viruses Article Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity Beatriz Vidaña 1,2,† , Pamela Martínez-Orellana 2,3,† , Jaime M. Martorell 4 , Massimiliano Baratelli 2,5 , Jorge Martínez 2,6 , Lourdes Migura-Garcia 2 , Lorena Córdoba 2 , Mónica Pérez 2 , Inmaculada Casas 7 , Francisco Pozo 7 , Lorenzo Fraile 8 , Natàlia Majó 2,6 and María Montoya 2,9, * 1 Bristol Veterinary School, Faculty of Health Sciences, University of Bristol, Langford, Bristol BS40 5DU, UK; beatriz.vidana@bristol.ac.uk 2 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain; pamela.martinez.phd@gmail.com (P.M.-O.); max2612@hotmail.it (M.B.); jorge.martinez@irta.cat (J.M.); lourdes.migura@irta.cat (L.M.-G.); lorena.cordoba@irta.cat (L.C.); monica.perez@irta.cat (M.P.); natalia.majo@uab.cat (N.M.) 3 Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy 4 Departament de Medicina i Cirurgia Animals, Facultat de Veterinaria, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; JaumeMiquel.Martorell@uab.cat 5 HIPRA Laboratories, Avda. la Selva 135, 17170 Amer (Girona), Spain 6 Departament de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain 7 Centro Nacional de Microbiología, Instituto de Salud Carlos III (ISCIII), 28220 Majadahonda, Spain; icasas@isciii.es (I.C.); pacopozo@isciii.es (F.P.) 8 Department de Ciència Animal, Universitat de Lleida, 25198 Lleida, Spain; lorenzo.fraile@prodan.udl.cat 9 Centro de Investigaciones Biológicas Margarita Salas (CIB-CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain * Correspondence: maria.montoya@cib.csic.es † These authors contributed equally to this work.  Received: 4 June 2020; Accepted: 21 July 2020; Published: 24 July 2020  Abstract: Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology. Keywords: influenza; immunopathology; pneumonia; pH1N1; resistance; oseltamivir Viruses 2020, 12, 794; doi:10.3390/v12080794 www.mdpi.com/journal/viruses Viruses 2020, 12, 794 2 of 20 1. Introduction The pandemic A (H1N1) 2009 virus (referred to hereafter as pdm09) caused the first influenza A virus (IAV) pandemic of the 21th century. Currently, the pdm09 virus causes a predominantly mild disease, but it develops into severe lung pathology associated with mortality in some cases [1]. The pdm09 matrix protein (MP) gene came from the Eurasian swine lineage [2], which contains the S31N mutation conferring resistance to adamantine antiviral therapeutics [3]. Therefore, the pharmacological therapy against pdm09 infection depends on neuraminidase inhibitor (NAIs) antivirals, namely oseltamivir and zanamivir [3,4]. A single amino acid substitution at several positions in or around the NA active site is able to strengthen virus resistance against NAIs [5]. Among such NA substitutions, a histidine-to-tyrosine substitution at position 275 (H275Y) is one of the best-characterized and most commonly found oseltamivir-resistant (OR) mutations [6–8]. In general, it is considered that resistant mutations in the NA enzymatic position compromise viral fitness [9,10], as NAI-resistant viruses have been associated with deficient viral replication and spread in seasonal H1N1 and H3N2 strains [11,12]. However, seasonal H1N1 with the H275Y mutation emerged and expanded globally without oseltamivir selection [13]. Such an event demonstrated that viral fitness was no longer compromised by the resistance mutation. Several investigations have revealed the presence of diverse “permissive” mutations (R222Q, V234M, and possibly D354G) in seasonal and pdm09 viruses that allowed the addition of H275Y without compromising viral fitness [14–16]. Overall, OR pdm09 viral infections have generally been described in immunocompromised patients who have been subjected to antiviral treatment [17–19]. Regardless, community clusters of OR pdm09 viruses containing the H275Y mutation have been reported [20–23]. Despite the fact that the frequency of OR in pdm09 viruses has remained low (<2%) [24–27], there are indicators which suggest that pdm09 could permanently acquire the NA H275Y mutation, as happened with its predecessor, the seasonal H1N1 [10,23]. The aim of this study was to compare the virulence of two OR pdm09 strains (R6 and R7) and a wild-type (Wt) virus (F) in vitro and in mice, and to characterize differential innate viral-host immune parameters associated with viral pathogenicity in ferrets as a human model of disease. 