infection with Streptococcus pneumoniae confirmed a role for specific members of the bacterial respiratory community in promoting IAV transmission. These results implicate a role for the bacterial respiratory flora in promoting airborne transmission of IAV.

microbiology↗

bioRxiv2019

HA stability regulates H1N1 influenza virus replication and pathogenicity in mice by modulating type I interferon responses in dendritic cells

Hemagglutinin (HA) stability, or the pH at which the HA is activated to cause membrane fusion, has been associated with the replicative fitness, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated several mechanisms by which a destabilizing HA mutation, Y17H (activation pH 6.0), may attenuate virus replication and pathogenicity in DBA/2 mice, compared to wild-type (WT; activation pH 5.5). Extracellular lung pH was measured to be near neutral (pH 6.9-7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate Y17H virus. The Y17H virus had accelerated single-step replication kinetics in MDCK, A549, and Raw264.7 cells. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced multistep replication in murine airway mNEC and mTEC cultures and attenuated virus replication, virus spread, severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in greater activation of antiviral responses, including type I IFN. These studies reveal HA stability may regulate pathogenicity by modulating IFN responses.\n\nImportanceDiverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza. Understanding HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.

microbiology↗