Influenza Virus: Small Molecule Therapeutics and Mechanisms of Antiviral Resistance

Abstract

Background:

Influenza viruses cause severe upper respiratory illness in children and the elderly during seasonal epidemics. Influenza viruses from zoonotic reservoirs can also cause pandemics with significant loss of life in all age groups. Although vaccination is one of the most effective methods to protect against seasonal epidemics, seasonal vaccines vary in efficacy, can be ineffective in the elderly population, and do not provide protection against novel strains. Small molecule therapeutics are a critical part of our antiviral strategies to control influenza virus epidemics and pandemics as well as to ameliorate disease in elderly and immunocompromised individuals.

Objective:

This review aims to summarize the existing antiviral strategies for combating influenza viruses, the mechanisms of antiviral resistance for available drugs, and novel therapeutics currently in development.

Methods:

We systematically evaluated and synthesized the published scientific literature for mechanistic detail into therapeutic strategies against influenza viruses.

Results:

Current IAV strains have developed resistance to neuraminidase inhibitors and nearly complete resistance to M2 ion channel inhibitors, exacerbated by sub-therapeutic dosing used for treatment and chemoprophylaxis. New tactics include novel therapeutics targeting host components and combination therapy, which show potential for fighting influenza virus disease while minimizing viral resistance.

Conclusion:

Antiviral drugs are crucial for controlling influenza virus disease burden, but their efficacy is limited by human misuse and the capacity of influenza viruses to circumvent antiviral barriers. To relieve the public health hardship of influenza virus, emerging therapies must be selected for their capacity to impede not only influenza virus disease, but also the development of antiviral resistance.

1. INTRODUCTION

Influenza viruses constitute a serious threat to human health, causing seasonal epidemics and occasional pandemics that result in significant morbidity and mortality worldwide. According to World Health Organization reports, each year seasonal influenza virus epidemics affect 3-5 million people and cause 250,000-500,000 deaths worldwide [1]. Influenza viruses cause an upper respiratory infection in humans affecting people of all ages; however, young children and the elderly are the most vulnerable to influenza virus infection. These two groups, in addition to immunocompromised individuals, are considered to be high-risk groups as influenza virus infections are severe in these populations [2]. Furthermore, seasonal influenza vaccines do not induce robust and protective immunity in these high-risk groups [3]. Antiviral drugs play a crucial role in impeding influenza virus disease progression and transmission, especially in these populations [2]. However, a major hurdle to treating influenza virus infections is the propensity of influenza viruses to develop antiviral resistance.

Influenza viruses are members of the Orthomyxoviridae family of negative-stranded RNA viruses containing genomes composed of 7-8 segments. The Orthomyxoviridae family includes seven genera: influenza viruses A-D, isavirus, thogotovirus, and quaranjavirus [4, 5]. Based on the serology of two viral glycoproteins, hemagglutinin (HA) and neuraminidase (NA), influenza A virus (IAV) can be further classified into subtypes such as H1N1, H2N2, H5N1 etc. Currently, there are at least 18 HA and 11 NA subtypes [6, 7]. IAV and influenza B viruses co-circulate and cause seasonal epidemics in humans. Of the seven genera, IAV displays broad host species tropism, infecting waterfowl, poultry, horses, pigs and seals; however, type B and C infections are usually restricted to humans and type D infections are limited to swine and cattle [8, 9]. Aquatic birds are considered to be the natural reservoirs for IAV as they harbor all subtypes of IAV strains [10]. IAV circulating in wildlife and domestic animals have crossed the species barrier and caused sporadic infection and even pandemics in humans. Zoonotic transmission of IAV has led to the last four influenza pandemics – the 1918 H1N1 Spanish flu, 1957 H2N2 Asian flu, 1968 H3N2 Hong Kong flu, and 2009 H1N1 swine flu [11]. On numerous occasions, IAV strains such as H5N1 and H7N9 that are endemic in domestic poultry have caused wide-spread infections in Southeast Asia with significant loss of human life [11, 12]. In contrast to IAV, influenza B viruses do not have an animal reservoir outside of humans and thus do not cause pandemics. As seasonal influenza vaccines contain antigens that induce immunity against seasonal strains but not against novel zoonotic strains, antivirals are the first line of defense against novel IAV strains until sufficient quantities of vaccines are made available.

