**5. Immunity and challenges of vaccination**

the H5N1 avian influenza virus isolated in 1997 (Hong Kong) both harbors the N66S mutation

Genetic reassortment in influenza viruses is yet another vital event that leads to sudden outbreaks of influenza. Influenza viruses have a segmented genome and thus, simultaneous infection with other IAVs results in reassortment event leading to formation of new viral progenies containing gene segments of mixed parental origin. Several pandemics have emerged in the past [54] and appears to be more frequent now than previously thought [55]. The reassortment event can be a result of errors during the replication of viral RNA polymerase, the host environment, the immune or evolutionary pressure [56]. The pandemics of 1957 and 1968 were caused by reassortant viral strains [57]. The HA, NA, and PB1 genes of the H2N2 1957 pandemic strain and the HA and PB1 fragments of the H3N2 1968 pandemic strain were both derived from avian influenza virus strains [57]. The HPAI subtype H5N1 isolated from geese in Guangdong province in 1996 evolved to produce H5N8 clades 2.3.4.4 Gs/GD HPAIV. A recent study on the evolution and pathogenicity of H5N2 avian influenza viruses isolated in H5N1 endemic areas in China revealed that these viral isolates were derived from reassortment events in which few isolates had the HA and NS derived from H5N1 while few had the HA derived from endemic H9N2 viruses [58]. A similar study from South Korea reported the emergence of novel reassortant H5N8 viruses in 2014 in ducks raised in breeder farms [59]. Since its first appearance, lineage of the HPAI H5N1 continues to circulate with lots of diversification of the HA gene into multiple genetic clades. The H5 clade 2.3.4.4 of the H5N8 subtype was subsequently detected in several countries of Europe by the end of 2014 and in summer of 2016, it was detected again in wild aquatic birds sampled in western Siberia [60]. A recent study has also shown that the reassortment event between the Gs/GD lineage H5N8 virus and North American origin viruses further resulted in the emergence of H5N1 and

Experimental observations have further revealed that reassortment between zoonotic and seasonal IAVs can result in production of airborne-transmissible viruses in mammals [62–65]. A study showed that a reassortant virus, comprising of the H5 hemagglutinin having 4 mutations from H5N1 avian virus and remaining seven segments from the 2009 pandemic H1N1 virus lead to reassortant H5 HA/H1N1 virus that gained the capacity of droplet transmission in ferret model [62]. Another experiment further showed that the avian H5N1 subtype viruses do have the potential to attain mammalian transmissibility by genetic reassortment [63]. The authors utilized reverse genetics to create several reassortant viruses between duck H5N1 (HA gene) and human-infective H1N1 virus to show that the new reassortant viruses could efficiently infect and sustained droplet transmission in guinea pigs without mortality [63]. Similar study reported that the avian-human H9N2 reassortant virus harboring the surface proteins of avian H9N2 in a human H3N2 backbone gained the ability of transmission through the respiratory droplets and caused clinical infection in ferrets similar to human influenza infections [64]. A recent study performed in a novel transfection-based inoculation system generated a reassortant H9N1 virus by transfecting the plasmids containing genes from H9N2 virus and pandemic H1N1 (pH1N1) virus into HEK 293 T cells. The resulting transfections gave rise to two reassortant viruses (H9N1) that had the capability of droplet-

in PB1-F2 which drastically enhanced the pathogenicity of these viruses [52].

H5N2 viruses in the US [61].

40 Influenza - Therapeutics and Challenges

transmissibility [65].

The isolation of influenza A/H1N1 in 1933 quickly ushered the development of the first generation of live-attenuated influenza vaccines (LAIV). The initially developed inactivated influenza vaccine (IIV) only targeted a single influenza strain (influenza A). Then, in 1942, a vaccine targeting both influenza A and B viruses were produced soon after the discovery of influenza B. Subsequently, scientists discovered that influenza viruses mutated, leading to antigenic changes (antigenic drift and antigenic shift). Since 1973, the World Health Organization (WHO) has been providing yearly recommendations for the composition of the influenza vaccine, based on results of the virological surveillance conducted by the WHO's Global Influenza Surveillance and Response System (GISRS). Later in 1978, the trivalent influenza vaccine was developed that included two influenza A strains and one influenza B strain. Currently, two influenza B lineages are circulating (Yamagata and Victoria) therefore, since 2013, the WHO recommendations suggested a second B strain to be added to make a quadrivalent influenza vaccine (QIV) [69]. Influenza vaccines protect against infection and can reduce illness and severity of infection especially in groups at risk for flu complications such as children, the elderly, pregnant women, and individuals with underlying medical conditions like asthma, HIV/AIDS, and chronic heart or lung diseases [70]. Frequent influenza infections at the human-animal interface may also warrant occupational vaccination for veterinarians, researchers, health care providers, farmers and animal traders who are more likely to be exposed to zoonotic influenza virus [71].

