Biology subjects

Nogales, A.

Publications and source records attributed to Nogales, A..

18 recordsLinked to original sources

bioRxiv2026

Genome-Scale Codon Deoptimization Enables Attenuation of Rift Valley Fever Virus

Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic pathogen responsible for severe disease in domestic and wild ungulates as well as humans, representing a major threat to livestock production and human public health. RVFV is endemic in many African countries and has the potential to spread to new geographical regions. Current vaccines have limitations in safety and efficacy, highlighting the need for strategies to develop new vaccines candidates. In this study, we explored the use of codon deoptimization (CD) as a novel attenuation approach for the development of live-attenuated vaccine (LAV) against RVFV. CD exploits the redundancy of the genetic code by replacing frequently used codons with synonymous, less-preferred codons, thereby reducing translational efficiency without altering the amino acid sequence. We recoded parts of the M and S genome segments of RVFV using the least frequently used codons in mammalian cells, ensuring complete preservation of protein functionality and immunogenicity. Using reverse genetics, we rescued a panel of recombinant (r)RVFV encoding codon-deoptimized S-segment NSs gene (rNScd), M-segment Gn/Gc genes (rMcd), or both (rMcd/NScd). These recombinant CD viruses were characterized in vitro in mammalian and insect cell lines and in vivo using wild-type and immunocompromised mice. Results demonstrated varying degrees of attenuation among the three CD rRVFV, with the one deoptimized in both viral segments, rMcd/NScd, as a promising LAV based on the safety profiles. This study provides proof of concept for the use of CD as a rational strategy to generate attenuated RVFV, for the development of next-generation vaccines against this zoonotic threat.

microbiology↗

bioRxiv2026

Enhanced Pathogenicity and Contact Transmissibility of Human-origin Avian Influenza H5N1 Clade 2.3.4.4b Genotype B3.13 Compared to D1.1 in Ferrets

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness. SignificanceInfluenza H5N1 viruses continue to diversify genetically while expanding into mammalian hosts, increasing opportunities for viral adaptation and zoonotic transmission, including humans. However, whether the predominant clade 2.3.4.4b genotype differs in its capacity to infect, transmit, and evolve in mammals remains poorly understood. Using the ferret model of influenza infection and transmission, we demonstrated that the currently circulating B3.13 and D1.1 genotypes exhibit distinct pathogenic and transmission characteristics despite retaining similar receptor-binding characteristics, NA functions, and antigenic profiles. While B3.13 readily infects and transmits in ferrets and does not develop further adaptive mutations associated with increased replication and transmission, D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential. These findings show that genotype-specific biological properties can influence zoonotic risk independently of antigenic similarity and emphasize the importance of integrating phenotypic characterization with genomic surveillance to improve pandemic preparedness and guide public health risk assessment.

microbiology↗

bioRxiv2026

A One-Shot Multivalent Live-Attenuated Candidate Influenza Vaccine against Divergent Zoonotic H5N1 Clades

