Humoral Response after a Fourth Dose with mRNA-1273 in Healthcare Workers with and without a History of SARS-CoV-2 Infection and Previously Vaccinated with Two Doses of BBIBP-CorV Plus BNT162b2 Vaccine.

There is limited information on the kinetics of the humoral response elicited by a fourth dose with a heterologous mRNA1273 booster in patients who previously received a third dose with BNT162b2 and two doses of BBIBP-CorV as the primary regimen. We conducted a prospective cohort study to assess the humoral response using Elecsys® anti-SARS-CoV-2 S (anti-S-RBD) of 452 healthcare workers (HCWs) in a private laboratory in Lima, Peru at 21, 120, 210, and 300 days after a third dose with a BNT162b2 heterologous booster in HCW previously immunized with two doses of BBIBP-CorV, depending on whether or not they received a fourth dose with the mRNA1273 heterologous vaccine and on the history of previous SARS infection -CoV-2. Of the 452 HCWs, 204 (45.13%) were previously infected (PI) with SARS-CoV-2, and 215 (47.57%) received a fourth dose with a heterologous mRNA-1273 booster. A total of 100% of HCWs presented positive anti-S-RBD 300 days after the third dose. In HCWs receiving a fourth dose, GMTs 2.3 and 1.6 times higher than controls were observed 30 and 120 days after the fourth dose. No statistically significant differences in anti-S-RBD titers were observed in those HCWs PI and NPI during the follow-up period. We observed that HCWs who received a fourth dose with the mRNA1273 and those previously infected after the third dose with BNT162b2 (during the Omicron wave) presented higher anti-S-RBD titers (5734 and 3428 U/mL, respectively). Further studies are required to determine whether patients infected after the third dose need a fourth dose.

Neutralizing Antibodies as Predictors of Vaccine Breakthrough Infection in Healthcare Workers Vaccinated with or without a Heterologous Booster Dose: A Cohort Study during the Third COVID-19 Wave in Peru.

We evaluated neutralizing antibody (NAbs) levels as a protective factor against vaccine breakthrough infection (VBI) in healthcare workers (HCWs) during the third COVID-19 wave in Peru. This retrospective cohort study employed the information from a private laboratory in Lima (Peru) of HCW who received only two BBIBP-CorV vaccines or (additionally) a heterologous booster with BNT162b2. We evaluated the association between the VBI and the levels of NAbs at 21, 90, 180, and 210 days after the BBIBP-CorV second dose. NAbs were calculated with the cPass™ SARS-CoV-2 Neutralization Antibody Detection kit (surrogate virus neutralization test (sVNT)) and the Elecsys® anti-SARS-CoV-2 S Test. Of the 435 HCW evaluated, 31.72% had an infection previous to vaccination, 68.28% received a booster dose, and 23.21% had a VBI during the third wave. The variables associated with a lower risk of VBI were male sex (aRR: 0.43) and those who had (180 days after BBIBP-CorV inoculation) NAbs levels ≥ 60% (aRR: 0.58) and ≥90% (aRR: 0.59) on cPass™, and ≥500 with Elecsys® (aRR: 0.58). HCW whose NAbs persisted at higher levels six months after the BBIBP-CorV showed a lower risk of suffering from a VBI during the third COVID-19 wave.

Chaperonin TRiC/CCT Participates in Mammarenavirus Multiplication in Human Cells via Interaction with the Viral Nucleoprotein.