The IAV genome is composed of eight negative-sense, single-stranded RNA segments encoding 11 or 12 viral proteins. Additional viral proteins are encoded through mRNA splicing or by utilizing alternate open reading frames for translation. Viral proteins polymerase basic protein 1 (PB1, RNA polymerase), polymerase basic protein 2 (PB2, cap binding protein), and polymerase acidic protein (PA, endonuclease) form a heterotrimeric RNA-dependent RNA polymerase (RdRp) complex that is involved in transcription and replication of the viral genome. Nucleoprotein (NP) binds to viral RNA and is critical for both transcription and replication processes. Three integral membrane proteins, HA, NA, and matrix 2 ion channel (M2), are present on the virion surface. HA, a type I membrane protein, facilitates the binding of virus particles to the cell surface receptor (sialic acid) and mediates subsequent entry into host cells. Under low pH conditions, HA undergoes a conformational change, activating it to fuse the viral envelope with the endosomal membrane. NA, a type II membrane protein, removes sialic acid to prevent the re-attachment of newly formed virions to the surface of infected cells and promote viral spread. M2, a small integral membrane protein, forms a proton pump on the virion membrane to facilitate the disassembly of the matrix protein from the ribonucleoprotein (RNP) complex. During the entry process, under low pH conditions in the late endosome, M2 pumps H⁺ into the virion. This acidification of the interior of the virion dissociates the matrix protein from the RNP complexes, thereby exposing the nuclear localization signals critical for the nuclear import of the RNP complexes. Upon entry into the nucleus, the RdRP proteins associated with the viral genomic RNA transcribe viral mRNA; then the newly synthesized RdRP proteins replicate viral genomic RNA. Nuclear export protein facilitates the export of newly formed RNP complexes to the plasma membrane for virion assembly and release. Viral proteins non-structural protein 1 and PB1-F2, expressed from a +1 ORF of PB1 mRNA, counteract host antiviral responses and enable efficient viral replication [8].

While vaccination is the best defensive strategy against seasonal influenza virus outbreaks, antivirals provide a complementary approach to fight influenza virus infection, especially in individuals who are unable to be vaccinated. Moreover, antivirals are the first line of defense for controlling novel influenza virus outbreaks, as little immunity is present in the human population and vaccine development is timeconsuming and laborious. The existing antivirals target the ion-channel activity of M2 or the sialidase activity of NA; however, a major hurdle to their efficacy is the development of resistance to these antivirals. Identification and characterization of antivirals with an expanded range of viral and host targets as well as advancing combination therapy are critical to generate novel and effective treatments with less potential for the emergence of resistant variants in order to ease the perpetual burden of influenza virus to public health.

2. FDA-APPROVED DRUGS AGAINST INFLUENZA VIRUSES

At present, there are five FDA-approved antivirals for the treatment of influenza virus infection that are categorized into two groups, M2 ion channel inhibitors (adamantanes) and NA inhibitors (NAIs). Due to widespread resistance of IAV to the adamantanes, the CDC currently recommends the three NA inhibitors as antiviral therapies [13]. The adamantane group consists of amantadine and rimantadine, which target M2 to inhibit influenza A virion disassembly. However, adamantanes are ineffective against the M2 of influenza B viruses because of dissimilarity in the amino acid sequences of the two proteins, resulting in polar residues lining the M2 pore for influenza B viruses rather than hydrophobic residues lining the M2 pore for IAV [14, 15]. NAIs include zanamivir, oseltamivir, and peramivir and function to prevent the release of nascent virions for both IAV and influenza B viruses. The currently circulating IAV strains are almost fully resistant to adamantanes, thus amantadine and rimantadine are no longer used for the treatment of IAV infection. In contrast, resistance to the available NAIs varies among seasonal strains, and this group of drugs is actively used for the treatment of influenza virus infections in humans [16].

