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Paragraph: However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .  Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as "carrageenan"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days.

Question: What percentage of people infected with influenza have a viral coinfection?
Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] .