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. 2024 Apr 18;345:199371. doi: 10.1016/j.virusres.2024.199371

Interaction and antiviral treatment of coinfection between SARS-CoV-2 and influenza in vitro

Danlei Liu a, Ka-Yi Leung b, Hoi-Yan Lam b, Ruiqi Zhang a, Yujing Fan a, Xiaochun Xie a, Kwok-Hung Chan b,c,d, Ivan Fan-Ngai Hung a,c,d,⁎
PMCID: PMC11047751  PMID: 38621598

Highlights

  • •

    The replication of SARS-CoV-2 was suppressed when coinfected with influenza.

  • •

    Molnupiravir and baloxavir monotherapy showed inhibitory effects on SARS-CoV-2 and influenza.

  • •

    Compared with monotherapies, the combination of molnupiravir and baloxavir can effectively against coinfection and reduce dosage of both drugs.

Keywords: SARS-CoV-2, Influenza, Coinfection, Antiviral, Molnupiravir, Baloxavir

Abstract

Background

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for three years. Coinfection with seasonal influenza may occur resulting in more severe diseases. The interaction between these two viruses for infection and the effect of antiviral treatment remains unclear.

Methods

A SARS-CoV-2 and influenza H1N1 coinfection model on Calu-3 cell line was established, upon which the simultaneous and sequential coinfection was evaluated by comparing the viral load. The efficacy of molnupiravir and baloxavir against individual virus and coinfection were also studied.

Results

The replication of SARS-CoV-2 was significantly interfered when the influenza virus was infected simultaneously or in advance (p < 0.05). On the contrary, the replication of the influenza virus was not affected by the SARS-CoV-2. Molnupiravir monotherapy had significant inhibitory effect on SARS-CoV-2 when the concentration reached to 6.25 μM but did not show any significant anti-influenza activity. Baloxavir was effective against influenza within the dosage range and showed significant effect of anti-SARS-CoV-2 at 16 μM. In the treatment of coinfection, molnupiravir had significant effect for SARS-CoV-2 from 6.25 μM to 100 μM and inhibited H1N1 at 100 μM (p < 0.05). The tested dosage range of baloxavir can inhibit H1N1 significantly (p < 0.05), while at the highest concentration of baloxavir did not further inhibit SARS-CoV-2, and the replication of SARS-CoV-2 significantly increased in lower concentrations. Combination treatment can effectively inhibit influenza H1N1 and SARS-CoV-2 replication during coinfection. Compared with molnupiravir or baloxavir monotherapy, combination therapy was more effective in less dosage to inhibit the replication of both viruses.

Conclusions

In coinfection, the replication of SARS-CoV-2 would be interfered by influenza H1N1. Compared with molnupiravir or baloxavir monotherapy, treatment with a combination of molnupiravir and baloxavir should be considered for early treatment in patients with SARS-CoV-2 and influenza coinfection.

1. Introduction

Coronavirus disease 2019 (COVID-19) pandemic has led to 772 million infection and 7 million deaths. The latest SARS-CoV-2 variants are associated with milder diseases, less severe cases which require hospitalization but is capable to escape from the hybrid immunity and more contagious than their earlier variants (Araf et al., 2022; Menni et al., 2022). COVID-19 patients are prone to the risk of coinfection with other pathogens like bacteria, fungi, and virus in clinical observation (Kim et al., 2020; Stowe et al., 2021). The proportion of viral coinfection was 3 % and commonly with respiratory syncytial virus (RSV) and influenza virus A (IAVs) (Lansbury et al., 2020). Some case reports have showed that SARS-CoV-2, coinfection with influenza could cause severe outcomes and higher mortality than single virus infection (Hashemi et al., 2020; Wu et al., 2020; Swets et al., 2022).