3. M2 INHIBITORS (ADAMANTANES)

3.1. Antiviral Mechanism

The first antivirals licensed for the treatment of influenza virus infection were amantadine and rimantadine, two small molecule inhibitors derived from the adamantane drug class (Fig. 1. These inhibitors act as molecular corks that plug the barrel of the M2 ion channel with a positively charged amino group, exerting electrostatic hindrance and preventing H⁺ flow through M2 into the virion (Fig. 3) [17, 18].

Fig. (1). Chemical structures of adamantanes.

Chemical structures were obtained from PubChem substance and compound database.

Fig. (3). Mechanisms of action of small molecule inhibitors of influenza viruses.

Step 1: Binding of influenza virus particle to sialic acid and uptake of virion via endocytosis. DAS181 removes cell surface sialic acids, thereby preventing viral infection. Step 2: Acidification of the virion in the endosome for virion disassembly. Adamantanes block the M2 ion-channel from pumping H⁺ into the virion and prevent virion disassembly. Step 3: HA-mediated fusion of viral membrane and endosomal membrane. Inhibitors such as RO5464466, arbidol, MBX2329, and MBX2546 block viral fusion. Step 4: Import of RNP complexes into the nucleus where viral transcription and replication occur. Step 5: Primary viral transcription by genome associated RdRP complex. VX-787 is a competitive inhibitor of PB2’s cap-binding activity, and L-742,001 and S-033188 inhibit the endonuclease activity of PA. These inhibitors block transcription of viral mRNA. Step 6a: Export of viral mRNA and synthesis of viral proteins. Nucleozin induces aggregation of NP molecules and blocks NP functions. Step 6b: Import of newly synthesized viral proteins and trafficking of HA, NA, and M2 to the plasma membrane. Nitazoxanide interferes with HA processing and maturation. Step 7: Replication of viral genome by newly synthesized RdRP proteins. Favipiravir and ribavirin are nucleoside analogs that interfere with RdRP activity. Naproxen and curcumin bind to the conserved RNA binding groove in NP and prevent RNA binding. Step 8: Export of RNP complexes from the nucleus to plasma membrane for virion assembly and release. Step 9: Release of virion from plasma membrane. NAIs inhibit the sialidase activity of NA and prevent the removal of cell surface sialic acids. This results in the aggregation of newly formed virions on the surface of infected cells and restriction of viral spread. Protease inhibitors block the extracellular cleavage and maturation of HA into a fusion-competent form, thereby preventing new rounds of infection.

3.2. M2 Resistance Mechanisms

Amantadine and rimantadine have become obsolete for inhibition of IAV due to widespread resistance among circulating human strains. Researchers first identified resistance to adamantanes in vitro as early as 1965 [19], and clinicians observed resistance in patients in the early 1980s [20, 21]. Mutations at amino acid positions 27, 30, 31, and 34 in the transmembrane domain of M2 confer resistance to adamantanes [22, 23]. S31N is the predominant resistance mutation, comprising 92% of resistant strains in the US (Table 1) [24]. Resistance mutations prevent adamantane inhibition via two mechanisms: reduction of adamantane binding by steric hindrance (S31N), and increased mobility of the pore, allowing for H⁺ flow through the pore in the presence of adamantane binding [22, 25]. As resistance mutations in the pore do not affect the function of the ion channel, mutations in M2 do not impede viral replication in vitro [26] and in the ferret model in vivo [27].

Table 1.

Antiviral resistance prevalence, resistance mutations, and compensatory mutations in clinically-relevant IAV strains.