2. Materials and Methods 2.1. Oseltamivir-Resistant and Wild-Type Viruses The three strains of human pdm09 viruses used in this study were isolated as previously described [28]. The A/Baleares/RR6121/2009 and A/Madrid/RR7495/2011 viruses bear the OR H275Y mutation and are hereafter referred to as R6 and R7, respectively. Both viruses were isolated from immunocompromised patients. The virus A/CastillaLaMancha/RR5911/2009, referred to here as F, was isolated from a fatal case of a patient without co-morbidities. Influenza viruses R6, R7, and F were grown in a Madin–Darby canine kidney (MDCK), as previously described [28]. R6 and R7 viral titers were 107.8 50% tissue culture infective dose (TCID50 )/mL and 107.3 TCID50 /mL, respectively. The F virus had a titer of 106.02 TCID50 /mL. Titers were determined using the Reed and Muench method [29]. Aminoacidic differences between viral strain-predicted proteins were identified. For each viral protein, the frequency of the amino acid at the mutated positions was calculated over an alignment greater than 1800 sequences of IAV recovered from the Global Initiative on Sharing Avian Influenza Data (GISAID) database. 2.2. In Vitro Infection and Viral Load MDCK cells were infected at 0.0001 multiplicity of infection (MOI) with OR pdm09 virus R6 and R7 and the Wt virus F. At 0, 6, 12, 24, and 48 h post-infection (hpi), supernatants were collected. Viral quantification was determined by TCID50 and a plaque assay determining PFU as previously described [28]. Virus infectivity in lung tissue samples from the mice virus was determined at 3, 5, and 7 dpi, as described by Almansa and colleagues [30]. Viruses 2020, 12, 794 3 of 20 In ferrets, viral secretion shedding in nasal swabs at 0, 1, 3, 6, and 10 dpi was determined by PFU for all available infected animals, as described before [28]. Nasal swabs were placed in 0.5 mL of DMEM (BioWhittaker® , Lonza, Verviers, Belgium) with 600 µg/mL penicillin and streptomycin, and frozen at −80 ◦ C until further processing. 2.3. Ethical Statement Experiments with mice and ferrets were performed following the animal use protocol approved by “Comissió d’Ètica en l’Experimentació Animal i Humana de la Universitat Autònoma de Barcelona” (Internal Register Number: 1124M2R) and the Ethical Animal Experimentation Commission of the Catalan Government (Register Number: 5767). Seven-week-old C57BL6/JOlaHsd (C57BL6) female mice (Harlan Laboratories, Barcelona, Spain) were housed in groups in experimental isolation cages at the biosafety level 3 (BSL3) facilities at the Centre de Recerca en Sanitat Animal (CReSA, Barcelona, Spain). Once mice were separated into different groups, they were kept for one week in acclimation. Animals were kept in standard housing cages and provided with commercial food pellets and tap water ad libitum throughout the experiment. Between eight and nine months of age, IAV seronegative ferrets (Euroferret, Denmark) were placed in BSL3 facilities at CReSA, in experimental isolation rooms, and then acclimated for 6 days. The animals were inhabited in standard housing cages for laboratory ferrets (F-SUITE Ferret Housing, Tecniplas, Italy) and were provided with commercial food pellets and tap water ad libitum throughout the experiment. 2.3.1. Mice Infection, Clinical Signs, and Sampling C57BL6/JOlaHsd female mice were separated into three groups of 20 animals. Mice were assigned to the following three groups: Uninfected control group (C); R6-infected group (R6); and R7-infected group (R7). Each virus was inoculated separately by an intranasal route at a dose of 104 PFU/mL. The control group remained uninfected. Mice were observed daily to record changes in body weight and clinical signs and any animal presenting severe weight loss of more than 20% compared to the weight at the start of the experiment, difficult labored breathing, or a prolonged inability to remain upright was humanely euthanized. Specimens of the serum and lung were acquired at 0, 