Early antiviral treatment for SARS-CoV-2 infection has demonstrated to reduce severe disease and death. Molnupiravir is a small molecule ribonucleoside prodrug of N-hydroxycytidine (NHC) that is active against both SARS-CoV-2 and some RNA viruses (Saravolatz et al., 2023). The broad-spectrum antiviral activity of molnupiravir may be due to its mutagenic mechanism applicable to a variety of viral polymerases (Kabinger et al., 2021). Remdesivir, as the first FDA-approved drug for the treatment of COVID-19, also has a broad-spectrum activity because it acts on the RdRp, but its effect in clinical application is controversial (Kabinger et al., 2021; Pan et al., 2021). Nirmatrelvir is a major protease inhibitor targeting the 3-chymotrypsin-like cysteine protease enzyme (3CLpro) of SARS-CoV-2, and also effective against other coronaviruses. When combined with ritonavir, nirmatrelvir can reduce the virus metabolism and increase serum level of drugs (Najjar-Debbiny et al., 2022; Hammond et al., 2022).

Neuraminidase inhibitors (NAIs) which prevents the release of the influenza virus by inhibiting the activity of neuraminidase on the virus surface have been extensively used over the years (Checkmahomed et al., 2020; Laborda et al., 2016). However, long-term antiviral treatment and virus mutations may result in gradual drug resistance which needs to be continuously monitored (Checkmahomed et al., 2020; Lackenby et al., 2018). In recent years, some new drugs which target the polymerase of influenza have shown promising clinical efficacy and been approved in some countries. Baloxavir was indicated to prevent the acidic protein (PA) endonuclease snatch cap, thereby inhibits vRNA synthesis (Mifsud et al., 2019), and showed efficacy and safety in clinical trials (Hayden et al., 2018; Ison et al., 2020).

Early antiviral treatment has been shown to reduce disease severity and death in real life studies. For SARS-CoV-2 and influenza coinfection however, there is not any known standard or recommended drug therapy. A single broad-spectrum antiviral drug or any drug combination may become a potential treatment option. In this study, we investigate the effect of SARS-CoV-2 and influenza during coinfection. We also studied the effect of molnupiravir and baloxavir against the SARS-CoV-2 and influenza H1N1 individually and as combination in vitro.

2. Materials and methods

2.1. Viruses, cell lines and compounds

The SARS-CoV-2 and influenza strains were isolated from patients, including the SARS-CoV-2 wild type (WT) strain (GISAID accession number: EPI_ISL_434571) and SARS-CoV-2 Omicron BA.5.2 (GISAID accession number: EPI_ISL_13777658). Influenza A/Guangdong-Maonan/SWL1536/2019 (H1N1) pdm09-like virus was isolated from a patient in Hong Kong in 2020 (20M406578). Titration was performed by plaque forming units (PFUs) in VeroE6 TMPRSS2 cell for SARS-CoV-2 viruses and in Madin-Darby canine kidney cells (MDCK) for influenza H1N1 virus. Human lung cancer cell (Calu-3) was used for all coinfection experiments. Molnupiravir (EIDD-2801, MedChemExpress, USA) and baloxavir (baloxavir acid, MedChemExpress, USA) were dissolved in DMSO and stored at -80 °C. All experiments involving live SARS-CoV-2 were performed in a Biosafety Level 3 facility at the University of Hong Kong (HKU).

2.2. SARS-CoV-2 and H1N1 coinfection

Calu-3 cells with growth medium were seeded to 24-well plates. The coinfection model was divided into three approaches, including simultaneous and sequential infection. The viruses used were influenza H1N1 strain and SARS-CoV-2 WT strain. Two viruses with an equal amount of 0.1 multiplicity of infection (MOI) were used. The cells were infected by mixing the two viruses in the simultaneous infection model, and one of the viruses was infected for 10 h in advance in the sequential infection model. Viruses with 300μL/well were inoculated and incubated for 1 h, then the wells were washed once in PBS and replaced the inoculum with fresh DMEM/F12. The supernatants were collected at baseline, 24 h, 48 h and 72 h after infection. All conditional infections were setup in triplicate and the experiments repeated at least twice.

2.3. Cell viability assay

Calu-3 cells with growth medium were seeded to 96-well plates. The dose range of molnupiravir was from 3.9 μM to 500 μM, with baloxavir from 0.78 μM to 100 μM. A two-fold dilution of the drugs with serum-free DMEM/F12 were prepared and 100μl was added into each well for 72 h. The supernatant after 72 h incubation was discarded, followed by adding 0.5 mg/ml 100ul MTT (Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich, Merck, St. Louis, MO, USA) into each well at 37 °C in an atmosphere of 5 % CO2 for 2 h in dark. Then added 100μl 0.01 M SDS-HCl into each well to dissolve the precipitated formazan crystals. Absorbance was read at 560 nm by plate reader (GloMax Explorer, Promega) and the cell viability was calculated by comparing the absorbance of the control group, the 50 % Cytotoxic concentration (CC50) was calculated based on the cell viability.