Influenza Subtype	M2 Ion Channel Inhibitors (Adamantanes)			NA Inhibitors
				Oseltamivir			Zanamivir
	% Resistant	Major M2 Mutation	Reference	% Resistant to NAIs	Major NA Mutations*	Compensatory Mutations	Major NA Mutation*	Refs.
H3N2	>96%	S31N	Deyde et al. [110]	0.2%	R292K E119V N294S	Unknown	Q136K	Hurt et al. [55] Kiso et al. [43]
Pre-2009 seasonal H1N1	16%	S31N	Deyde et al. [110]	>99% (oseltamivir)	H274Y	V234M, R222Q, K329E, D344N, D354G	Q136K	Dharan et al. [60] Hauge et al. [38] Bloom et al. [52] Hurt et al. 49
2009 pandemic H1N1	100% (USA)	S31N	MMWR30 Rungrotmongkol et al. [111]	0.5%	H274Y I223R	V240I, N368K	I223R	Hurt et al. [55] Butler et al. [53]
H5N1	Varies by clade	S31N V27A	Hill et al. [66] Baranovich et al. [112]	Rare	H274Y N294S	Unknown	Unknown	Hill et al. [66] Baranovich et al. [112]

^*NA amino acids are represented using N2 numbering.

3.3. Emergence of Adamantane-Resistant Strains

Upon licensing of adamantanes for use to inhibit IAV in humans, resistance was quickly observed in treated patients [19]. Early studies demonstrated that 30-50% of patients shed resistant viruses as soon as 2-3 days post adamantane treatment [28]. However, the majority of circulating strains remained sensitive to adamantanes for decades [20]. Resistance worldwide increased drastically from 1.8% in 2002 to 13.3% in 2003 [29]. By 2005, 92% of H3N2 strains had acquired the S31N mutation that confers resistance to adamantanes [24]. During the 2009 H1N1 outbreak, pH1N1 and H3N2, which comprised almost 100% of circulating strains, were completely resistant to adamantanes [30]. Currently, over 99% of circulating IAV strains are resistant to adamantanes (Table 1) [31]. However, limited stocks of adamantanes are maintained for rapid response against the emergence of adamantane-sensitive IAV strains.

4. NEURAMINIDASE INHIBITORS

4.1. Antiviral Mechanism

Since the emergence of adamantane-resistant viruses, inhibitors targeting the neuraminidase of influenza viruses have been developed. The three FDA-approved NAIs, zanamivir, oseltamivir, and peramivir, are derivatives of N-acetyl-neuraminic acid (2,3-dehydro-2-deoxy-N-acetylneuraminic acid, “DANA”, Fig. 2. These NAIs bind to the active site of NA, competitively inhibiting binding of NA to its substrate, N-acetyl-neuraminic acid. Due to this inhibition, NA can no longer cleave N-acetyl-neuraminic acid from host proteins, preventing virion release (Fig. 3). Zanamivir is structurally similar to DANA, with the exception of a 4-guanidino group replacing the C4-OH [32]. The crucial addition of the large basic group increased binding to NA compared to DANA by 10,000 fold [33]. Oseltamivir contains an amine group and pentyl ether side chain in place of the C4-OH and glycerol side chain of DANA, respectively [34]. Peramivir features a cyclopentane ring, the 4-guanidino group of zanamivir, and the pentyl ether side chain of oseltamivir [35]. The pentyl ether side chain favors binding to the hydrophobic pocket of NA over the glycerol side chain in DANA [34]. Because of the similarity of the NAIs to the substrate of NA, these inhibitors are particularly effective against influenza viruses and developing resistance to these inhibitors is more complex [31].

Fig. (2). Chemical structures of neuraminidase inhibitors.

Chemical structures were obtained from PubChem Substance and Compound database.

4.2. NA Resistance Mechanisms

Due to slight differences in the structure of NAIs, resistance is usually drug and strain specific [2]. H274Y is the primary mutation responsible for oseltamivir and peramivir resistance in H1N1 and H5N1 strains, [36–41] although the presence of this mutation in H5N1 strains is rare (Table 1; N2 numbering). Along with R292K and N294S resistance mutations, H274Y blocks E276 from accommodating the pentyl ether side chain of oseltamivir [42]. R292K and E119V are the most frequent oseltamivir resistance mutations in H3N2 strains [43]. R292 is one of the residues in the catalytic triad, necessary for binding sialic acid and NAIs. Pandemic H1N1 can contain H274Y, I222V, or I222R, and even a combination of these mutations, resulting in oseltamivir resistance and sometimes multi-drug resistance to all three NAIs [44–47].