The continued emergence of genetically diverse high pathogenicity avian influenza (HPAI) H5N1 viruses with zoonotic potential highlights the urgent need for developing vaccines capable of providing broad protection against multiple circulating clades. Here, we developed a one-shot, multivalent, live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive (ts), cold-adapted (ca), and attenuated (att) influenza A/Ann Arbor/6/1960 master donor virus (MDV) that incorporates the hemagglutinin (HA) and neuraminidase (NA) glycoproteins from representative clades 2.3.4.4b (A/Louisiana/12/2024), 2.3.2.1a (A/Victoria/149/2024), and 2.3.2.1e (A/Cambodia/2302009/2023) H5N1 viruses. A single intranasal (IN) immunization of C57BL/6 mice with the multivalent LAIV elicited robust humoral immune responses, with immune sera exhibiting broad cross-reactivity against antigens from all three H5N1 clades included in the vaccine. Following homologous viral challenge, vaccinated C57BL/6 mice were completely protected from disease, demonstrating the immunogenicity and protective efficacy of the multivalent LAIV. By simultaneously targeting antigenically distinct H5N1 lineages with pandemic potential, this strategy expands antigenic coverage within a single LAIV to confirm pan-H5N1 protection. Together, these findings support the development and implementation of this multivalent LAIV as a broadly protective pan-H5N1 LAIV for pandemic preparedness. SignificanceThe increasing genetic diversity of zoonotic H5N1 viruses complicates vaccine design. We developed a multivalent live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive, cold-adapted, and attenuated (ts, ca, att) master donor virus (MDV) influenza A/Ann Arbor/6/1960 backbone that expresses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins of H5N1 clades 2.3.4.4b, 2.3.2.1a, and 2.3.2.1e. A single intranasal (IN) immunization with the multivalent LAIV induced broadly cross-reactive neutralizing antibody (NAb) responses and protected experimental vaccinated animals against homologous lethal viral challenge, demonstrating the feasibility of the multivalent LAIV to protect against H5N1 clades of highest concern to humans. These findings demonstrate the feasibility of developing and implementing this multivalent LAIV as a broad protective pan-H5N1 LAIV against divergent H5N1 viruses for human use.

microbiology↗

bioRxiv2026

Neuraminidase-Based Cross-Protective Immunity against H5N1 Influenza Viruses in Humans

Highly pathogenic avian influenza A(H5N1) virus continues to expand globally across wild birds, poultry, and multiple mammalian hosts, and the detection of genetic features associated with mammalian adaptation underscores the need to better define preexisting human immunity relevant to pandemic preparedness. Although serologic and vaccine studies have historically emphasized hemagglutinin (HA), neuraminidase (NA) is a key antigen capable of providing protective immunity. Here, we performed a retrospective analysis of NA-specific antibodies in human sera collected across ten influenza seasons spanning the pre- and post-2009 pandemic periods. A total of 749 paired human serum samples (1,498 total) were obtained prior to seasonal inactivated influenza vaccination (T1) and 28 days after vaccination (T2). NA-inhibiting antibodies were measured by enzyme-linked lectin assay (ELLA) using N1 antigens representing three genotypes (B.3.13, D1.1, and EA-2021-AB) from clade 2.3.4.4b A(H5N1) viruses. In parallel, HA-directed responses were assessed by hemagglutination inhibition (HAI). In addition, luciferase-based microneutralization assays were performed to assess the presence of neutralizing antibodies. NA-inhibiting antibodies were detectable against all three genotypes across seasons. Seasonal vaccination was associated with a modest but reproducible increase in NA-inhibiting titers, most apparent from the 2009-2010 season onward, coincident with the introduction of A(H1N1)pdm09 into seasonal vaccine formulations. In contrast, HAI activity against H5 was generally low or undetectable, despite detectable HA-reactive antibodies by ELISA. These data indicate that cross-reactive NA-specific antibodies are present in human sera and can be boosted by seasonal vaccination, supporting increased consideration of NA in influenza serosurveillance and vaccine evaluation. IMPORTANCEThe continued spread of A(H5N1) viruses and reports of mammalian adaptation markers heighten concerns about potential zoonotic transmission into humans. Most studies of human antibody responses focus on influenza HA, but NA is also an important immune target. By analyzing paired sera from humans collected over ten influenza seasons, we show that NA-inhibiting antibodies cross-reacting with multiple contemporary genotypes from clade 2.3.4.4b A(H5N1) viruses are detectable and can be modestly boosted seasonal inactivated influenza vaccination, particularly after the 2009 pandemic. These findings highlight the importance of inducing NA-specific antibodies during influenza vaccination and in including NA in surveillance and in the assessment of vaccines intended to improve preparedness for emerging influenza viruses.