The eukaryotic chaperonin containing tailless complex polypeptide 1 ring complex (CCT, also known as TCP-1 Ring Complex, TRiC/CCT) participates in the folding of 5% to 10% of the cellular proteome and has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses, but the mechanisms by which the TRiC/CCT complex contributes to virus multiplication remain poorly understood. Here, we document that the nucleoprotein (NP) of the mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a substrate of the human TRiC/CCT complex, and that pharmacological inhibition of TRiC/CCT complex function, or RNAi-mediated knockdown of TRiC/CCT complex subunits, inhibited LCMV multiplication in human cells. We obtained evidence that the TRiC/CCT complex is required for the production of NP-containing virus-like particles (VLPs), and the activity of the virus ribonucleoprotein (vRNP) responsible for directing replication and transcription of the viral genome. Pharmacological inhibition of the TRIC/CCT complex also restricted multiplication of the live-attenuated vaccine candidates Candid#1 and ML29 of the hemorrhagic fever causing Junin (JUNV) and Lassa (LASV) mammarenaviruses, respectively. Our findings indicate that the TRiC/CCT complex is required for mammarenavirus multiplication and is an attractive candidate for the development of host directed antivirals against human-pathogenic mammarenaviruses. IMPORTANCE Host-directed antivirals have gained great interest as an antiviral strategy to counteract the rapid emergence of drug-resistant viruses. The chaperonin TRiC/CCT complex has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses. Here, we have provided evidence that the chaperonin TRiC/CCT complex participates in mammarenavirus infection via its interaction with the viral NP. Importantly, pharmacological inhibition of TRiC/CCT function significantly inhibited multiplication of LCMV and the distantly related mammarenavirus JUNV in human cells. Our findings support that the TRiC/CCT complex is required for multiplication of mammarenaviruses and that the TRiC/CCT complex is an attractive host target for the development of antivirals against human-pathogenic mammarenaviruses.

Mammarenavirus Genetic Diversity and Its Biological Implications.

Members of the family Arenaviridae are classified into four genera: Antennavirus, Hartmanivirus, Mammarenavirus, and Reptarenavirus. Reptarenaviruses and hartmaniviruses infect (captive) snakes and have been shown to cause boid inclusion body disease (BIBD). Antennaviruses have genomes consisting of 3, rather than 2, segments, and were discovered in actinopterygian fish by next-generation sequencing but no biological isolate has been reported yet. The hosts of mammarenaviruses are mainly rodents and infections are generally asymptomatic. Current knowledge about the biology of reptarenaviruses, hartmaniviruses, and antennaviruses is very limited and their zoonotic potential is unknown. In contrast, some mammarenaviruses are associated with zoonotic events that pose a threat to human health. This review will focus on mammarenavirus genetic diversity and its biological implications. Some mammarenaviruses including lymphocytic choriomeningitis virus (LCMV) are excellent experimental model systems for the investigation of acute and persistent viral infections, whereas others including Lassa (LASV) and Junin (JUNV) viruses, the causative agents of Lassa fever (LF) and Argentine hemorrhagic fever (AHF), respectively, are important human pathogens. Mammarenaviruses were thought to have high degree of intra-and inter-species amino acid sequence identities, but recent evidence has revealed a high degree of mammarenavirus genetic diversity in the field. Moreover, closely related mammarenavirus can display dramatic phenotypic differences in vivo. These findings support a role of genetic variability in mammarenavirus adaptability and pathogenesis. Here, we will review the molecular biology of mammarenaviruses, phylogeny, and evolution, as well as the quasispecies dynamics of mammarenavirus populations and their biological implications.

Molecular Engineering of a Mammarenavirus with Unbreachable Attenuation.

Several mammarenaviruses cause severe hemorrhagic fever (HF) disease in humans and pose important public health problems in their regions of endemicity. There are no United States (US) Food and Drug Administration (FDA)-approved mammarenavirus vaccines, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that has limited efficacy. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames (ORF) separated by a noncoding intergenic region (IGR). The large (L) segment encodes the RNA dependent RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). In the present study, we document the generation of a recombinant form of the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) expressing a codon deoptimized (CD) GPC and containing the IGR of the S segment in both the S and L segments (rLCMV/IGR-CD). We show that rLCMV/IGR-CD is fully attenuated in C57BL/6 (B6) mice but able to provide complete protection upon a single administration against a lethal challenge with LCMV. Importantly, rLCMV/IGR-CD exhibited an unbreachable attenuation for its safe implementation as a live-attenuated vaccine (LAV). IMPORTANCE Several mammarenaviruses cause severe disease in humans and pose important public health problems in their regions of endemicity. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenaviral therapy is limited to an off-label use of ribavirin whose efficacy is controversial. Here, we describe the generation of recombinant version of the prototypic mammarenavirus lymphocytic choriomeningitis virus (rLCMV) combining the features of a codon deoptimized (CD) GPC and the noncoding intergenic region (IGR) of the S segment in both S and L genome segments, called rLCMV/IGR-CD. We present evidence that rLCMV/IGR-CD has excellent safety and protective efficacy features as live-attenuated vaccine (LAV). Importantly, rLCMV/IGR-CD prevents, in coinfected mice, the generation of LCMV reassortants with increased virulence. Our findings document a well-defined molecular strategy for the generation of mammarenavirus LAV candidates able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including unbreachable attenuation.