Oseltamivir or zanamivir resistance mutations usually cannot confer cross-resistance to each other. While H274Y is specific to oseltamivir and peramivir resistance due to the shared pentyl ether group, Q136K is important for zanamivir resistance in H3N2 and H1N1 strains [48, 49]. However, Q136K arises rapidly during passaging in cell culture. Therefore, it is often unclear whether the resistance mutation is initially present in the clinical isolate or emerges as a result of selection, thus hindering NAI resistance evaluation and interpretation [50].

NAI resistance mutations can result in reduced viral fitness; however, in the 2007-2008 influenza season, an oseltamivir-resistant H1N1 strain without any defects in viral fitness emerged, spread, and swiftly became the dominant circulating strain. Unlike the previous resistant strains with the single H274Y mutation, this oseltamivir resistant strain contained additional mutations, V234M, R222Q, K329E, D344N, and D354G. These mutations compensated for the loss of fitness caused by the H274Y mutation, restoring full NA function and viral replication [51, 52]. However, the 2009 novel H1N1 pandemic virus (pH1N1), which was sensitive to NAIs, replaced the 2007-2008 seasonal H1N1 oseltamivir-resistant strain. Although present at low levels, some strains of pH1N1 containing the H274Y mutation can transmit efficiently within communities with compensatory mutations V240I and N368K [44, 45, 53].

4.3. Emergence of NAI-Resistant Strains

Oseltamivir resistance emerges more readily in H1N1 strains (27%) than H3N2 strains (3%), as shown in a study conducted from 2005-2007 [54]. Indeed, currently circulating H3N2 strains are 0.2% resistant to oseltamivir, while pre-pandemic seasonal H1N1 strains reached over 99% resistance to oseltamivir (Table 1) [31, 55]. Incidences of resistance emergence are most often seen in children and immunocompromised individuals on oseltamivir regimens, potentially due to prolonged periods of viral replication, higher viral loads, and sub-optimal doses of oseltamivir [43, 56, 57]. However, resistance also emerges in individuals undergoing chemoprophylaxis or even in individuals with no prior exposure to antivirals or drug-resistant strains [58, 59]. As peramivir is the newest FDA-approved NAI, there is not much data on peramivir resistance. Due to the shared pentyl ether group and the predominant resistance mutation H274Y, cross-resistance to oseltamivir and peramivir is frequently seen [33].

Global circulation of oseltamivir-resistant viruses containing the H274Y mutation remained low before 2007, due to weakened viral replication [39]. Unfortunately, in the 2007-2008 IAV season, an H274Y H1N1 strain with unimpaired viral fitness quickly overwhelmed antiviral defenses in Europe, swiftly spreading to North America and Asia [36, 38, 60]. By 2008-2009, 98.5% of seasonal H1N1 strains contained the H274Y mutation [60]. In a turn of events, a pandemic H1N1 strain emerged in 2009, replacing the circulating seasonal H1N1 strains, even though the pandemic strain was susceptible to oseltamivir. Sporadic cases of H274Y and oseltamivir resistance in pH1N1 have emerged since 2010, and resistance can develop within 48 hours of treatment [2]. As with seasonal H1N1, oseltamivir resistance emerges most prominently in immunocompromised individuals [2]. In the 2013-2014 season, 1.9% of circulating H1N1 strains were resistant to oseltamivir [61]. In the 2014-2015 season, oseltamivir-resistant pH1N1 decreased to 0.6%, similar to levels in the 2012-2013 season [55]. Similar levels of oseltamivir-resistant pH1N1 (0.7% in the USA) persisted in the 2015-2016 season [62]. However, community outbreaks with a greater proportion of multi-drug resistant pH1N1 have occurred in Australia (15%) and Japan (29%), raising the question of whether these strains will persist and transmit on a global scale [44, 63]. These mutations also confer cross-resistance to peramivir [31, 63].