immunology↗

bioRxiv2025

Bioluminescent reporter influenza A viruses to track viral infections

Influenza A viruses (IAV) infect a wide range of mammal and bird species and are responsible for seasonal outbreaks and occasional pandemics. Studying IAV requires methods to detect the presence of the virus in infected cells or animal models. Recombinant IAV expressing fluorescent proteins have allowed monitoring viral infection in cultured cells and ex vivo in the organs of infected animals. However, fluorescent-expressing IAV are often attenuated and are not suited for the imaging of infected animals using in vivo imaging systems (IVIS). To overcome this limitation, we generated a recombinant A/California/04/2009 H1N1 (pH1N1) expressing nanoluciferase (Nluc) from the non-structural (NS) viral segment (pH1N1-Nluc) that replicates efficiently in vitro, with growth kinetics and plaque morphology comparable to wild-type pH1N1 (pH1N1-WT). We used this pH1N1-Nluc to demonstrate its ability to identify neutralizing antibodies and antivirals, with neutralization and inhibition results comparable to pH1N1-WT. In mice, pH1N1-Nluc was able to induce similar body weight loss and mortality, and viral titers comparable to pH1N1-WT, results that were recapitulated in a ferret model of IAV infection. Using IVIS, pH1N1-Nluc enabled non-invasive, real-time tracking of viral infection in vivo and ex vivo following infection of mice with viral titers comparable to pH1N1-WT. The flexibility of this approach was further demonstrated by the generation of a Nluc-expressing recombinant A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Altogether, our results demonstrate that Nluc-expressing recombinant IAV represent a valuable tool for in vitro and in vivo studies, including the identification of antivirals and/or neutralizing antibodies, and to assess protective efficacy of vaccines. IMPORTANCEDespite the availability of recombinant influenza A viruses (IAV) expressing fluorescent reporter genes to track viral infections in vitro and ex vivo, these viruses are often attenuated and do not represent the best option for imaging entire animals using in vivo imaging systems (IVIS). To solve this limitation, we generated recombinant influenza pandemic A/California/04/2009 H1N1 expressing nanoluciferase (pH1N1-Nluc) from the viral non-structural (NS) segment and demonstrate how expression of Nluc does not affect viral replication in vitro or viral pathogenesis in vivo. Importantly, we demonstrate the feasibility of detecting pH1N1-Nluc infection in vivo using IVIS. We also validate the flexibility of this approach by generating an influenza A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Our results support the feasibility of using these recombinant IAVs expressing Nluc from the NS segment for in vitro and in vivo studies, including the identification of neutralizing antibodies and/or antivirals, and to assess protective efficacy of vaccines.

microbiology↗

bioRxiv2025

Targeting ROCK Signaling Mitigates Influenza Virus-Induced Fibrogenesis in Human Airway Organoids

Influenza A virus (IAV) pandemics continue to pose serious global health threats, particularly to immunocompromised individuals, children, and the elderly. IAV infections trigger inflammation and tissue damage, promoting lung fibrosis. Unraveling these mechanisms is key to preventing and treating viral-induced pulmonary fibrosis and its lasting impact on respiratory health. Despite available antivirals and vaccines, there is a lack of FDA-approved therapeutics for severe or prolonged IAV pathogenesis. We modeled infection with a recombinant highly pathogenic human A/Texas/37/2024 H5N1 (rHPh-TX H5N1) strain using human airway organoids (HAO) to investigate viral replication, innate immune response, infection-induced fibrogenesis, and therapeutic approaches. The rHPh-TX H5N1 replicated efficiently, triggering a potent interferon (IFN) response and pro-inflammatory cytokine expression in HAO. Prolonged infection led to increased fibroblast-like cells surrounding infected areas, characterized by increased alpha-smooth muscle actin (-SMA) expression and upregulation of transforming growth factor-beta (TGF-{beta}), which caused fibroblast activation and extracellular matrix remodeling. Fibrosis-associated markers (FN, COL1A, COL3A, MMP2, MMP9) were significantly higher than in HAO infected with a pandemic recombinant A/California/04/09 H1N1 (pH1N1). Notably, Rho-associated coiled-coil-forming protein kinase (ROCK) pathway inhibition reduced fibrogenesis, with ROCK1 inhibition proving more effective than ROCK2 inhibition. These findings highlight the potential of targeting ROCK signaling to mitigate IAV-induced lung fibrosis.