The Pan-ErbB tyrosine kinase inhibitor afatinib inhibits multiple steps of the mammarenavirus life cycle.

The mammarenavirus Lassa virus (LASV) causes a life-threatening acute febrile disease, Lassa fever (LF). To date, no US Food and Drug Administration (FDA)-licensed medical countermeasures against LASV are available. This underscores the need for the development of novel anti-LASV drugs. Here, we screen an FDA-approved drug library to identify novel anti-LASV drug candidates using an infectious-free cell line expressing a functional LASV ribonucleoprotein (vRNP), where levels of vRNP-directed reporter gene expression serve as a surrogate for vRNP activity. Our screen identified the pan-ErbB tyrosine kinase inhibitor afatinib as a potent inhibitor of LASV vRNP activity. Afatinib inhibited multiplication of lymphocytic choriomeningitis virus (LCMV) a mammarenavirus closely related to LASV. Cell-based assays revealed that afatinib inhibited multiple steps of the LASV and LCMV life cycles. Afatinib also inhibited multiplication of Junín virus vaccine strain Candid#1, indicating that afatinib can have antiviral activity against a broad range of human pathogenic mammarenaviruses.

Genetic studies to determine the risk of non-ischemic sudden death: review article / Estudios genéticos para determinar el riesgo de muerte súbita no isquémica: artículo de revisión

Heart disease is the cause of sudden death in more than 80% of cases. Ischemic heart disease is the cause for 90% of all sudden cardiac deaths, while in the remaining 10% of cases, heart diseases have a hereditary origin and comprise a wide spectrum of disorders that include cardiomyopathies and channelopathies. The aim of this review is to highlight the importance of genetic counseling for patients with hereditary cardiovascular disease and its evaluation by a multidisciplinary team.

La cardiopatía es la causa de muerte súbita en más del 80% de casos. La cardiopatía isquémica es responsable en el 90% del total de las muertes súbitas cardiacas, mientras que en el 10% de casos restantes, las cardiopatías tienen un origen hereditario y comprenden un amplio espectro de trastornos que incluyen a las cardiomiopatías y las canalopatías. El objetivo de esta revisión es poner en evidencia la importancia del asesoramiento genético de los pacientes con enfermedad cardiovascular hereditaria y su evaluación a través de un equipo multidisciplinario.

Humoral Immune Response Induced by the BBIBP-CorV Vaccine (Sinopharm) in Healthcare Workers: A Cohort Study.

Insufficient data have been reported about the effect of the inactivated SARS-CoV-2 vaccine (BBIBP-CorV) on the humoral response through time in healthcare workers (HCW). This retrospective cohort studied the information of 252 HCW from a private laboratory, comparing the antibody-mediated response provoked by BBIBP-CorV between HCW previously infected with SARS-CoV-2 (PI) and not previously infected (NPI), employing the Elecsys® anti-SARS-CoV-2 S and the cPass™ SARS-CoV-2 Neutralization Antibody Detection kit at intervals of 21, 90, and 180 days after vaccination. The presence of neutralizing antibodies in HCW 21 days after full vaccination was 100% in PI and 91.60% in NPI. We observed a progressive decrease in antibody levels over time in both groups. Comparing HCW PI with NPI, PI had a 10.9, 14.3, and 8.6-fold higher antibody titer with the Elecsys® anti-SARS-CoV-2 S at 21 (p < 0.001), 90 (p< 0.001) and 180 days (p < 0.001) respectively, compared to NPI. Using the percent of signal inhibition (PSI) of the antibody neutralization cPass™, HCW PI showed a level of 1.3, 2.0, and 3.1 times more antibodies, at 21 (p < 0.001), 90 (p < 0.001), and 180 days (p < 0.001) respectively, compared to NPI. We determined a progressive decrease in humoral immunity over time, particularly higher in those NPI.

Evaluation of the Humoral Immune Response of a Heterologous Vaccination between BBIBP-CorV and BNT162b2 with a Temporal Separation of 7 Months, in Peruvian Healthcare Workers with and without a History of SARS-CoV-2 Infection.