2.4. Monotherapy for simultaneous coinfection

Calu-3 cells with growth medium were seeded to 24-well plates. Influenza H1N1 and SARS-CoV-2 WT were both diluted to 0.5 MOI and mixed together before infecting the cells. The wells were replaced the drugs with four-fold serial dilution after 1 h inoculation. The dose range of molnupiravir was from 1.56 M to 100 μM and baloxavir from 0.25 μM to 16 μM. The supernatants were collected at 72 h for viral load determination. The results were used to calculate 50 % effective concentration (EC50) and 90 % effective concentration (EC90).

2.5. Combination therapy for simultaneous coinfection

Calu-3 cells with growth medium were seeded to 96-well plates. Influenza H1N1 and SARS-CoV-2 (WT or Omicron BA.5.2) with 0.5 MOI were mixed and inoculated into cells. Combination drugs were added to cells instead of inoculum post 1 h inoculation. The dose range of molnupiravir was from 0.10 μM to 25 μM with four-fold serial dilution and the baloxavir was fixed at 4 μM. The supernatants were collected at 72 h for viral load determination. All conditional infections were set in triplicate and the experiments repeated at least twice.

2.6. Viral RNA copies quantification

RNA extraction was performed by QIAamp Viral RNA Mini Kit (Qiagen, Germany) in SARS-CoV-2 and the H1N1 coinfection part, and Viral DNA/RNA Extraction Kits (Tianlong, China) was used in the antiviral treatment part. qRT-PCR for SARS-CoV-2 and Influenza H1N1 detection was performed by QuantiNova Probe RT-PCR kit (Qiagen, Germany) and standard curve type of the StepOne Real-Time PCR system (Applied Biosystems, USA). Primers used in the detection targeted SARS-CoV-2 RdRp/Hel and FluA M genes respectively.

2.7. Immunofluorescence staining for coinfection

The infected cells were harvested and coated on the slide. Ten percent of PBS-buffered formaldehyde was used for cell fixation. The primary antibodies were mouse anti-Flu A nucleoprotein (NP) and rabbit anti-SARS-CoV-2 nucleocapsid (N) respectively. The secondary antibodies were goat anti-mouse IgG (FITC) and goat anti-rabbit IgG (Alexa Fluor 594). The image was observed by immunofluorescence microscope (Olympus BX53F).

2.8. Statistical analysis

All data were analyzed by GraphPad Prism 9.4.1 software. Nonlinear regression was used to determine the CC50, EC50 and EC90. Student t-test was used to compare the viral load between groups. The p < 0.05 was considered statistically significant.

3. Results

3.1. SARS-CoV-2 and influenza H1N1 stain coinfection in vitro

To explore the interaction between SARS-CoV-2 and influenza virus, they were simultaneously and sequentially infected on Calu-3 cell, the results showed that replication of SARS-CoV-2 was inhibited to varying degrees.

The single infection of SARS-CoV-2 WT and H1N1 was tested initially. The immunofluorescence and PCR results showed that the replication of H1N1 was faster than SARS-CoV-2 WT and reached the peak at 48 h. The SARS-CoV-2 WT reached the peak at 72 h, and the replication of SARS-CoV-2 on Calu-3 was higher than H1N1 at 0.1 MOI infection (Fig. 1a-b).

Fig. 1.

Fig 1

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Single infection of SARS-CoV-2 and Influenza H1N1. a, Immunofluorescence images of single infection at 24 h, 48 h, 72 h, 96 h and 120 h. The green (FITC) represented H1N1 and the red (Alexa Fluor 594) represented SARS-CoV-2 WT. b, The infected cell counting and viral load of single infection.

In the simultaneous coinfection model, replication of SARS-CoV-2 started to be inhibited by H1N1 after 24 h. The viral load of the SARS-CoV-2 WT in coinfection was significantly lower than WT in single infection at 72 h (p = 0.0098). The influenza H1N1 showed no significant difference in viral load upon coinfection or single infection (Fig. 2).