For over half a century now, WHO has been collaborating with scientists, epidemiologists, and policymakers to create an integrated approach to manufacture, test, and approve influenza vaccine research and development efforts, including their proper use and efficient distribution. Since the virus mutates frequently, WHO, GISRS network and collaborating centers predict the strains that are expected to circulate in the following season because of the time required to manufacture vaccines. This happens twice a year, one for the northern hemisphere and another for the southern hemisphere [70]. But the virus can mutate during the time it takes to develop the vaccine, resulting in a mismatch between circulating virus and the vaccine.

the production timeline for both IIV and LAIV [78]. Production of IIV and LAIV require the use of embryonated eggs. Disadvantages for egg-based flu vaccine production include being contraindicated in people with severe allergies to eggs, and in the event of a pandemic where the virus is pathogenic to poultry, embryonated eggs may be in short supply [69]. Currently, licensed influenza vaccines focus on the production of antibodies against the viral HA protein, which binds host receptors to mediate viral entry. Strain-specific antibodies produced against the HA neutralize the virus and prevent infection. However, the HA is under positive

Preventing Zoonotic Influenza

43

http://dx.doi.org/10.5772/intechopen.76966

Vaccine-induced HAI antibody titer is currently accepted as the correlate of protection against influenza. An HAI titer of ≥1:40 in healthy adults is the titer at which approximately 50% of individuals are protected from infection. However, some studies have indicated that a higher HAI titer may be required in children and that T cells may be a better indicator for protection in the elderly [79, 80]. Also, serum HAI antibody titer is not a reliable correlate of protection for seasonal and pandemic LAIV vaccines. LAIV has been shown to be effective in the absence of a robust serum antibody response [77]. The HAI antibody titer also fails to take into account other aspects of immune memory against the virus, including the contribution of non-neutralizing antibodies and T cell responses to protection. The immune response to influenza is complicated, and there could be several correlates of protection apart from HAI antibodies. A more comprehensive immunological analysis and an integrative genomic analysis of the human immune response [81] using the different influenza vaccines could further define other correlates of protection to better interpret influenza vac-

Influenza A viruses (IAVs) infects human, swine, and domestic poultry; therefore; interspecies and intercontinental spread make IAV more complicated. Vaccination of domestic poultry (including chicken and turkey) is common against the HPAI, H5/H7 LPAI, and H9N2 LPAI worldwide. In the past, emergency vaccination against HPAI to control epizootics has occurred. Areas include Mexico (H5N1, 1995), Pakistan (H7N3, 1995–2004), Asia/Africa/ Europe (H5N1, 1996–continuing), and North Korea (H7N7, 2005) to aid in stamping out programmes [83]. Poultry vaccines are manufactured inexpensively and are not filtered and purified like human vaccines and usually contain a whole virus, and not just HA antigen. Mineral oil, which induces a strong immune reaction and causes inflammation and abscesses, is added

Usage of vaccine to control swine influenza virus (SIV) varies by countries; some countries use vaccination strategies, while others do not. For examples, SIV vaccination is conducted extensively in Europe and North America. In Korea, on the other hand, vaccines for SIV control are rarely used despite availability in the market. Because of the genetic diversity of circulating SIV strains, most commercial vaccines consist of multiple strains of subtype H1N1, H1N2, and H3N2. Nevertheless, the rapid evolution of circulating viruses could surpass the updates of commercial vaccines. Combining the herd-specific autogenous vaccine with other commercialized vaccines occurs in some countries; about 20% of pig farms in the United States used autogenous vaccines in 2006. However, compared to avian influenza viruses, vaccines against SIVs have not been used extensively by swine veterinarians in many countries because other major pathogens including the porcine reproductive syndrome virus and porcine circovirus

selection for antigenic escape from neutralization by pre-existing antibodies [70].

cine efficacy [82].

as an adjuvant to poultry vaccines.

Although the effectiveness of the flu vaccine varies from year to year depending mainly on the match of the strain in the vaccine and the circulating strain, most provide modest to high protection against influenza [72]. The US-CDC has reported that flu vaccination reduces medical visits, flu illness, hospitalizations, and deaths [73]. Vaccination is still the most efficient way to prevent infection and severe outcomes caused by influenza viruses.