microbiology↗

bioRxiv2025

A Live Attenuated Vaccine Candidate against Emerging Highly Pathogenic Cattle-Origin 2.3.4.4b H5N1 Viruses

Influenza viruses present a significant public health risk, causing substantial illness and death in humans each year. Seasonal flu vaccines must be updated regularly, and their effectiveness often decreases due to mismatches with circulating strains. Furthermore, inactivated vaccines do not provide protection against shifted influenza viruses that have the potential to cause a pandemic. The highly pathogenic avian influenza H5N1 clade 2.3.4.4b is prevalent among wild birds worldwide and is causing a multi-state outbreak affecting poultry and dairy cows in the United States (US) since March 2024. In this study, we have generated a NS1 deficient mutant of a low pathogenic version of the cattle-origin human influenza A/Texas/37/2024 H5N1, namely LPhTXdNS1, and validated its safety, immunogenicity, and protection efficacy in a prime vaccination regimen against wild-type (WT) A/Texas/37/2024 H5N1. The attenuation of LPhTXdNS1 in vitro was confirmed by its reduced replication in cultured cells and inability to control IFN{beta} promoter activation. In C57BL/6J mice, LPhTXdNS1 has reduced viral replication and pathogenicity compared to WT A/Texas/37/2024 H5N1. Notably, LPhTXdNS1 vaccinated mice exhibited high immunogenicity that reach its peak at weeks 3 and 4 post-immunization, leading to robust protection against subsequent lethal challenge with WT A/Texas/37/2024 H5N1. Altogether, we demonstrate that a single dose vaccination with LPhTXdNS1 is safe and able to induce protective immune responses against H5N1. Both safety profile and protection immunity suggest that LPhTXdNS1 holds promise as a potential solution to address the urgent need for an effective vaccine in the event of a pandemic for the treatment of infected animals and humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/646033v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ca0d0borg.highwire.dtl.DTLVardef@1e5232org.highwire.dtl.DTLVardef@a6694corg.highwire.dtl.DTLVardef@14b0832_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

A Human H5N1 Influenza Virus Expressing Bioluminescence for Evaluating Viral Infection and Identifying Therapeutic Interventions

A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in dairy cows was first reported on March 25, 2024, in the United States (US), marking the first discovery of HPAIV H5N1 in cattle. Soon after, a dairy worker on an affected dairy farm became the first human case linked directly to this outbreak. Studies with influenza A virus (IAV) require secondary methods to detect the virus in infected cells or animal models of infection. We modified the non-structural (NS) genome segment of the human A/Texas/37/2024 (HPhTX) H5N1 virus to create a recombinant virus expressing nanoluciferase (HPhTX NSs-Nluc), enabling the tracking of virus in cultured cells and mice via in vitro, ex vivo, and in vivo imaging systems (IVIS). In vitro, HPhTX NSs-Nluc showed growth and plaque characteristics similar to its wild-type (WT) counterpart. In vivo, HPhTX NSs-Nluc allowed tracking viral infection in the entire animals and in the organs of infected animals using in vivo and ex vivo IVIS, respectively. Importantly, the morbidity, mortality, and replication titers of HPhTX NSs-Nluc were comparable to those of the WT HPhTX. In vitro, HPhTX NSs-Nluc was inhibited by Baloxavir acid (BXA) to levels observed with WT HPhTX. We also demonstrate the feasibility of using HPhTX NSs-Nluc to evaluate the antiviral activity of BXA in vivo. Our findings support that HPhTX NSs-Nluc represents an excellent tool for tracking viral infections, including the identification of prophylactics or therapeutics for the treatment of the HPAIV H5N1 responsible of the outbreak in dairy cows.

microbiology↗

bioRxiv2025

Generation and characterization of an ovine cell line derived from peripheral blood and its potential use to study livestock and zoonotic viral infections

