Publications

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new member of the Coronaviridae family responsible for the coronavirus disease 19 (COVID-19) pandemic. To date, SARS-CoV-2 has been accountable for over 624 million infection cases and more than 6.5 million human deaths. The development and implementation of SARS-CoV-2 reverse genetics approaches have allowed researchers to genetically engineer infectious recombinant (r)SARS-CoV-2 to answer important questions in the biology of SARS-CoV-2 infection. Reverse genetics techniques have also facilitated the generation of rSARS-CoV-2 expressing reporter genes to expedite the identification of compounds with antiviral activity in vivo and in vitro. Likewise, reverse genetics has been used to generate attenuated forms of the virus for their potential implementation as live-attenuated vaccines (LAV) for the prevention of SARS-CoV-2 infection. Here we describe the experimental procedures for the generation of rSARS-CoV-2 using a well-established and robust bacterial artificial chromosome (BAC)-based reverse genetics system. The protocol allows to produce wild-type and mutant rSARS-CoV-2 that can be used to understand the contribution of viral proteins and/or amino acid residues in viral replication and transcription, pathogenesis and transmission, and interaction with cellular host factors.

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has become a global threat to human health. Although ZIKV has been known to circulate for decades causing mild febrile illness, the more recent ZIKV outbreaks in the Americas and the Caribbean have been associated with severe neurological disorders and congenital abnormalities. The development of ZIKV reverse genetics approaches have allowed researchers to address key questions on the biology of ZIKV by genetically engineering infectious recombinant (r)ZIKV. This has resulted in a better understanding of the biology of ZIKV infections, including viral pathogenesis, molecular mechanisms of viral replication and transcription, or the interaction of viral and host factors, among others aspects. In addition, reverse genetics systems have facilitated the identification of anti-ZIKV compounds and the development of new prophylactic approaches to combat ZIKV infections. Different reverse genetics strategies have been implemented for the recovery of rZIKV. All these reverse genetics systems have faced and overcome multiple challenges, including the viral genome size, the toxicity of viral sequences in bacteria, etc. In this chapter we describe the generation of a ZIKV full-length complementary (c)DNA infectious clone based on the use of a bacterial artificial chromosome (BAC) and the experimental procedures for the successful recovery of rZIKV. Importantly, the protocol described in this chapter provides a powerful method for the generation of infectious clones of other flaviviruses with genomes that have stability problems during bacterial propagation.

Minigenomes (MGs) have greatly advanced research on the viral life cycle, including viral replication and transcription, virus‒host interactions, and the discovery of antivirals against RNA viruses. However, an MG for infectious bursal disease virus (IBDV) has not been well established. Here, we describe the development of IBDV MG, in which the entire coding sequences of viral genomic segments A and B are replaced with Renilla luciferase (Rluc) or enhanced green fluorescent protein (EGFP) reporter genes. Under the control of the RNA polymerase I promoter, the translation of IBDV MG is controlled by the viral proteins VP1 and VP3. Interestingly, IBDV B MG shows greater activity than does IBDV A MG. Moreover, the sense IBDV B MG was expressed at a higher level than the antisense IBDV B MG. In agreement with our previous findings, the translation of IBDV B MG controlled by VP1 and VP3 is independent of the cellular translation machinery components eukaryotic initiation factor (eIF)4E and eIF4G, but intact VP1 polymerase activity, VP3 dsRNA-binding activity, and the interaction between VP1 and VP3 are indispensable for both sense and antisense IBDV B MG activity. In addition, ribavirin, which inhibits IBDV replication, inhibits IBDV B MG activity in a dose-dependent manner. Collectively, the IBDV MG established in this study provides a powerful tool to investigate IBDV intracellular replication and transcription and virus‒host interactions and facilitates high-throughput screening for the identification of IBDV antivirals.

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. By increasing herd immunity, current vaccines have improved infection outcomes for many. However, prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a differential staining strategy with a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and a native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan-1, the more recent Omicron JN.1 strain, and SARS-CoV. 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain. Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

Since the emergence of highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.3.4.4b as a novel reassortant virus from subtype H5N8, the virus has led to a massive number of outbreaks worldwide in wild and domestic birds. Compared to the parental HPAIV H5N8 clade 2.3.4.4b, the novel reassortant HPAIV H5N1 displayed an increased ability to escape species barriers and infect multiple mammalian species, including humans. The virus host range has been recently expanded to include ruminants, particularly dairy cattle in the United States, where cattle-to-cattle transmission was reported. As with the avian 2.3.4.4.b H5N1 viruses, the cattle-infecting virus was found to transmit from cattle to other contact animals including cats, raccoons, rodents, opossums, and poultry. Although replication of the virus in cows appears to be mainly confined to the mammary tissue, with high levels of viral loads detected in milk, infected cats and poultry showed severe respiratory disease, neurologic signs, and eventually died. Furthermore, several human infections with HPAIV H5N1 have also been reported in dairy farm workers and were attributed to exposures to infected dairy cattle. This is believed to represent the first mammalian-to-human transmission report of the HPAIV H5N1. Fortunately, infection in humans and cows, as opposed to other animals, appears to be mild in most cases. Nevertheless, the H5N1 bovine outbreak represents the largest outbreak of the H5N1 in a domestic mammal close to humans, increasing the risk that this already mammalian adapted H5N1 further adapts to human-to-human transmission and starts a pandemic. Herein, we discuss the epidemiology, evolution, pathogenesis, and potential impact of the recently identified HPAIV H5N1 clade 2.3.4.4b in dairy cattle in the United States. Eventually, interdisciplinary cooperation under a One Health framework is required to be able to control this ongoing HPAIV H5N1 outbreak to stop it before further expansion of its host range and geographical distribution.

Lassa virus (LASV) is a rodent-borne mammarenavirus that causes tens to hundreds of thousands of human infections annually in Western Africa. Approximately 20% of these infections progress to Lassa fever (LF), an acute disease with case-fatality rates from ≈20-70%. Currently, there are no approved vaccines or specific therapeutics to prevent or treat LF. The LASV genome consists of a small (S) segment that has two genes, GP and NP, and a large (L) segment that has two genes, L and Z. In both segments, the two genes are separated by non-coding intergenic regions (IGRs). Recombinant LASVs (rLASVs), in which the L segment IGR was replaced with the S segment IGR or in which the GP gene was codon-deoptimized, lost fitness in vitro, were highly attenuated in vivo, and, when used as vaccines, protected domesticated guinea pigs from otherwise lethal LASV exposure. Here, we report the generation of rLASV/IGR-CD, which includes both determinants of attenuation and further enhances the safety of the vaccine compared with its predecessors. rLASV/IGR-CD grew to high titers in Vero cells, which are approved for human vaccine production, but did not cause signs of disease or pathology in guinea pigs. Importantly, guinea pigs vaccinated with rLASV/IGR-CD were completely protected from disease and death after a typically lethal exposure to wild-type LASV. Our data support the development of rLASV/IGR-CD as a live-attenuated LF vaccine with stringent safety features.

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress. Meanwhile, heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and the SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect the Z-mediated virus budding and GP2-mediated fusion activity that is required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan-NMT inhibitor DDD85646 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlates with reduced Z budding activity and GP2-mediated fusion activity as well as with proteasome-mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic-fever-causing Junin (JUNV) and Lassa (LASV) mammarenaviruses. Our results support the exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.