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Herpes simplex virus 1 (HSV-1) is responsible for herpes simplex virus encephalitis (HSE), associated with a 70% mortality rate in the absence of treatment. Despite intravenous treatment with acyclovir, mortality remains significant, highlighting the need for new anti-herpetic agents. Herein, we describe a novel neurovirulent recombinant HSV-1 (rHSV-1), expressing the fluorescent tdTomato and Gaussia luciferase (Gluc) enzyme, generated by the Clustered regularly interspaced short palindromic repeats (CRISPR)—CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) system. The Gluc activity measured in the cell culture supernatant was correlated (P = 0.0001) with infectious particles, allowing in vitro monitoring of viral replication kinetics. A significant correlation was also found between brain viral titers and Gluc activity in plasma (R2 = 0.8510, P

Human metapneumovirus (hMPV) is an important respiratory pathogen especially in young children and elderly subjects. Our objective was to assess the immunogenicity and protection conferred by predominant pre- and post-fusion (F) hMPV-F constructs in Balb/C mice. Immunizations without adjuvant were not immunogenic whereas alum-adjuvanted hMPV-F proteins, regardless of their conformations, generated comparable neutralizing antibody titers with undetectable pulmonary viral titers following viral challenge. In conclusion, we found no apparent advantage for mixtures of predominant pre-fusion F proteins over post-fusion conformations for hMPV vaccination in opposite to recent data obtained with the human respiratory syncytial virus.

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml−1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

Background Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. Methods Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. Results I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. Conclusion The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.

The prolonged treatment of immunosuppressed (IS) individuals with anti-influenza monotherapies may lead to the emergence of drug-resistant variants. Herein, we evaluated oseltamivir and polymerase inhibitors combinations against influenza A/H3N2 infections in an IS mouse model. Mice were IS with cyclophosphamide and infected with 3 &times, 103 PFU of a mouse-adapted A/Switzerland/9715293/2013 (H3N2) virus. Forty-eight hours post-infection, the animals started oseltamivir, favipiravir or baloxavir marboxil (BXM) as single or combined therapies for 10 days. Weight losses, survival rates and lung viral titers (LVTs) were determined. The neuraminidase (NA) and polymerase genes from lung viral samples were sequenced. All untreated animals died. Oseltamivir and favipiravir monotherapies only delayed mortality (the mean day to death (MDD) of 21.4 and 24 compared to 11.4 days for those untreated) while a synergistic improvement in survival (80%) and LVT reduction was observed in the oseltamivir/favipiravir group compared to the oseltamivir group. BXM alone or in double/triple combination provided a complete protection and significantly reduced LVTs. Oseltamivir and BXM monotherapies induced the E119V (NA) and I38T (PA) substitutions, respectively, while no resistance mutation was detected with combinations. We found that the multiple dose regimen of BXM alone provided superior benefits compared to oseltamivir and favipiravir monotherapies. Moreover, we suggest the potential for drug combinations to reduce the incidence of resistance.