BackgroundMesenchymal stromal cells (MSCs) are a population of undifferentiated non-hematopoietic fibroblast-like cells isolated from several tissues with multipotent differentiation capacity in vitro. This study focused on the establishment and characterization of a mesenchymal stromal cell line derived from ovine peripheral blood mononuclear cells (PBMCs). MethodsMSC were isolated from ovine blood and used to develop a cell line. The characterization of the cell line to confirm its mesenchymal origin was carried out by different assays. First at all, cells were characterized by flow cytometry (FACS) using monoclonal antibodies specific for MSC and hematopoietic non-stromal cell surface antigens. Real-time quantitative PCR (RT-qPCR) was performed using gene primer pairs specific for hematopoietic and mesenchymal cell surface markers. In addition, we assayed the potential of the cell line to differentiate in vitro to other lineages such as osteoblasts and neurons and confirmed that by specific staining with Alizarin Red (AR) and RT-qPCR and immunofluorescence (IFA). Finally, we assayed the susceptibility of the cell line to various livestock, animal and human viruses. ResultsFACS and RT-qPCR analyses revealed that the ovine cell line expressed mesenchymal markers and was negative for hematopoietic markers assayed. MSC were able to differentiate to osteoblasts and neurons, these results were confirmed by quantitative analyses of AR staining and RT-qPCR and IFA assays. The cell line showed susceptibility to infection with all viruses assayed confirmed by IFA asssays. ConclusionsIn this work, we used isolated MSCs from peripheral blood to establish a new ovine cell line. The characterization and the osteogenic and neuronal differentiation carried out confirmed that the cell line had a mesenchymal stromal origin. In fact, the new established ovine cell line was permissive to all viruses studied in this work. The new sheep cell line could be a useful tool for isolating and characterizing viruses and studying virus-host interactions.

cell biology↗

bioRxiv2024

Are we serologically prepared against an avian influenza pandemic and could seasonal flu vaccines help us?

The current situation with H5N1 highly pathogenic avian influenza virus (HPAI) is causing a worldwide concern due to multiple outbreaks in wild birds, poultry, and mammals. Moreover, multiple zoonotic infections in humans have been reported. Importantly, HPAI H5N1 viruses with genetic markers of adaptation to mammals have been detected. Together with HPAI H5N1, avian influenza viruses H7N9 (high and low pathogenic) stand out due to their high mortality rates in humans. This raises the question of how prepared we are serologically and whether seasonal vaccines are capable of inducing protective immunity against these influenza subtypes. An observational study was conducted in which sera from people born between years 1925-1967, 1968-1977, and 1978-1997 were collected before or after 28 days or 6 months post-vaccination with an inactivated seasonal influenza vaccine. Then, haemagglutination inhibition, viral neutralization, and immunoassays were performed to assess the basal protective immunity of the population as well as the ability of seasonal influenza vaccines to induce protective responses. Our results indicate that subtype-specific serological protection against H5N1 and H7N9 in the representative Spanish population evaluated was limited or nonexistent. However, seasonal vaccination was able to increase the antibody titers to protective levels in a moderate percentage of people, probably due to cross-reactive responses. These findings demonstrate the importance of vaccination and suggest that seasonal influenza vaccines could be used as a first line of defense against an eventual pandemic caused by avian influenza viruses, to be followed immediately by the use of more specific pandemic vaccines. ImportanceInfluenza A viruses (IAV) can infect and replicate in multiple mammalian and avian species. Avian influenza virus (AIV) is a highly contagious viral disease that occurs primarily in poultry and wild water birds. Due to the lack of population immunity in humans and ongoing evolution of AIV, there is a continuing risk that new IAV could emerge and rapidly spread worldwide causing a pandemic, if the ability to transmit efficiently among humans was gained. The aim of this study is to analyze the basal protection and presence of antibodies against IAV H5N1, and H7N9 subtypes in the population from different ages. Moreover, we have evaluated the h humoral response after immunization with a seasonal influenza vaccine. This study is strategically important to evaluate the level of population immunity that is a major factor when assessing the impact that an emerging IAV strain would have, and the role of seasonal vaccines to mitigate the effects of a pandemic.