Fig. 2.

Fig 2

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Interaction of SARS-CoV-2 and Influenza H1N1 in simultaneous and sequential coinfection. The experiments were performed on Calu-3 cells, and the supernatants were collected to detect the viral load of two viruses respectively. *p < 0.05, **p < 0.01, *** p < 0.001. The viral load is from three independent experiments performed in triplicate.

In the sequential infection model, SARS-CoV-2 WT was significantly inhibited if pre-infected with H1N1 virus. The viral load of the SARS-CoV-2 WT strain in sequential infection was lower than WT in single infection at 72 h (p = 0.0078). However, the viral load of H1N1 was unaffected by sequential infection (Fig. 2). If pre-infected with SARS-CoV-2 WT, the trend of SARS-CoV-2 WT being suppressed could also be seen at 72 h. The replication of H1N1 was not significantly affected by pre-infection with SARS-CoV-2 (Fig. 2).

In coinfection group, double immunofluorescence staining showed the percentage of cells infected by H1N1 was higher than SARS-CoV-2 at 24 h and comparable at 48 h, then H1N1 began to decline while SARS-CoV-2 persistently increased at 72 h. Compared with coinfection and single infection groups, the replication rate of SARS-CoV-2 single infection was higher than coinfection after 24 h, but there was no difference in H1N1 infection (Fig. 3).

Fig. 3.

Fig 3

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Double immunofluorescence images of simultaneous coinfection. Immunofluorescence images of simultaneous coinfection at 24 h, 48 h and 72 h. The green (FITC) represented H1N1 and the red (Alexa Fluor 594) represented SARS-CoV-2 WT.

3.2. Single antiviral drug against simultaneous coinfection

The simultaneous coinfection model was selected to further evaluate the therapeutic effect of antiviral drugs. The CC50 values of molnupiravir and baloxavir were 542.20 μM and 28.36 μM. respectively, and the relative cell viabilities were above 90 % at 159.22 μM and 17.02 μM, respectively. Molnupiravir and baloxavir have been tested for antiviral effects against SARS-CoV-2 and influenza H1N1 in both single and coinfection. The EC50 and EC90 values were indicated in Table 1.

Table 1.

Inhibitory effect of molnuiravir and baloxavir on SARS-CoV-2 and Influenza H1N1 coinfection.

Infection group Molnupiravir
 Baloxavir
EC50 (μM) EC90 (μM) EC50 (μM) EC90 (μM)
Coinfection (WT) <1.56 3.76 ± 3.06 >16.00 >16.00
Coinfection (H1N1) 68.41 ± 46.27 >100.00 <0.25 <0.25
WT 2.98 ± 3.09 9.81 ± 2.66 8.46 ± 4.33 >16.00
H1N1 55.39 ± 58.62 >100.00 <0.25 <0.25
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Compared with the non-treated group, the dose of molnupiravir ranged from 6.25 μM to 100 μM against single infection or coinfection with SARS-CoV-2 WT revealed significant inhibition (p < 0.05), and the H1N1 can also be suppressed at 100 μM (p = 0.0271) in coinfection group (Fig. 4a). Baloxavir was found significantly inhibited SARS-CoV-2 WT at 16 μM in single infection (p < 0.05) but did not inhibit SARS-CoV-2 WT in coinfection. Meanwhile, the replication of SARS-CoV-2 WT in coinfection significantly increased (p < 0.05) and was close to single infection when the baloxavir was below 16 μM (Fig. 4b). The replication of H1N1 was significantly inhibited in the treatment with all dose range of baloxavir in both coinfection and single infection group (Fig. 4b).

Fig. 4.

Fig 4

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Single drug activity in SARS-CoV-2 and influenza H1N1 coinfection. *p < 0.05, **p < 0.01, *** p < 0.001. The viral load is from three independent experiments performed in triplicate.