3, 5, 7, and 14 dpi. In order to minimize suffering, blood samples were obtained under 5% isofluorane anesthesia. Euthanasia of mice was performed under anesthesia with 5% isoflurane with an intraperitoneal inoculation of penthobarbital. Subsequently, lungs were dissected by surgical methods. Lung tissues were weighed, homogenized, dry ice frozen, and stored at −80 ◦ C until further use. 2.3.2. Ferret Infection, Clinical Signs, and Sampling Sixteen ferrets were randomly assigned to three different experimental groups. The control group included three ferrets. The R6 and F group included eight ferrets each, intratracheally inoculated with 105 TCID50 /mL of the R6 or F pdm09 strain, respectively. Ferrets (Euroferret, Denmark) were neutered males between eight and nine months old and seronegative for IAV (influenza A antibody competition multi-species ELISA, ID Screen® , France). Intratracheal inoculation was performed under deep sedation of the animals with ketamine (5–10 mg/kg) (Imalgene 1000® Merial, S.A., Spain) and medetomidine (0.05 mg/kg) (Domtor® Pfiser, S.A., Spain), both administered subcutaneously, and after inoculation, animals were reverted from anesthesia with atipemazol (0.25 mg/kg), administered subcutaneously. Animals were monitored by a specialized veterinarian until they had fully recovered from sedation. Ferrets were monitored daily for clinical signs following a clinical score modified from [28]. Control animals’ clinical signs were monitored during acclimatization and at 0 dpi. Animals were euthanized by an intravenous injection of sodium pentobarbital (100 mg/kg) under anesthesia with ketamine (5–10 mg/kg/SC) (Imalgene 1000® Merial, S.A., Spain) and medetomidine Viruses 2020, 12, 794 4 of 20 (0.05 mg/kg/SC) (Domtor® Pfiser, S.A., Spain). Necropsies were performed on 1, 3, and 10 days post infection (dpi). Ferret Acute Phase Proteins Ferret serum APP haptoglobin (Hp) and serum amyloid A (SAA) were measured and quantified by an ELISA Haptoglobin Assay and Multispecies Serum Amyloid A Immunoassay, (Tridelta Development Ltd., County Kildare, Ireland). Sera samples were taken under sedation of the animals with butorphanol 0.5 mg/kg administrated subcutaneously, from the cranial vena cava at 0 dpi in control animals and all infected animals available at 0, 1, 3, 6, and 10 dpi. All samples were collected in 1 mL blood-heparine tubes. Heparine blood samples were centrifugated at 3000 rpm for 10 min at 4 ◦ C to separate plasma samples. Plasma samples were store at −20 ◦ C. Animals were individually monitored by a specialized veterinarian until they had completely recovered from sedation. Sampling at 0 dpi in all animals was performed while animals were anesthetized for viral inoculation, in order to minimize the handling and anesthetic procedures in the animals. Viral RNA Detection and IAV Antigen Expression and Quantification in Ferret Lungs Viral RNA was extracted with the NucleoSpin® RNA Virus Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions, using a 5 mm square sample of the proximal mediastinic right caudal lobe of each ferret. The IAV M gene was quantified by TaqMan one-step quantitative real time RT-PCR (rRT-PCR) using primers, a probe, and the amplification conditions described in [31] using Fast7500 equipment (Applied Biosystems, Foster City, CA, USA). Individual samples were run in duplicate and each assay contained known positive and negative amplification controls that were also run in duplicate. Threshold cycle (Ct) values ≤40 were considered positive for IAV. The IAV antigen was also quantified in the lung by immunohistochemistry (IHC). Fixed paraffin-embedded tissues were stained with a primary antibody against the influenza A nucleoprotein and IAV antigen immune staining was quantified as previously described [32]. Ferret Innate Immune Gene Expression Profiles The gene expressions of IFNα, IFNβ, IFNγ, TNFα, IL-6, IL-8, CXCL10, and RIG-I were detected by two-step RT-PCR, as previously described [32]. 