Information on the effects of a heterologous booster in adult patients first vaccinated with the BBIBP-CorV vaccine is limited. This prospective cohort study evaluated the humoral response of 152 healthcare workers (HCWs) from a private laboratory in Lima (Peru) before and after receiving the BNT162b2 vaccine, with a seven-month interval since the BBIBP-CorV doses. We employed the Elecsys® anti-SARS-CoV-2 S and the cPass™ SARS-CoV-2 Neutralization Antibody (NAbs) assays to evaluate anti-S-RBD IgG and NAbs, respectively. Of the 152 HCWs, 79 (51.98%) were previously infected (PI) with SARS-CoV-2 and 73 (48.02%) were not previously infected (NPI). The proportion of HCWs with positive NAbs, seven months after the BBIBP-CorV immunization, was 49.31% in NPI and 92.40% in PI. After the booster, this ratio increased to 100% in both groups. The anti-S-RBD IgG and NAbs in the HCWs' NPI increased by 32.7 and 3.95 times more, respectively. In HCWs' PI, this increment was 5 and 1.42 times more, respectively. There was no statistical association between the history of previous SARS-CoV-2 infection and the titer of anti-S-RBD IgG and NAbs after the booster. The humoral immunity presented a robust increase after receiving the BNT162b2 booster and was more pronounced in NPI.

A Bifluorescent-Based Assay for the Identification of Neutralizing Antibodies against SARS-CoV-2 Variants of Concern In Vitro and In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the U.S. Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2-neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here, we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and an rSARS-CoV-2 strain expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta [ß]) VoC in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 strain and the parental wild-type (WT) rSARS-CoV-2 WA-1 strain had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of hMAbs with neutralizing activity against SARS-CoV-2, including VoC in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses, demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Inhibitors of anti-apoptotic Bcl-2 family proteins exhibit potent and broad-spectrum anti-mammarenavirus activity via cell cycle arrest at G0/G1 phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their pro-apoptotic properties, but rather their ability of inducing cell arrest at G0/G1 phase. OLX and ABT-737 mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, siRNA-mediated knock down of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE: Anti-apoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors mediated cell cycle arrest at the G0/G1 phase, rather than their pro-apoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate anti-mammarenavirus activity in vivo , and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals.

Progress in Anti-Mammarenavirus Drug Development.

Mammarenaviruses are prevalent pathogens distributed worldwide, and several strains cause severe cases of human infections with high morbidity and significant mortality. Currently, there is no FDA-approved antiviral drugs and vaccines against mammarenavirus and the potential treatment option is limited to an off-label use of ribavirin that shows only partial protective effect and associates with side effects. For the past few decades, extensive research has reported potential anti-mammarenaviral drugs and their mechanisms of action in host as well as vaccine candidates. This review describes current knowledge about mammarenavirus virology, progress of antiviral drug development, and technical strategies of drug screening.

Novel Dihydroorotate Dehydrogenase Inhibitors with Potent Interferon-Independent Antiviral Activity against Mammarenaviruses In Vitro.

Mammarenaviruses cause chronic infections in rodents, which are their predominant natural hosts. Human infection with some of these viruses causes high-consequence disease, posing significant issues in public health. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenavirus drugs are limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe side effects. Dihydroorotate dehydrogenase (DHODH) inhibitors, which block de novo pyrimidine biosynthesis, have antiviral activity against viruses from different families, including Arenaviridae, the taxonomic home of mammarenaviruses. Here, we evaluate five novel DHODH inhibitors for their antiviral activity against mammarenaviruses. All tested DHODH inhibitors were potently active against lymphocytic choriomeningitis virus (LCMV) (half-maximal effective concentrations [EC50] in the low nanomolar range, selectivity index [SI] > 1000). The tested DHODH inhibitors did not affect virion cell entry or budding, but rather interfered with viral RNA synthesis. This interference resulted in a potent interferon-independent inhibition of mammarenavirus multiplication in vitro, including the highly virulent Lassa and Junín viruses.

Reverse genetics approaches for the development of mammarenavirus live-attenuated vaccines.