3.3. Drug combination against simultaneous coinfection

According to the results of individual treatment, the highest tested concentrations of molnupiravir and baloxavir were used below the EC50 against the coinfection. The dosage of molnupiravir ranging from 0.10 μM to 25 μM and baloxavir at 4 μM were then selected. There was no difference between molnupiravir and baloxavir combination or molnupiravir monotherapy in the treatment of SARS-CoV-2 WT or Omicron BA5.2 in single infection. On the contrary, lower dose of molnupiravir (from 0.10 μM to 6.25 μM) monotherapy was more effective against coinfection (p < 0.05), molnupiravir/baloxavir combination suppressed the SARS-CoV-2 WT and Omicron BA5.2 effectively when the molnupiravir drug concentration reached 25uM. Suppression of the influenza H1N1 is entirely due to the baloxavir effect in both single infection and coinfection (Fig. 5).

Fig. 5.

Fig 5

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Drug combination activity in SARS-CoV-2 and influenza H1N1 coinfection. The drugs were added after 1 h virus inoculation and incubated with Calu-3 cell for 72 h to quantify the viral load in the supernatants. In molnupiravir and baloxavir combination group, the concentration of baloxavir was fixed at 4 μM. The dose range of molnupiravir was from 0.10 μM to 25 μM in all groups. The viral load of H1N1 only showed the results of SARS-CoV-2 WT coinfection, as he results and trends are similar with Omicron BA5.2. T-test was used to compare the viral load between single and combination treatment groups, *p < 0.05, **p < 0.01, *** p < 0.001. The viral load is from three independent experiments performed in triplicate.

4. Discussion

SARS-CoV-2 and influenza coinfection could result in higher viral load, worsened cytokine storm, and organ damage, leading to more severe disease (Hashemi et al., 2020; Zhang et al., 2021; Achdout et al., 2021). Early antiviral treatment plays an important role to prevent the disease progression. In order to understand the interaction between the two viruses, we studied the individual virus replication and in coinfection, and also the effects of the antivirals molnupiravir and baloxavir separately and as combination treatment. Interestingly, coinfection of the SARS-CoV-2 and influenza H1N1 resulted in suppression of the replication of SARS-CoV-2. This is consistent with the results of some studies (Zhang et al., 2021; Achdout et al., 2021). The interference mechanism during coinfection is not yet clear, some researchers believe that one virus can be prior to activate the innate immune response, thereby inhibiting the infection and replication of the other virus (Achdout et al., 2021; McAfee et al., 2015). In animal models of IAVs infection, the peak of interferon (IFN) stimulated gene (ISGs) level at 3-day post infection (dpi) (Oishi et al., 2022). In contrast, innate immunity is delayed in SARS-CoV-2 infection, and the peak of ISGs level occurs at 5 dpi (Oishi et al., 2022; Horiuchi et al., 2021). Calu-3 is an IFN type I/III responsive cell used in our study, it is hypothesized that IAV induces early activation of IFN response, thereby suppressing the replication of the SARS-CoV-2 without antiviral treatment. The results of immunofluorescence in this study intuitively showed that the percentage of cells infected by H1N1 was higher at early stage of infection (post 24 h infection), which cannot be ruled out by the above mechanism.

The virus-virus interaction makes the drug treatment become more complicated. In our study, single therapy with either molnupiravir or baloxavir was investigated at first. The results showed that they have high efficacy against SARS-CoV-2 and influenza H1N1 respectively (Fig. 4). These two drugs at high concentration also showed cross-antiviral effects and that concentrations did not show any cytotoxicity on Calu-3. Molnupiravir is, as a broad-spectrum drug against RNA viruses (Kabinger et al., 2021), our results showed that SARS-CoV-2 is high susceptible to the drug's inhibitory effect. On the other hand, baloxavir at 16 μM also showed inhibitory activity against SARS-CoV-2. The mechanism remains unclear. Some studies indicated that baloxavir prevents the transcription of viral mRNA by targeting the nucleic acid endonuclease of viral PA polymerase (Mifsud et al., 2019), and molecular docking showed that baloxavir has a potential activity against RdRp, PLpro and Mpro of SARS-CoV-2 (Mandal et al., 2021). When compared the coinfection and single infection of SARS-CoV-2 models, the coinfection against WT exhibited lower viral load in molnupiravir treatment in general. The reason may be attributed to the interference by influenza H1N1 infection. At the lower dose of baloxavir treatment, the replication of SARS-CoV-2 increased in the coinfection, possibly due to the interference by H1N1 was decreased.