2.3.3. Histopathology Mice and ferret lung sections were taken for histological examination, according to standard protocols, and stained with hematoxylin and eosin (HE) for examination under light microscopy. Cross sections of each mouse’s right lung lobes were separately evaluated, and semiquantitative assessments of IAV-associated microscopic lesions in the lungs were performed. The histopathological scoring grades assigned were ranked as follows: 0 (no lesions); 1 (mild to moderate necrotizing bronchiolitis); 2 (bronchointerstitial pneumonia characterized by necrotizing bronchiolitis and inflammation of close surrounding alveoli); and 3 (necrotizing bronchiolitis and diffuse inflammation of the alveolar parenchyma in the section). Microscopic lesion scores were assigned for each lobe, and the means were used for the final histopathological score for each animal. 2.3.4. Hemagglutination Inhibition (HI) Assay Antibodies against IAV were measured using an HI assay, as previously described [28]. Briefly, mice and ferret sera were treated overnight with four volumes of Receptor Destroying Enzyme (RDE) (Sigma-Aldrich SA, Madrid, Spain) solution (100 U/mL) at 37 ◦ C. Then, five volumes of 1.5% sodium citrate were added to serum samples and incubated for 30 min at 56 ◦ C. Treated sera were diluted 1:2 in PBS. Finally, sera were incubated for 1 h at 4 ◦ C with 4 hemagglutination units of pdm09 Viruses 2020, 12, x FOR PEER REVIEW 5 of 19 sera were Viruses diluted 2020, 12, 1:2 in PBS. Finally, sera were incubated for 1 h at 4 °C with 4 hemagglutination 794 5 of 20 units of pdm09 and one volume of a 50% suspension of RBC. Controls of positive and negative sera were added to the assay. Positive results were considered when HI titers were >40. and one volume of a 50% suspension of RBC. Controls of positive and negative sera were added to the assay. 2.4. results were considered when HI titers were >40. PositiveAnalyses Statistical 2.4. Statistical GraphPadAnalyses Prism 6 (GraphPad Software, La Jolla, CA, USA) was used to perform data visualization. All statistical analysis was carried out using SPSS 15.0 software (SPSS Inc., Chicago, IL, GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) was used to perform data visualization. USA). For all analyses, mice and ferrets were used as the experimental unit. The significance level All statistical analysis was carried out using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). For all (α) was established at 0.05. The Shapiro Wilk and Levene’s test were used to evaluate the normality analyses, mice and ferrets were used as the experimental unit. The significance level (α) was of the distribution of the analysed quantitative variables and the homogeneity of variances, established at 0.05. The Shapiro Wilk and Levene’s test were used to evaluate the normality of the respectively. A normal distribution was not detected for any continuous variable. Therefore, a distribution of the analysed quantitative variables and the homogeneity of variances, respectively. non-parametric test (Wilcoxon test) employing the U Mann–Whitney test to compare each pair of A normal distribution was not detected for any continuous variable. Therefore, a non-parametric values was used to confront the different values collected for all of the parameters (weight loss, test (Wilcoxon test) employing the U Mann–Whitney test to compare each pair of values was used to clinical signs, acute phase proteins, histopathology, viral load, and cytokine and viral sensing gene confront the different values collected for all of the parameters (weight loss, clinical signs, acute phase expression profiles) between groups (control, F, and R6 and R7 influenza virus) at all time points. proteins, histopathology, viral load, and cytokine and viral sensing gene expression profiles) between groups (control, F, and R6 and R7 influenza virus) at all time points. 