Resistance to zanamivir has remained low, most likely due to similarities in structure to DANA and sialic acid, the substrate of NA. Mutations to escape zanamivir would presumably influence sialic acid binding and interfere with the function of NA [33]. Additionally, since zanamivir is used much less frequently than oseltamivir to treat IAV infections, lack of exposure to zanamivir could also explain the absence of resistance [31]. Zanamivir resistance, conferred through the mutation Q136K, was found in under 2.3% of seasonal H1N1 and H3N2 strains in Australasia and Southeast Asia, and isolated zanamivir resistance was also observed in pH1N1 [47–49]. In the 2015-2016 season, all circulating strains in the US remained susceptible to zanamivir [62].

Avian IAV strains are predominantly susceptible to NAIs [64, 65]. Although a few H5N1 clades show resistance in nature, resistance also emerges readily during treatment, mostly through acquisition of the H274Y mutation [40, 41, 66]. H7N9 strains have been shown to develop the resistance mutation R294K during treatment, conferring resistance to oseltamivir and peramivir, and demonstrating reduced sensitivity to zanamivir [65, 67].

5. FACTORS DRIVING ANTIVIRAL RESISTANCE

Many elements contribute to the emergence of antiviral resistance, including several intrinsic properties of influenza viruses. Due in part to its RNA genome and RdRP, which lacks proof-reading activity, influenza virus is highly mutagenic, resulting in the production of “quasispecies,” a population of genetically distinct viral variants that cluster around a central genomic sequence. This assortment of sequences allows influenza virus to overcome selection pressures, such as antiviral treatment. Antigenic drift occurs when mutations arise that allow influenza virus to evade immune detection and control. The surface glycoproteins are also constantly fluctuating to escape immune detection, resulting in antigenically distinct influenza virus strains. In addition, due to the segmented nature of the genome, when multiple different strains of IAV infect a host, genome shuffling can occur, whereby segments of multiple strains mix to produce novel strains. This process, known as genetic reassortment, may also play a role in disseminating antiviral resistance if a sensitive strain acquires a segment containing resistant properties. These perpetual modes of diversifying the genome allow influenza viruses to adapt to selective pressures including antiviral restriction.

Human characteristics and practices can also impact the development of antiviral resistance. Using antivirals as a prophylactic measure contributes to the emergence of antiviral resistance. Influenza virus transmission to an individual undergoing chemoprophylaxis can cause selection of resistant variants, promoting the spread of resistance. This process is compounded if the individual is on a sub-optimal dose of antivirals, as there is not enough concentration to inhibit viral replication, but enough pressure to select for resistant variants. Also, already infected patients on sub-optimal dosages of antivirals will provide conditions for resistant variants to emerge [59, 68]. Moreover, resistance develops during cases of prolonged viral replication and shedding caused by virulent and pandemic strains or infection of individuals in high risk groups that are unable to clear the infection efficiently. Due to higher viral burden, longer duration of replication, and selection pressure from antivirals in these circumstances, there is an increased likelihood for resistant variants to emerge and dominate. As seen by these influenza virus-driven and host-contributed factors, an intricate network of components drives antiviral resistance (Table 2).

Table 2.

Factors contributing to antiviral resistance.

Virus-driven Factors		Host-contributed Factors
Issue	Potential Solution	Issue	Solution
Error-prone viral polymerase, quasispecies, antigenic drift	Target host pathways	Chemoprophylaxis, use of sub-therapeutic doses	Confined and checked use of chemoprophylaxis with full dosage
Antigenic shift	Vigilant surveillance	Prolonged shedding due to virulent strain or infection of high risk group	Hospitalized isolation, treatment and maintenance to prevent nosocomial transmission

6. COMBATING ANTIVIRAL RESISTANCE

Antivirals should be considered second in the line of defense in fighting influenza virus epidemics. Vaccination is our best recourse for containing influenza virus outbreaks and limiting emergence and transmission of antiviral resistant strains [2]. While the protection afforded by the seasonal influenza virus vaccine varies from year to year, in this past season almost 100% of influenza virus isolates were antigenically similar to the 2015-2016 Northern Hemisphere vaccine [62]. Antivirals could provide additional protection for high-risk groups and individuals who are unable to be vaccinated [2, 69].

