Publications by Year: 2025

The family Poxviridae comprises multiple viruses with large double-stranded (ds) DNA genomes that can infect numerous vertebrate and invertebrate hosts, including humans. The development of genetic engineering methods for Vaccinia virus (VACV), the prototypic member in the family, have allowed the manipulation of the genomes of poxviruses for the generation of recombinant (r)VACV expressing easily traceable luciferase and/or fluorescent reporter genes. These recombinant viruses have significantly contributed to progress in the field of poxvirus research and accelerated the development of novel prophylactic vaccines and therapeutic antiviral treatments. Recently, we described two reporter rVACV expressing luciferase (Nluc) and fluorescent (GFP or Scarlet) proteins to easily track viral infections in different systems, overcoming the limitations associated with the use of rVACV expressing a single luciferase or fluorescent reporter gene. Here, we describe the experimental procedures to carry out in vitro, in vivo and ex vivo studies using these novel bireporter-expressing rVACV, which also represent an excellent option to study the biology of VACV, including the use of these reporter viruses for testing new antivirals and vaccines, using cultured cells and/or well-characterized animal models of infection.

The ongoing monkeypox (mpox) disease outbreak has spread to multiple countries in Central Africa and evidence indicates it is driven by a more virulent clade I monkeypox virus (MPXV) strain than the clade II strain associated with the 2022 global mpox outbreak, which led the WHO to declare this mpox outbreak a public health emergency of international concern. The FDA-approved small molecule antiviral tecovirimat (TPOXX) is recommended to treat mpox cases with severe symptoms, but the limited efficacy of TPOXX and the emergence of TPOXX resistant MPXV variants has challenged this medical practice of care and highlighted the urgent need for alternative therapeutic strategies. In this study we have used vaccinia virus (VACV) as a surrogate of MPXV to assess the antiviral efficacy of combination therapy of TPOXX together with mycophenolate mofetil (MMF), an FDA-approved immunosuppressive agent that we have shown to inhibit VACV and MPXV, or the N-myristoyltransferase (NMT) inhibitor IMP-1088. Both MMF and IMP-1088 drugs exhibited strong dose-dependent antiviral activity against VACV and mpox, and potent synergistic effects in conjunction with TPOXX. Our findings support combination therapy of direct-acting (TPOXX) and host-targeted (MMF and IMP-1088) antivirals as a promising approach to treat mpox and prevent the emergence and spread of TPOXX-resistant MPXV variants.

Rift Valley fever (RVF) is an arboviral zoonotic disease affecting many African countries with the potential to spread to other geographical areas. In this chapter we describe the use of a replication-competent recombinant (r)RVFV expressing NanoLuc Luciferase (Nluc) for in vitro studies. The determination of parameters such as neutralizing antibodies in serum samples, or the antiviral activity of drugs is usually carried out using standard assays based on the assessment of cytopathic effect on cell cultures. The use of a virus encoding a traceable reporter protein allows to correlate the presence or absence of infection with the detection of the product in the infected cultures, thus tracking the level of RVFV infection in an objective, quantitative manner. In addition to this quantitative measurement of results, our protocol offers two other advantages, such as a shorter time to read, given that 48 h post-infection the production of the reporter protein is enough to give an accurate result, and the use of an attenuated virus, which reduces the risk of exposure.

The host range of HPAIV H5N1 was recently expanded to include ruminants, particularly dairy cattle in the United States (US). Shortly after, human H5N1 infection was reported in a dairy worker in Texas following exposure to infected cattle. Herein, we rescued the cattle-origin influenza A/bovine/Texas/24-029328-02/2024(H5N1, rHPbTX) and A/Texas/37/2024(H5N1, rHPhTX) viruses, identified in dairy cattle and human, respectively, and their low pathogenic forms, rLPbTX and rLPhTX, with monobasic HA cleavage sites. Intriguingly, rHPhTX replicated more efficiently than rHPbTX in mammalian and avian cells. Still, variations in the PA and NA proteins didn't affect their antiviral susceptibility to PA and NA inhibitors. Unlike rHPbTX and rLPbTX, both rHPhTX and rLPhTX exhibited higher pathogenicity and efficient replication in infected C57BL/6J mice. The lungs of rHPhTX-infected mice produced higher inflammatory cytokines/chemokines than rHPbTX-infected mice. Our results highlight the potential risk of HPAIV H5N1 virus adaptation in human and/or dairy cattle during the current multistate/multispecies outbreak in the US.

UNLABELLED: 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 to use 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 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.

IMPORTANCE: Rift 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 (r)RVFV that express fluorescent or luminescent proteins to track viral infection. All the generated reporter-expressing rRVFVs were able to propagate in mammalian or insect cells and 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.

UNLABELLED: 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 to use 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 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.

IMPORTANCE: Rift 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 (r)RVFV that express fluorescent or luminescent proteins to track viral infection. All the generated reporter-expressing rRVFVs were able to propagate in mammalian or insect cells and 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.

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, hemagglutination 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 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.

The host range of HPAIV H5N1 was recently expanded to include ruminants, particularly dairy cattle in the United States (US). Shortly after, human H5N1 infection was reported in a dairy worker in Texas following exposure to infected cattle. Herein, we rescued the cattle-origin influenza A/bovine/Texas/24-029328-02/2024(H5N1, rHPbTX) and A/Texas/37/2024(H5N1, rHPhTX) viruses, identified in dairy cattle and human, respectively, and their low pathogenic forms, rLPbTX and rLPhTX, with monobasic HA cleavage sites. Intriguingly, rHPhTX replicated more efficiently than rHPbTX in mammalian and avian cells. Still, variations in the PA and NA proteins didn't affect their antiviral susceptibility to PA and NA inhibitors. Unlike rHPbTX and rLPbTX, both rHPhTX and rLPhTX exhibited higher pathogenicity and efficient replication in infected C57BL/6J mice. The lungs of rHPhTX-infected mice produced higher inflammatory cytokines/chemokines than rHPbTX-infected mice. Our results highlight the potential risk of HPAIV H5N1 virus adaptation in human and/or dairy cattle during the current multistate/multispecies outbreak in the US.