immunology↗

bioRxiv2024

Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic disease that causes severe disease in both domestic and wild ungulates and humans, making it a significant threat to livestock and public health. The RVFV genome consists of three single-stranded, negative-sense RNA segments differing in size: Small (S), Medium (M) and Large (L). Segment S encodes the virus nucleoprotein N and the virulence-associated factor non-structural (NSs) protein in opposite orientations, separated by an intergenic region (IGR). To overcome the current need of using secondary techniques to detect the presence of RVFV in infected cells, we used T7-driven polymerase plasmid-based reverse genetics to generate replication-competent recombinant (r)RVFV expressing Nanoluciferase (Nluc) or Venus fluorescent proteins. These reporter genes were used as valid surrogates to track the presence of RVFV in mammalian and insect cells. Notably, we explored the genome plasticity of RVFV and compared four different strategies by modifying the viral segment S in order to introduce the reporter gene foreign sequences. The reporter-expressing rRVFV were stable and able to replicate in cultured mammalian and insect cells, although to a lesser extent than the recombinant wild-type (WT) counterpart. Moreover, rRVFV expressing reporter genes were validated to identify neutralizing antibodies or compounds with antiviral activity. In vivo, all mice infected with the reporter-expressing rRVFV displayed an attenuated phenotype, although at different levels. These rRVFV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of RVFV, and represent an excellent biotechnological tool for developing new therapeutics against RVFV infections. IMPORTANCERift Valley fever virus (RVFV) is a mosquito-borne virus and zoonotic agent threat that can be deadly to domestic or wild ungulates, and humans. In this work, we used reverse genetics approaches to explore the genome plasticity of RVFV by generating a set of recombinant rRVFV that express fluorescent or luminescent proteins to track viral infection. All the generated reporter-expressing rRVFV were able to propagate in mammalian or insect cells, and in a mouse model of infection. Our studies may contribute to advances in research on RVFV and other bunyaviruses and pave the way for the development of novel vaccines and the identification of new antivirals for the prophylactic and therapeutic treatment, respectively, of RVFV infections.

microbiology↗

bioRxiv2023

Antivirals against monkeypox infections

Monkeypox virus (MPXV) infection in humans are historically restricted to endemic regions in Africa. However, in 2022, an alarming number of MPXV cases have been reported globally with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. MPXV vaccines are limited and only two antivirals, tecovirimat and brincidofovir, approved by the United States (US) Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit Orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (Scarlet or GFP) and luciferase (Nluc) reporter genes to identify compounds with anti-Orthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed antiviral activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating the broad-spectrum antiviral activity against Orthopoxviruses and their potential to be used for the antiviral treatment of MPXV, or other Orthopoxvirus, infections. IMPORTANCEDespite the eradication of smallpox, some Orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, there is presently limited access to those vaccines. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV, and other potentially zoonotic Orthopoxvirus infections. Here, we show that thirteen compounds, derived from two different libraries, previously found to inhibit several RNA viruses, exhibit also antiviral activity against VACV. Notably, eleven compounds also displayed antiviral activity against MPXV, demonstrating their potential to be incorporated into the therapeutic armamentarium to combat Orthopoxvirus infections.

microbiology↗

bioRxiv2022

Vaccinia virus attenuation by codon deoptimization of the A24R gene for vaccine development