Combination therapy is a promising treatment method with little emergence of resistance when compared to treatment with single antivirals. Combining multiple NAIs may result in competitive antagonism of the inhibitors, but combining NAIs with other classes of antivirals may act synergistically to improve disease outcome and reduce the emergence of resistance [70–72]. Interestingly, a study performed in the mouse model demonstrated a dose-dependent protective effect of amantadine against amantadine-resistant H1N1 when present in a triple-combination therapy with oseltamivir and ribavirin [73]. Although more clinical trials are needed, treating influenza virus infections with multiple antivirals targeting distinct stages of the viral life cycle shows promise.

To increase the success of antivirals, chemoprophylaxis should be prescribed cautiously to lower exposure of influenza virus to antivirals. Indeed, drug resistance and transmission can still emerge in individuals who have undergone chemoprophylaxis, especially when given sub-therapeutic doses [59, 68]. The CDC generally recommends chemoprophylaxis be reserved for individuals at higher risk for influenza virus complications who may have exposure from infected family members or close contacts [2]. By limiting chemoprophylaxis, we can reduce drug resistance and increase antiviral availability and efficacy for those whose threat of influenza virus-related complications is more imminent. In addition, careful surveillance, management, and swift action must be utilized to keep drug-resistant strains from circulating.

7. NOVEL ANTIVIRAL INHIBITORS

The emergence of antiviral resistance is concerning, highlighting the need to develop novel antiviral drugs with lowered potential for resistance (Fig. 3. Many IAV strains, including oseltamivir-resistant strains, have shown strong, long-lasting susceptibility to laninamivir, another DANA-derived NAI licensed for use in Japan [74, 75]. However, some strains of IAV containing the R292K mutation, including H3N2 and H7N9, show reduced sensitivity to laninamivir [67, 76]. Other promising strategies against influenza viruses that are in development include inhibitors to the RdRP components, HA, and NP, as well as inhibitors targeting host processes.

7.1. Polymerase Inhibitors

Favipiravir and ribavirin are nucleoside analogs that specifically inhibit the RdRP activity of diverse families of RNA viruses, including influenza viruses. Favipiravir demonstrates strong inhibition of all circulating strains of IAV, with effective concentration 50 (EC₅₀) values in the nanomolar range [77]. Both of these broad-spectrum antivirals have shown potential in combination therapy [72, 73, 78]. Favipiravir is licensed for limited use in Japan and has recently completed a Phase 3 clinical trial in the US [31, 72]. In a 2016 study conducted in Japanese patients, favipiravir treatment resulted in low levels of mutations derived from the RdRP, without any changes in sensitivity to favipiravir [79]. Although attenuated, strains containing the mutation V43I in the PB1 subunit of the RdRP confer resistance to ribavirin in vitro and in mice [80].

L-742,001, S-033188, and VX-787 are small molecular inhibitors targeting subunits of the RdRP [81]. L-742,001 blocks the PA endonuclease by acting as a metal-chelating agent [82, 83]. Drug selection studies performed with L-742,001 indicate that resistance is extremely low and resistance did not arise organically in cell culture experiments [84]. S-033188 also inhibits PA and a Phase 3 clinical trial of S-033188 is ongoing [81] VX-787 is a competitive inhibitor of PB2’s cap-binding activity that confers almost full protection to IAV challenge upon treatment up to 96 hours after infection in the mouse model [85, 86]. Unfortunately, low levels of resistant variants have already developed in a human study, although these strains showed reduced viral fitness [87].