The role of a signaling pathway through macrophage colony-stimulating factor (MCSF) and its receptor, macrophage colony-stimulating factor 1 receptor (CSF1R), during experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE) was studied by two different approaches. First, we evaluated the effect of stimulation of the MCSF/CSF1R axis before infection. Exogenous MCSF (40 μg/kg of body weight intraperitoneally [i.p.]) was administered once daily to BALB/c mice on days 4 and 2 before intranasal infection with 2,500 PFU of HSV-1. MCSF treatment significantly increased mouse survival compared to saline (50% versus 10%; P = 0.0169). On day 6 postinfection (p.i.), brain viral titers were significantly decreased, whereas beta interferon (IFN-β) was significantly increased in mice treated with MCSF compared to mice treated with saline. The number of CD68(+) (a phagocytosis marker) microglial cells was significantly increased in MCSF-treated mice compared to the saline-treated group. Secondly, we conditionally depleted CSF1R on microglial cells of CSF1R-loxP-CX3CR1-cre/ERT2 mice (in a C57BL/6 background) through induction with tamoxifen. The mice were then infected intranasally with 600,000 PFU of HSV-1. The survival rate of mice depleted of CSF1R (knockout [KO] mice) was significantly lower than that of wild-type (WT) mice (0% versus 67%). Brain viral titers and cytokine/chemokine levels were significantly higher in KO than in WT animals on day 6 p.i. Furthermore, increased infiltration of monocytes into the brains of WT mice was seen on day 6 p.i., but not in KO mice. Our results suggest that microglial cells are essential to control HSE at early stages of the disease and that the MCSF/CSF1R axis could be a therapeutic target to regulate their response to infection. IMPORTANCE Microglia appear to be one of the principal regulators of neuroinflammation in the central nervous system (CNS). An increasing number of studies have demonstrated that the activation of microglia could result in either beneficial or detrimental effects in different CNS disorders. Hence, the role of microglia during herpes simplex virus encephalitis (HSE) has not been fully characterized. Using experimental mouse models, we showed that an early activation of the MCSF/CSF1R axis improved the outcome of the disease, possibly by inducing a proliferation of microglia. In contrast, depletion of microglia before HSV-1 infection worsened the prognosis of HSE. Thus, an early microglial response followed by sustained infiltration of monocytes and T cells into the brain seem to be key components for a better clinical outcome. These data suggest that microglia could be a potential target for immunomodulatory strategies combined with antiviral therapy to better control the outcome of this devastating disease.

After 6 years of circulation in humans, a novel antigenic variant of influenza A(H1N1)pdm09 (i.e., A/Michigan/45/2015) emerged in 2015-16 and has predominated thereafter worldwide. Herein, we compared in vitro and in vivo properties of 2016 wild-type (WT) A/Michigan/45/15-like isolate and its H275Y neuraminidase (NA) variant to the original A/California/07/09-like counterparts. The H275Y mutation induced comparable levels of resistance to oseltamivir and peramivir without altering zanamivir susceptibility in both 2009 and 2016 isolates. In vitro, the two WT isolates had comparable replicative properties. The 2016-H275Y isolate had lower titers at 36 h post-inoculation (PI) (P 0.05) while the 2009-H275Y titers were lower at both 24 h (P 0.01) and 36 h PI (P 0.001) vs the respective WTs. In mice, the 2016-WT isolate caused less weight losses (P 0.001) and lower lung viral titers (LVTs) (P 0.01) vs the 2009-WT. The LVTs of 2016-WT and 2016-H275Y groups were comparable whereas the 2009-H275Y LVTs were lower vs the respective WT (P 0.01). Ferrets infected with the 2016-WT isolate and their contacts had higher nasal viral titers (NVTs) at early time points vs the 2009-WT group (P 0.01). Also, NVTs of 2016-H275Y animals were lower vs the 2016-WT group at early time points in both infected (P 0.01) and contact animals (P 0.001). In conclusion, while the H275Y mutation similarly impacts the A/California/07/2009- and A/Michigan/45/2015-like A(H1N1)pdm09 NAs, the fitness of these isolates differs according to animal models with the 2016 virus being less virulent in mice but slightly more virulent in ferrets, potentially reflecting a period of cumulative changes in surface and internal genes.

BACKGROUND: Zika virus (ZIKV) infection has been associated with prolonged viral excretion in human semen and causes testicular atrophy and infertility in 10-week-old immunodeficient mice. METHODS: Male IFNAR(−/−) mice, knockout for type I interferon receptor, were immunized with GLS-5700, a deoxyribonucleic acid-based vaccine, before a subcutaneous ZIKV challenge with 6 × 10(5) plaque-forming units at 13 weeks of age. On day 28 postinfection, testes and epididymides were collected in some mice for histological and functional analyses, whereas others were mated with naive female wild-type C57BL/6J. RESULTS: Although all mice challenged with ZIKV developed viremia, most of them were asymptomatic, showed no weight loss, and survived infection. On day 28 postinfection, none of the unvaccinated, infected mice (9 of 9) exhibited abnormal spermatozoa counts or motility. However, 33% (3 of 9) and 36% (4 of 11) of mated males from this group were infertile, from 2 independent studies. Contrarily, males from the noninfected and the vaccinated, infected groups were all fertile. On days 75 and 207 postinfection, partial recovery of fertility was observed in 66% (2 of 3) of the previously infertile males. CONCLUSIONS: This study reports the effects of ZIKV infection on male fertility in a sublethal, immunodeficient mouse model and the efficacy of GLS-5700 vaccination in preventing male infertility.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The &Delta, SH and &Delta, G deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the &Delta, G-85473 and especially &Delta, SH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