Poxviruses have large DNA genomes and they are able to infect multiple vertebrate and invertebrate animals, including humans. Despite the eradication of smallpox, poxvirus infections still remain a significant public health concern. Vaccinia virus (VV) is the prototypic member in the poxviridae family and it has been used extensively for different therapeutic applications, including the generation of vaccines against multiple infectious diseases and/or for oncolytic treatment. Many attempts have been pursued to develop novel attenuated forms of VV with improved safety profiles for their implementation as vaccines and/or vaccines vectors. We and others have previously demonstrated how RNA viruses encoding codon-deoptimized viral genes are attenuated, immunogenic and able to protect, upon a single administration, against challenge with parental viruses. In this study, we employed the same experimental approach based on the use of misrepresented codons for the generation of a recombinant (r)VV encoding a codon-deoptimized A24R gene, which is a key component of the viral RNA polymerase. Similar to our previous studies with RNA viruses, the A24R codon-deoptimized rVV (v-A24cd) was highly attenuated in vivo but able to protect, after a single intranasal dose administration, against an otherwise lethal challenge with parental VV. These results indicate that poxviruses can be effectively attenuated by synonymous codon deoptimization and open the possibility of using this methodology alone or in combination with other experimental approaches for the development of attenuated vaccines for the treatment of poxvirus infection, or to generate improved VV-based vectors. Moreover, this approach could be applied to other DNA viruses. IMPORTANCEThe family poxviridae includes multiple viruses of medical and veterinary relevance, being vaccinia virus (VV) the prototypic member in the family. VV was used during the smallpox vaccination campaign to eradicate variola virus (VARV), which is considered a credible bioterrorism threat. Because of novel innovations in genetic engineering and vaccine technology, VV has gained popularity as a viral vector for the development of vaccines against several infectious diseases. Several approaches have been used to generate attenuated VV for its implementation as vaccine and/or vaccine vector. Here, we generated a rVV containing a codon-deoptimized A24R gene (v-A24cd), which encodes a key component of the viral RNA polymerase. v-A24cd was stable in culture cells and highly attenuated in vivo but able to protect against a subsequent lethal challenge with parental VV. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated VV and/or vaccine vectors.

microbiology↗

bioRxiv2022

A single amino acid substitution in PB1 of pandemic H1N1 with A/Ann Arbor/6/60 master donor virus mutations as a novel live-attenuated influenza virus vaccine

Influenza A viruses (IAV) remain emerging threats to human public health. Live-attenuated influenza vaccines (LAIV) are one of the most effective prophylactic options to prevent disease caused by influenza infections. However, licensed LAIV remain restricted for use in 2- to 49-year old healthy and non-pregnant people. Therefore, development of LAIV with increased safety, immunogenicity, and protective efficacy is highly desired. The United States (U.S.) licensed LAIV is based on the master donor virus (MDV) A/Ann Arbor/6/60 H2N2 backbone, which was generated by adaptation of the virus to growth at low temperatures. Introducing the genetic signature of the U.S. MDV into the backbone of other IAV strains resulted in varying levels of attenuation. While the U.S. MDV mutations conferred an attenuated phenotype to other IAV strains, the same amino acid changes did not significantly attenuate the pandemic A/California/04/09 H1N1 (pH1N1) strain. To attenuate pH1N1, we replaced the conserved leucine at position 319 with glutamine (L319Q) in PB1 and analyzed the in vitro and in vivo properties of pH1N1 viruses containing either PB1 L319Q alone or in combination with the U.S. MDV mutations using two animal models of influenza infection and transmission, ferrets and guinea pigs. Our results demonstrated that L319Q substitution in the pH1N1 PB1 alone or in combination with the mutations of the U.S. MDV resulted in reduced pathogenicity (ferrets) and transmission (guinea pigs), and an enhanced temperature sensitive phenotype. These results demonstrate the feasibility of generating an attenuated MDV based on the backbone of a contemporary pH1N1 IAV strain. IMPORTANCEVaccination represents the most effective strategy to reduce the impact of seasonal IAV infections. Although LAIV are superior in inducing protection and sterilizing immunity, they are not recommended for many individuals who are at high risk for severe disease. Thus, development of safer and more effective LAIV are needed. A concern with the current MDV used to generate the U.S. licensed LAIV is that it is based on a virus isolated in 1960. Moreover, mutations that confer the temperature sensitive, cold-adapted, and attenuated phenotype of the U.S. MDV resulted in low level of attenuation in the contemporary pandemic A/California/04/09 H1N1 (pH1N1). Here, we show that introduction of PB1 L319Q substitution, alone or in combination with the U.S. MDV mutations, resulted in pH1N1 attenuation. These findings support the development of a novel LAIV MDV based on a contemporary pH1N1 strain as a medical countermeasure against currently circulating H1N1 IAV.