7.2. HA Inhibitors

HA is the major surface glycoprotein imperative for virion attachment and membrane fusion. HA is synthesized as a precursor protein, HA₀, which is fusion incompetent. Proteolytic cleavage by trypsin-like proteases in the respiratory tract converts HA₀ into the fusion-competent form containing the HA₁ and HA₂ subunits linked by a di-sulfide bond. The HA₁ subunit facilitates the binding of virus to sialic acid and the HA₂ subunit mediates fusion of viral and endosomal membranes under low-pH conditions. As this is one of the first steps of influenza virus infection, HA is a popular target for the development of antiviral drugs [88]. Several compounds, including RO5464466, arbidol, MBX2329, and MBX2546, obstruct HA fusion by binding to the pre-fusion state and preventing low pH-mediated fusion [88–91]. Broadly-neutralizing antibodies also show anti-influenza virus activity by binding to the stem region of HA to prevent the conformational change necessary for fusion or by binding to the receptor binding face to block virion attachment [88, 92, 93]. Nitazoxanide is a small molecule inhibitor with broad spectrum activity against IAV strains and other RNA and DNA viruses [94]. The mechanism of nitazoxanide involves blocking post-translational HA maturation and interfering with egress of nascent virions [95]. Postexposure treatment of IAV-infected patients with nitazoxanide decreased viral titers and reduced the time to alleviation of flu-like symptoms in a Phase 2b/3 clinical trial [94, 96].

7.3. NP Inhibitors

NP is required for viral transcription, genome synthesis, and intracellular trafficking of RNP complexes. In addition, oligomerized NP binds viral RNA, concealing it from host surveillance. The inhibitor nucleozin induces NP aggregation, preventing efficient RNP packaging [72, 97]. However, pre-existing resistance against nucleozin has already been identified [98]. Naproxen and curcumin are two inhibitors that may circumvent pre-existing resistance by inhibiting the conserved RNA binding groove in NP, preventing NP-RNA interactions [98–100].

7.4. Host Targeting

Antiviral drugs targeting viral proteins can become ineffective due to the ability of the RdRP to mutate the viral genome and generate resistant variants. An alternative strategy is to target the host proteins and pathways critical for the life cycle of influenza virus [101, 102]. DAS181 is a sialidase fusion protein that cleaves α2’-6’ and α2’-3’ sialic acid from epithelial cells, potently blocking influenza virus binding and entry into cells [103–105]. Currently in clinical trials, DAS181 is administered as an inhalant to cleave sialic acid from the airway epithelium [103, 106]. In addition, multiple protease inhibitors that block cleavage and activation of fusion competent HA are also being assesed [107–109]. Moreover, genome-wide screens have led to the identification of cellular components critical for IAV replication and have elucidated the viral-host interactome. This novel information can be used to develop new host-directed antivirals with reduced predisposition for the development of antiviral resistance [101, 102].

CONCLUSION

Antivirals are critical tools in our fight against influenza virus infection and disease. However, achieving the right balance between antiviral treatment and limited resistance is a complex and daunting issue. Antivirals should be used as a supplemental measure secondary to vaccination, and antiviral treatment should generally be reserved for individuals in high-risk groups. During pandemics, antivirals can be employed to counter infection, disease, and transmission of pandemic strains in all age groups. We should monitor the emergence of predominant resistance mutations (Table 1) and take necessary measures to reduce transmission of resistant strains of IAV. Circulation of drug-resistant pH1N1 has not yet occurred, partially due to enhanced monitoring and implementation of restrained antiviral treatment of influenza virus infections, after experiences from the rapid emergence and dissemination of oseltamivir-resistant H1N1 in the 2007-2008 seasons. Multipronged approaches such as new viral-targeting drugs, combination therapies, and host-directed drugs will be necessary to continue to combat influenza virus infections.

LIST OF ABBREVIATIONS

DANA

2,3-dehydro-2-deoxy-N-acetylneuraminic acid

HA

Hemagglutinin

IAV

Influenza A virus

M2

Matrix 2 ion channel

NA

Neuraminidase

NAI

NA inhibitor

NP

Nucleoprotein

PA

Polymerase acidic protein, endonuclease

PB1

Polymerase basic protein 1

PB2

Polymerase basic protein 2, cap binding protein

RdRP

RNA-dependent RNA polymerase

RNP

Ribonucleoprotein