According to the results of monotherapy in this study, the drug doses were adjusted for testing in combination therapy, the concentration of baloxavir against SARS-CoV-2 was fixed to below EC50. Compared with molnupiravir alone, combination therapy showed lower efficacy in inhibiting SARS-CoV-2 in coinfection. It was suspected that in the monotherapy, molnupiravir did not significantly inhibit H1N1 in coinfection, so the inhibition of SARS-CoV-2 was considered to be the combined effect of molnupiravir and H1N1 infection. However, in the molnupiravir/baloxavir combination treatment, baloxavir effectively inhibited the replication of H1N1 and reduced its viral load to near baseline level, leading to the replication of SARS-CoV-2 in coinfection was similar to single infection (Fig. 5). When concentration of molnupiravir reached at 25 μM, the inhibitory effect of combination and monotherapy against SARS-CoV-2 are similar. As mentioned above, the significant inhibition of H1N1 by baloxavir influenced the effect of molnupiravir against coinfection with SARS-CoV-2. Single drug treatment for coinfection requires to balance the effectiveness for both viruses, the effective concentration of baloxavir against both viruses simultaneously is at 16 μM (Fig. 4b) while molnupiravir exceeds at least 50 μM according to the results of EC50 (Table 1) and monotherapy (Fig. 4a). Although the monotherapies with high concentrations did not show the cytotoxicity on cells, the combination therapy can reduce the dosage of both drugs at the same time. In our study, the combination therapy showed no synergistic antiviral effect, but its inhibitory effect on SARS-CoV-2 and influenza was significantly higher than that of monotherapies. Molnupiravir therapy alone only had inhibitory effect on H1N1 at 100 μM, the decrease of viral load was less than one log10 in coinfection. Similarly, high concentration of baloxavir at 16 μM showed more significant effect on SARS-CoV-2 and the decrease of viral load was close to one log10 and did not show further inhibition of SARS-CoV-2 in coinfection group. On the contrary, both drugs can reduce to concentration at 25 μM molnupiravir / 4 μM baloxavir in combination therapy, the replication levels of two viruses in coinfection would reduce over 2 log10. Therefore, it was believed that the combination therapy can be more effective against coinfection when compared to single drug therapy.

There are some limitations in our study. Firstly, we only evaluated the viral load without comparing viral titers. The reason is that the two virus titers need to be tested separately. Such large sample size and high workload associated with the drug study, especially in combination therapy renders viral titer test infeasible for the time being. Secondly, the study cannot show reliable cell viability by coinfection because the two viruses have different sensitivity to cause cytopathic effect (CPE) on Calu-3 cell, resulting the cell viability test may not be accurate. The overall impact of coinfection and drug treatment will be further tested and confirmed by animal model.

In summary, we found that during coinfection, SARS-CoV-2 would be interfered by influenza H1N1, resulting in reduced replication capacity. Although the virus-virus-drug interaction is complex, the combination therapy of molnupiravir and baloxavir can effectively work well against coinfection and reduce the dosage of two drugs when compared with monotherapy in vitro, and the results may provide a clinical reference for drug and dosage selection.

CRediT authorship contribution statement

Danlei Liu: Formal analysis, Data curation, Conceptualization, Methodology, Writing – original draft. Ka-Yi Leung: Methodology, Validation. Hoi-Yan Lam: Methodology, Validation. Ruiqi Zhang: Methodology. Yujing Fan: Methodology. Xiaochun Xie: Methodology. Kwok-Hung Chan: Supervision, Writing – review & editing. Ivan Fan-Ngai Hung: Supervision, Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Health and Medical Research Fund (HMRF) Commissioned Research on COVID-19 (Grant No.: COVID1903010) and Commissioned Study on Influenza Vaccine (Grant No.: IMQ) in Hong Kong; Research Grants Council Research Fund (Grant No.: 17119820) in Hong Kong. We sincerely thank all staff and students involved in this study. We also would like to acknowledge Dr. Man-Chun Chiu and Mr. Jian-Piao Cai for technical support.

Data availability

  • Data will be made available on request.

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