Several mammarenaviruses can cause severe hemorrhagic fever disease with a very high case fatality rate, representing important threats to human health within the viruses' endemic regions. To date, there are no United States (US) Food and Drug Administration (FDA)-licensed vaccines available to combat mammarenavirus infections in humans, and current anti-mammarenavirus therapy is limited to off-label use of the guanosine analog ribavirin, which has limited efficacy and has been associated with significant side effects. Vaccination is one of the most effective ways to prevent viral diseases, and live-attenuated vaccines (LAVs) have been shown to often provide long-term protection against a subsequent natural infection by the corresponding virulent form of the virus. The development of mammarenavirus reverse genetics systems has provided investigators with a powerful approach for the investigation of the molecular and cell biology of mammarenaviruses and also for the generation of recombinant viruses containing predetermined mutations in their genome for their implementation as LAVs for the treatment of mammarenavirus infections. In this review, we summarize the current knowledge on the mammarenavirus molecular and cell biology, and the use of reverse genetic approaches for the generation of recombinant mammarenaviruses. Moreover, we briefly discus some novel LAV approaches for the treatment of mammarenavirus infections based on the use of reverse genetics approaches.

Identification and Characterization of Novel Compounds with Broad-Spectrum Antiviral Activity against Influenza A and B Viruses.

Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC50) = 3.80 nM to 1.73 µM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC50 = 0.14 µM to 0.55 µM; SI-MTT = 70.12 to >357.14) or cotreatment (EC50 = 34.69 nM to 7.52 µM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections.IMPORTANCE Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.

The Glycoprotein of the Live-Attenuated Junin Virus Vaccine Strain Induces Endoplasmic Reticulum Stress and Forms Aggregates prior to Degradation in the Lysosome.

Argentine hemorrhagic fever is a potentially lethal disease that is caused by Junin virus (JUNV). There are currently around 5 million individuals at risk of infection within regions of endemicity in Argentina. The live attenuated vaccine strain Candid #1 (Can) is approved for use in regions of endemicity and has substantially decreased the number of annual Argentine hemorrhagic fever (AHF) cases. The glycoprotein (GPC) gene is primarily responsible for attenuation of the Can strain, and we have shown that the absence of an N-linked glycosylation motif in the subunit G1 of the glycoprotein complex of Can, which is otherwise present in the wild-type pathogenic JUNV, causes GPC retention in the endoplasmic reticulum (ER). Here, we show that Can GPC aggregates in the ER of infected cells, forming incorrect cross-chain disulfide bonds, which results in impaired GPC processing into G1 and G2. The GPC fails to cleave into its G1 and G2 subunits and is targeted for degradation within lysosomes. Cells infected with the wild-type Romero (Rom) strain do not produce aggregates that are observed in Can infection, and the stress on the ER remains minimal. While the mutation of the N-linked glycosylation motif (T168A) is primarily responsible for the formation of aggregates, other mutations within G1 that occurred earlier in the passage history of the Can strain also contribute to aggregation of the GPC within the ER.IMPORTANCE The development of vaccines and therapeutics to combat viral hemorrhagic fevers remains a top priority within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. The Can strain, derived from the pathogenic XJ strain of JUNV, has been demonstrated to be both safe and protective against AHF. While the vaccine strain is approved for use in regions of endemicity within Argentina, the mechanisms of Can attenuation have not been elucidated. A better understanding of the viral genetic determinants of attenuation will improve our understanding of the mechanisms contributing to disease pathogenesis and provide critical information for the rational design of live attenuated vaccine candidates for other viral hemorrhagic fevers.

Correction: Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses.

[This corrects the article DOI: 10.1371/journal.ppat.1006892.].

A cell-based, infectious-free, platform to identify inhibitors of lassa virus ribonucleoprotein (vRNP) activity.

The mammarenavirus Lassa (LASV) is highly prevalent in West Africa where it infects several hundred thousand individuals annually resulting in a high number of Lassa fever (LF) cases, a febrile disease associated with high morbidity and significant mortality. Mounting evidence indicates that the worldwide-distributed prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. There are not Food and Drug Administration (FDA) licensed vaccines and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective and can cause significant side effects. Therefore, there is an unmet need for novel antiviral drugs to combat LASV. This task would be facilitated by the implementation of high throughput screens (HTS) to identify inhibitors of the activity of the virus ribonucleoprotein (vRNP) responsible for directing virus RNA genome replication and gene transcription. The use of live LASV for this purpose is jeopardized by the requirement of biosafety level 4 (BSL4) containment. We have developed a virus-free cell platform, where expression levels of reporter genes serve as accurate surrogates of vRNP activity, to develop cell-based assays compatible with HTS to identify inhibitors of LASV and LCMV mammarenavirus vRNP activities.