The WHO and CDC recommend yearly vaccination for nearly everyone over 6 months of age, especially those at higher risk of influenza complications and mortality [70, 73]. The European Centre for Disease Prevention and Control (ECDC) also recommends yearly vaccination of high risk groups: older adults and all persons (over 6 months of age) with chronic medical conditions including those with diseases of the respiratory system (e.g. asthma), cardiovascular system (e.g. coronary artery disease), endocrine system (e.g. diabetes), hepatic system (e.g. liver cirrhosis), renal system (e.g. chronic renal failure), neurological/ neuromuscular conditions (e.g. parkinsonism), any condition compromising respiratory functions e.g. morbid obesity (BMI > 40), physical handicap in children and adults, and immunosuppression due to disease or treatment including due to hematological conditions and HIV infection [74].

Currently licensed flu vaccines include inactivated influenza vaccine (IIV), live attenuated influenza vaccine (LAIV), and recombinant HA vaccines [75]. These vaccines are either trivalent or quadrivalent with components representing influenza A and B viruses predicted to circulate in the next influenza season. The IIV is either a split virion or subunit vaccine containing 15 μg of each purified HA protein administered intramuscularly, or 9 μg of each purified HA protein administered intradermally [75]. A higher dose version with 60 μg of each HA antigen is available for older adults aged 65 years and above. The IIV induces a strain-specific serum IgG antibody response. A vaccine with an oil-in-water adjuvant MF59 also enhances the immunogenicity of IIV in the elderly [76].

The LAIV contains live viruses with temperature-sensitive and attenuating mutations [77] and is a combination of the same four strains as the QIV. The LAIV is administered intranasally as a spray. The mutations in the LAIV strains allow the viruses to replicate at the cooler temperature of the nasal cavity but prohibit replication at the temperature of the lower respiratory tract. The LAIV results in the production of strain-specific serum IgG as well as mucosal IgA and T cell responses [77]. The recombinant HA vaccine with HA proteins expressed in insect cells from baculovirus vectors is currently licensed only for adults aged 18 to 49 years and are recommended for individuals who are allergic to eggs [75]. The manufacturing process for the recombinant HA vaccine is shorter than the IIV and LAIV, which would be important in case of a pandemic. The 2009 pandemic showed the challenges in production and distribution of vaccines against a newly emerged virus within a short timeframe given the production timeline for both IIV and LAIV [78]. Production of IIV and LAIV require the use of embryonated eggs. Disadvantages for egg-based flu vaccine production include being contraindicated in people with severe allergies to eggs, and in the event of a pandemic where the virus is pathogenic to poultry, embryonated eggs may be in short supply [69]. Currently, licensed influenza vaccines focus on the production of antibodies against the viral HA protein, which binds host receptors to mediate viral entry. Strain-specific antibodies produced against the HA neutralize the virus and prevent infection. However, the HA is under positive selection for antigenic escape from neutralization by pre-existing antibodies [70].

influenza vaccine research and development efforts, including their proper use and efficient distribution. Since the virus mutates frequently, WHO, GISRS network and collaborating centers predict the strains that are expected to circulate in the following season because of the time required to manufacture vaccines. This happens twice a year, one for the northern hemisphere and another for the southern hemisphere [70]. But the virus can mutate during the time it takes to develop the vaccine, resulting in a mismatch between circulating virus and

Although the effectiveness of the flu vaccine varies from year to year depending mainly on the match of the strain in the vaccine and the circulating strain, most provide modest to high protection against influenza [72]. The US-CDC has reported that flu vaccination reduces medical visits, flu illness, hospitalizations, and deaths [73]. Vaccination is still the most efficient way to

The WHO and CDC recommend yearly vaccination for nearly everyone over 6 months of age, especially those at higher risk of influenza complications and mortality [70, 73]. The European Centre for Disease Prevention and Control (ECDC) also recommends yearly vaccination of high risk groups: older adults and all persons (over 6 months of age) with chronic medical conditions including those with diseases of the respiratory system (e.g. asthma), cardiovascular system (e.g. coronary artery disease), endocrine system (e.g. diabetes), hepatic system (e.g. liver cirrhosis), renal system (e.g. chronic renal failure), neurological/ neuromuscular conditions (e.g. parkinsonism), any condition compromising respiratory functions e.g. morbid obesity (BMI > 40), physical handicap in children and adults, and immunosuppression due to disease or treatment including due to hematological conditions