microbiology↗

bioRxiv2021

Bi-reporter vaccinia virus for tracking viral infections in vitro and in vivo

Recombinant viruses expressing reporter genes allow visualization and quantification of viral infections and can be used as valid surrogates to identify the presence of the virus in infected cells and animal models. However, one of the limitations of recombinant viruses expressing reporter genes is the use of either fluorescent or luciferase proteins that are used alternatively for different purposes. Vaccinia virus (VV) is widely used as a viral vector, including recombinant (r)VV singly expressing either fluorescent or luciferase reporter genes that are useful for specific purposes. In this report, we engineered two novel rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes from different loci in the viral genome. In vitro, these bi-reporter expressing rVV have similar growth kinetics and plaque phenotype than those of the parental WR VV isolate. In vivo, rVV Nluc/Scarlet and rVV Nluc/GFP effectively infected mice and were easily detected using in vivo imaging systems (IVIS) and ex vivo in the lungs from infected mice. We used these bi-reporter expressing rVV to assess viral pathogenesis, infiltration of immune cells in the lungs, and to directly identify the different subsets of cells infected by VV in the absence of antibody staining. Collectively, these rVV expressing two reporter genes open the feasibility to study the biology of viral infections in vitro and in vivo, including host-pathogen interactions and dynamics or tropism of viral infections. Moreover, they represent an excellent approach for the discovery of new prophylactics and/or therapeutics for the treatment of poxvirus infections. IMPORTANCEDespite the eradication of variola virus (VARV), the causative agent of smallpox, poxviruses still represent an important threat to human health due to their possible use as bioterrorism agents and the emergence of zoonotic poxvirus diseases. Recombinant vaccinia viruses (rVV) expressing easily traceable fluorescent or luciferase reporter genes have significantly contributed to the progress of poxvirus research. However, rVV expressing one marker gene have several constraints for in vitro and in vivo studies, since both fluorescent and luciferase proteins impose certain limitations for specific applications. To overcome these limitations, we generated optimized rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes to easily track viral infection in vitro and in vivo. This new generation of double reporter-expressing rVV represent an excellent option to study viral infection dynamics in cultured cells and validated animal models of infection, and for the discovery of new poxvirus antiviral treatments.

microbiology↗

bioRxiv2021

Amino acid residues involved in inhibition of host gene expression by influenza A/Brevig Mission/1/1918 PA-X

Influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and a plasmid encoding the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-Xs shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression.

microbiology↗

bioRxiv2020

A broad and potent H1-specific human monoclonal antibody produced in plants prevents influenza virus infection and transmission in guinea pigs

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift, respectively. Previously, we have described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV) in vitro, and prophylactic and therapeutic activities in vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrate how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian produced KPF1-HEK hMAb. KPF1-Antx hMAb shows broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, that are comparable to those observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrates, for the first time, that plant-produced influenza hMAbs have similar in vitro and in vivo biological properties to those produced in mammalian cells. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.

immunology↗

bioRxiv2019

Influenza Viruses in Mice: Deep Sequencing Analysis of Serial Passage and Effects of Sialic Acid Structural Variation

Influenza A viruses have regularly jumped to new hosts to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus, but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here we examine the processes of influenza A virus infection and evolution in mice by comparing deep sequence variation of a human H1N1 pandemic virus, a seasonal H3N2 virus, and a H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with that seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc.\n\nSIGNIFICANCEMice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals, but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not effect influenza evolution in this model. Overall this shows that mice provide a useful animal model for influenza, but that host passage evolution will vary depending on the virus being tested.

microbiology↗