Neuraminidase (NA) inhibitors (NAIs), including oseltamivir and zanamivir, play an important therapeutic role against influenza infections in immunocompromised patients. In such settings, however, NAI therapy may lead to the emergence of resistance involving mutations within the influenza surface genes. The aim of this study was to investigate the evolution of hemagglutinin (HA) and NA genes of influenza A(H1N1)pdm09 virus in an immunocompromised patient receiving oseltamivir then zanamivir therapies. Nasopharyngeal swabs (NPS) samples were collected between 01-27-2018 and 04-20-2018 from a hematopoietic stem cell transplant recipient. These included 11 samples collected either pre-therapy, during oseltamivir and zanamivir as well as after therapy. The A(H1N1)pdm09 HA/NA genes were sequenced. The H275Y NA substitution was quantified by droplet digital RT-PCR assay. A(H1N1)pdm09 recombinant viruses containing HA mutations were tested by HA elution experiments to investigatein vitrobinding properties. Oseltamivir rapidly induced the H275Y NA mutation which constituted 98.33% of the viral population after 15 days of oseltamivir treatment. The related HA gene contained S135A and P183S substitutions within the receptor-binding site. After a switch to zanamivir, 275H/Y and 119E/G/D mixed populations were detected. In the last samples, the double H275Y-E119G NA variant dominated with S135A and P183S HA substitutions. This report confirms that oseltamivir can rapidly induce the emergence of the H275Y substitution in A(H1N1)pdm09 viruses and subsequent switch to zanamivir can lead to additional substitutions at codon E119 resulting in multi-drug resistance. Such data highlight the need for novel antiviral agents.

Background Neuraminidase (NA) inhibitors (NAIs), including oseltamivir and zanamivir, play an important therapeutic role against influenza infections in immunocompromised patients. In such settings, however, NAI therapy may lead to the emergence of resistance involving mutations within the influenza surface genes. The aim of this study was to investigate the evolution of NA and haemagglutinin (HA) genes of influenza A(H1N1) pdm09 virus in an immunocompromised patient receiving oseltamivir then zanamivir therapies. Methods Nasopharyngeal swab (NPS) samples were collected between 27 January 2018 and 11 April 2018 from a haematopoietic stem cell transplant recipient. These include 10 samples collected either pre-therapy, during oseltamivir and zanamivir treatment as well as after therapy. The A(H1N1)pdm09 HA/NA genes were sequenced. The H275Y NA substitution was quantified by droplet digital RT-PCR assay. A(H1N1)pdm09 recombinant viruses containing HA mutations were tested by HA elution experiments to investigate in vitro binding properties. Results Oseltamivir rapidly induced the H275Y NA mutation which constituted 98.33% of the viral population after 15 days of oseltamivir treatment. The related HA gene contained S135A and P183S substitutions within the receptor-binding site. After a switch to zanamivir, 275H/Y and 119E/G/D mixed populations were detected. In the last samples, the double H275Y-E119G NA variant dominated with S135A and P183S HA substitutions. Conclusions This report confirms that oseltamivir can rapidly induce the emergence of the H275Y substitution in A(H1N1)pdm09 viruses and subsequent switch to zanamivir can lead to additional substitutions at codon E119 resulting in multi-drug resistance. Such data additionally suggest a potential compensatory role for HA substitutions near the receptor binding site.