Currently licensed flu vaccines include inactivated influenza vaccine (IIV), live attenuated influenza vaccine (LAIV), and recombinant HA vaccines [75]. These vaccines are either trivalent or quadrivalent with components representing influenza A and B viruses predicted to circulate in the next influenza season. The IIV is either a split virion or subunit vaccine containing 15 μg of each purified HA protein administered intramuscularly, or 9 μg of each purified HA protein administered intradermally [75]. A higher dose version with 60 μg of each HA antigen is available for older adults aged 65 years and above. The IIV induces a strain-specific serum IgG antibody response. A vaccine with an oil-in-water adjuvant MF59

The LAIV contains live viruses with temperature-sensitive and attenuating mutations [77] and is a combination of the same four strains as the QIV. The LAIV is administered intranasally as a spray. The mutations in the LAIV strains allow the viruses to replicate at the cooler temperature of the nasal cavity but prohibit replication at the temperature of the lower respiratory tract. The LAIV results in the production of strain-specific serum IgG as well as mucosal IgA and T cell responses [77]. The recombinant HA vaccine with HA proteins expressed in insect cells from baculovirus vectors is currently licensed only for adults aged 18 to 49 years and are recommended for individuals who are allergic to eggs [75]. The manufacturing process for the recombinant HA vaccine is shorter than the IIV and LAIV, which would be important in case of a pandemic. The 2009 pandemic showed the challenges in production and distribution of vaccines against a newly emerged virus within a short timeframe given

prevent infection and severe outcomes caused by influenza viruses.

also enhances the immunogenicity of IIV in the elderly [76].

the vaccine.

42 Influenza - Therapeutics and Challenges

and HIV infection [74].

Vaccine-induced HAI antibody titer is currently accepted as the correlate of protection against influenza. An HAI titer of ≥1:40 in healthy adults is the titer at which approximately 50% of individuals are protected from infection. However, some studies have indicated that a higher HAI titer may be required in children and that T cells may be a better indicator for protection in the elderly [79, 80]. Also, serum HAI antibody titer is not a reliable correlate of protection for seasonal and pandemic LAIV vaccines. LAIV has been shown to be effective in the absence of a robust serum antibody response [77]. The HAI antibody titer also fails to take into account other aspects of immune memory against the virus, including the contribution of non-neutralizing antibodies and T cell responses to protection. The immune response to influenza is complicated, and there could be several correlates of protection apart from HAI antibodies. A more comprehensive immunological analysis and an integrative genomic analysis of the human immune response [81] using the different influenza vaccines could further define other correlates of protection to better interpret influenza vaccine efficacy [82].

Influenza A viruses (IAVs) infects human, swine, and domestic poultry; therefore; interspecies and intercontinental spread make IAV more complicated. Vaccination of domestic poultry (including chicken and turkey) is common against the HPAI, H5/H7 LPAI, and H9N2 LPAI worldwide. In the past, emergency vaccination against HPAI to control epizootics has occurred. Areas include Mexico (H5N1, 1995), Pakistan (H7N3, 1995–2004), Asia/Africa/ Europe (H5N1, 1996–continuing), and North Korea (H7N7, 2005) to aid in stamping out programmes [83]. Poultry vaccines are manufactured inexpensively and are not filtered and purified like human vaccines and usually contain a whole virus, and not just HA antigen. Mineral oil, which induces a strong immune reaction and causes inflammation and abscesses, is added as an adjuvant to poultry vaccines.

Usage of vaccine to control swine influenza virus (SIV) varies by countries; some countries use vaccination strategies, while others do not. For examples, SIV vaccination is conducted extensively in Europe and North America. In Korea, on the other hand, vaccines for SIV control are rarely used despite availability in the market. Because of the genetic diversity of circulating SIV strains, most commercial vaccines consist of multiple strains of subtype H1N1, H1N2, and H3N2. Nevertheless, the rapid evolution of circulating viruses could surpass the updates of commercial vaccines. Combining the herd-specific autogenous vaccine with other commercialized vaccines occurs in some countries; about 20% of pig farms in the United States used autogenous vaccines in 2006. However, compared to avian influenza viruses, vaccines against SIVs have not been used extensively by swine veterinarians in many countries because other major pathogens including the porcine reproductive syndrome virus and porcine circovirus are considered more important [83]. Nevertheless, successful application of influenza vaccines in animals may contribute in reducing zoonotic transmission.