3. Results 3. Results 3.1. Oseltamivir-Sensitive (F) and Oseltamivir-Resistant (R6) Replicate Faster Than the R7 Virus in Vitro 3.1. Oseltamivir-Sensitive Viral replication kinetics (F) andfor Oseltamivir-Resistant R6, R7, and F viruses (R6) Replicate Faster than were examined bythea R7 Virus In regular Vitro virological method (PFU Viral plaquekinetics replication assay and TCID for R6, 50)and R7, andFexpressed viruses were in titers examinedat different time virological by a regular points (Figure 1A method and (PFUB). For PFU plaque assayplaque and TCID assay50 ) results, and viral expressed titers in from titers at R6-infected different timecells (2.4 points × 10 (Figure5 PFU/mL) were 1A,B). For PFU similar to thatresults, plaque assay for theviral F virus titers(1.6from× 10 R6-infected cells (2.4 × 105 higher 5 PFU/mL), but 24 times PFU/mL) than thesimilar were one from R7-infected to that for the F cells virus(1(1.6 × 10 4 × 10 5 PFU/mL) at 24but PFU/mL), hpi24 (Figure times1A). Nothan higher differences the onewere fromobserved at 48cells R7-infected hpi.(1 × 104viral When titers PFU/mL) were measured at 24 hpi (Figureby1A).the No TCID 50 assay (Figure differences 1B), the progeny were observed at 48 hpi.from When F virus viralwas detected titers as early asby were measured 6 hpi, whereas the one from the R6 virus was firstly detected at 12 hpi. the TCID50 assay (Figure 1B), the progeny from F virus was detected as early as 6 hpi, whereas the At 24 hpi, higher viral titers were observed one from the R6invirus F- (10was 6.8 TCID50/mL and R6- (106.9 TCID50/mL) infected cells when compared with R7 firstly detected at 12 hpi. At 24 hpi, higher viral titers were observed in F- 5.8 TCID50 /mL and R6- (106.9Viral (106.8TCID 50 /mL) (Figure 1B). TCID 50 /mL) infected production was undetectable before 24 cells when compared hpiR7 with in (10 R7-infected 50 /mL) 5.8 TCID cells. However, (Figure 1B).both Viral OR viruses (R6 production was and R7) reachedbefore undetectable similar24titers hpi inatR7-infected 48 hpi, with values cells. of 106.1 and However, both10 6.8 OR 50 /mL, TCID viruses50/mL, (R6 and respectively. R7) reachedInsimilar conclusion, titers atthe R6 virus 48 hpi, seems of with values 6.1 to10replicate 6.8 and 10faster TCIDthan the respectively. R7 virus in vitro. In conclusion, the R6 virus seems to replicate faster than the R7 virus in vitro. Figure 1. In vitro virus kinetics of pdm09 OR viruses (R6 and R7) were compared with a non-resistance Figure 1. In vitro virus kinetics of pdm09 OR viruses (R6 and R7) were compared with a pdm09 virus (F) and the viral titer was determined at 0, 6, 12, 24, and 48 h post-infection (hpi) by a non-resistance pdm09 virus (F) and the viral titer was determined at 0, 6, 12, 24, and 48 h plaque assay (A) and by 50% tissue culture infective dose (TCID50 ) (B). post-infection (hpi) by a plaque assay (A) and by 50% tissue culture infective dose (TCID50) (B). 3.2. R6-Infected Mice Presented Earlier Clinical Signs and Higher Mortality Rates than R7-Infected Mice 3.2. R6-Infected Mice Presented Earlier Clinical Signs and Higher Mortality Rates Than R7-Infected Mice C57BL6 mice were intranasally infected with 104 PFU of R6 or R7 viruses. The control group was not subjectedmice C57BL6 were intranasally to intranasal infected inoculation, withuntreated. remaining 104 PFU ofAnimals R6 or R7 viruses. were The control monitored group daily for two was weeks to control the body weight and survival (Figure 2A,B). Loss of body weight was not observedfor not subjected to intranasal inoculation, remaining untreated. Animals were monitored daily in Viruses 2020, 12, x FOR PEER REVIEW 6 of 19 Viruses 2020, 12, 794 6 of 20 two weeks to control the body weight and survival (Figure 2A and B). Loss of body weight was not observed in untreated mice. The R6-infected group experienced a peak of weight loss at day 4 pi and R7-infected untreated mice.mice The showed R6-infecteda similar peak but thereafter, group experienced a peak of at day 7loss weight pi.atDuring day 4 pithe and first 2 dpi, the R7-infected R6-infected mice showed agroupsimilarshowed peak but a thereafter, statisticallyatsignificant (p < 0.05) day 7 pi. During higher the first percentage 2 dpi, of weight the R6-infected group loss compared showed to R7-infected a statistically significant < 0.05) mice.(pBoth infected highergroups percentageexperienced of weightalossstatistically compared significant to R7-infectedhigher percentage mice. of weight Both infected groupsloss from days experienced 3 to 7 pi compared a statistically to uninfected significant higher percentageanimals (p