Baloxavir marboxil (BXM) is a potent inhibitor of the polymerase acidic (PA) protein of influenza viruses. However, clinical trials predominantly involving influenza A(H1N1) and A(H3N2) infections showed that BXM exhibited a low barrier of resistance. Contrasting with influenza A viruses, BXM-resistant influenza B variants remain poorly documented. We evaluated the impact of I38 T/M and E23K PA substitutions, previously reported in influenza A viruses, on in vitro properties and virulence of contemporary influenza B recombinant viruses. Influenza B/Phuket/3073/2013 recombinant wild-type (WT) virus and the I38T, I38M and E23K PA mutants were assessed for their susceptibility to baloxavir acid (BXA), the active metabolite of BXM, by plaque reduction assays in ST6GalI-MDCK cells. Luciferase-based minigenome tests were performed to determine polymerase activity. Replication kinetics and genetic stability were evaluated in ST6GalI-MDCK cells. Virulence was evaluated in BALB/c mice. The I38T, I38M and E23K substitutions increased BXA IC50s values by 12.6-, 5.5-, and 2.6-fold, respectively, compared to the WT. Minigenome assays revealed a 46% loss of polymerase activity for the E23K substitution vs the WT while the I38T and I38M PA variants retained ≈80% of activity. Peak viral titers were comparable for the WT, I38T and I38M recombinants (7.95 ± 0.5, 7.45 ± 0.25 and 8.11 ± 0.28 logTCID50/mL), respectively, whereas it was significantly lower for the E23K mutant (6.28 ± 0.28 logTCID50/mL; P

Thrombin has been demonstrated to be involved in several viral diseases including human metapneumovirus (hMPV) infections. We previously showed that immediate administration of thrombin inhibitor argatroban post-infection protected mice against hMPV disease. This current work aims at determining whether warfarin and heparin, two other anticoagulants inhibiting thrombin formation and activities, may also be used for treatment against hMPV in vivo. We found that immediate injections of argatroban, warfarin or heparin after virus challenge protected mice against hMPV infection, as evidenced by decreased or no mortality, less weight loss, reduced viral load and attenuated inflammation. However, delayed treatments starting 1 day post-infection with argatroban or warfarin almost did not impact the survival whereas delayed treatment with heparin induced an increased mortality during infection. Moreover, these treatments also did not reduce weight loss, viral replication and inflammation. In agreement with these results, thrombin generation was decreased upon immediate anticoagulant treatments but was unaltered upon delayed treatments. Thus, thrombin generation occurs at the onset of hMPV infection and thrombin inhibition may be only useful for the treatment of this disease when initiated in the early stage. In this case, heparin is not recommended because of its reduced efficacy on mortality in infected mice whereas argatroban and warfarin appear as safe and effective drugs for the treatment of hMPV disease. The antiviral and anti-inflammatory effects of argatroban occur via thrombin-dependent pathways whereas the mechanisms by which warfarin exerts its beneficial effects against hMPV infection were not elucidated and need to be further studied.

Three neuraminidase inhibitors (NAIs: Oseltamivir, zanamivir and peramivir) are currently approved in many countries for the treatment of influenza A and B infections. The emergence of influenza B viruses (IBVs) containing mutations of cross-resistance to these NAIs constitutes a serious clinical threat. Herein, we used a reverse genetics system for the current B/Phuket/3073/2013 vaccine strain to investigate the impact on in vitro properties and virulence of H136N, R152K, D198E/N, I222T and N294S NA substitutions (N2 numbering), reported by the World Health Organization (WHO) as clinical markers of reduced or highly-reduced inhibition (RI/HRI) to multiple NAIs. Recombinant viruses were tested by NA inhibition assays. Their replicative capacity and virulence were evaluated in ST6GalI-MDCK cells and BALB/c mice, respectively. All NA mutants (excepted D198E/N) showed RI/HRI phenotypes against &ge, 2 NAIs. These mutants grew to comparable titers of the recombinant wild-type (WT) IBV in vitro, and some of them (H136N, I222T and N294S mutants) induced more weight loss and mortality in BALB/c mice in comparison to the recombinant WT IBV. These results demonstrate that, in contemporary IBVs, some NA mutations may confer RI/HRI phenotypes to existing NAIs without altering the viral fitness. This reinforces the need for development of novel antiviral strategies with different mechanisms of action.