to oseltamivir [92]. A study showed that the amino acid changes at residue 223 (I → R/V) conferred reduced inhibition to oseltamivir and zanamivir [93]. The N2 subtype has been associated with oseltamivir resistance due to mutation at E119V and R292K. The R292K has also been linked to zanamivir resistance [94]. Studies have demonstrated that the most frequent mutation conferring the oseltamivir resistance in NA of the H1N1 and H5N1 subtypes was H274Y, while the E119V and R292K mutations were more common among the H3N2 and H7N9 subtypes [95]. Another study showed that R292K mutation in NA protein in the H7N9 virus strains were detected in patients after drug treatment. This substitution promoted resistance against oseltamivir [96]. Similarly oseltamivir resistance was associated with the H274Y NA mutation in H5N1 influenza viruses detected in patients during treatment or prophylaxis [97]. Few other studies have reported that the Egyptian H5N1avian influenza isolates from humans had N294S NA mutation [98]. Boltz et al. reported that H5N1 viruses of clade 2.3.2 isolated from the Republic of Laos in 2006–2008 had V116A, I222L, and S246 N mutations in NA [99]. The ongoing concerns about influenza A viruses and increasing antiviral resistance needs immediate attention, better antiviral surveillance for better management and control of

Preventing Zoonotic Influenza

45

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**7. Infection control, advances in vaccines and therapeutics**

at several crucial steps, such as entry, signaling, assembly, and egress [1].

[78]. The main areas of research and development in flu vaccines involve:

Celvapan) are also being used to overcome this issue [101].

**1.** Creation of vaccines with protective immunity lasting more than one season,

**2.** Shortening of the production time to allow a virological assessment nearer the upcoming influenza season. Cell-culture-based vaccines (e.g., Optaflu, Flucelvax, Preflucel, and

Generally, people infected with the flu are advised to stay home and rest, both to recover and to avoid infecting others. In severe cases, or for individuals at high risk of complications, physicians may prescribe antiviral medication. The antiviral drugs currently available against influenza viruses are adamantane derivatives (amantadine and rimantadine) and neuraminidase (NA) inhibitors (zanamivir, oseltamivir and peramivir). A viral infection can be inhibited

Oseltamivir, works by blocking neuraminidase that enables newly made influenza virus to escape from an infected cell. Zanamivir (inhaled), peramivir (intravenous), and inavir (inhaled) operate in a similar way. Baloxavir, discovered in Osaka, received preliminary approval in Japan in January 2018 and will be filed for regulatory review in the US and Europe thereafter. Baloxavir requires a single dose, unlike oseltamivir which is taken twice a day for 5 days [100]. Efforts to improve currently available vaccines have been explored over the last 2 decades such as: increasing the antigen dose, intradermal route of administration to activate other arms of the immune system, and adding immunostimulating compounds such as adjuvants

future influenza pandemics.

#### **6. Antiviral resistance mutants**

Antiviral resistance in influenza viruses is a global concern and the number of resistant mutants is increasing year after year. The antiviral drugs have been formulated mainly against the M2 ion channel (amantadine and rimantadine) and the neuraminidase proteins (oseltamivir and zanamivir) of influenza viruses. These FDA approved drugs are currently used for prophylaxis and treatment of influenza A infections and are effective against the HPAI H5N1 viruses [84]. The effectiveness of these drugs ranges from 80 to 90% if the treatment had begun within 48 hours of infection [85]. The antiviral resistance in influenza may develop during disease treatment and occasionally spreads widely to replace the susceptible strains in the absence of drug pressure. An example of this event is the global spread of adamantaneresistant H3N2 viruses in the year 2003, oseltamivir-resistant seasonal H1N1 viruses since 2007 and more recently the adamantane-resistant pandemic A (H1N1) viruses in 2009. Such events show the highly unpredictable nature of influenza viruses and increase the challenge of its management. Sometimes a single reassortment event or mutations leads to emergence of variant influenza viruses such as the pandemic 2009 or seasonal A (H1N1) viruses that becomes completely unresponsive to most antiviral drugs. The amantadine resistance was soon observed after the discovery of the drug in early 1960s and studies subsequently reported that a single point mutation in the M2 protein lead to the emergence of high-level resistant mutant viruses showing resistance to both amantadine and rimantadine [86]. Other studies also suggested that resistance to M2 blockers (amantadine/rimantadine) can be achieved by only a few substitutions in the codon L26, L27, A30, A31 and G34 of the M2 gene [87] and these mutants retain the virulence and are transmissible between humans [88]. A study showed that adamantane resistance emerged in about 30% of patients post few days of treatment [89]. Another study has shown the synergistic antiviral effects of amantadine-oseltamivir combination chemotherapy [90]. The adamantanes were very effective for almost 4 decades after which the frequency of adamantine resistance among influenza A H3N2 viruses started to increase. The global resistance among H3N2 virus was as low as 0.8% between the periods 1991 to 1995. The adamantine resistance has now been reported for human H1N1, H3N2 and H5N1 avian influenza viruses. The frequency of resistance further increased to 28% during 2004–2005 and to 72% in 2005–2006 for H1N1 variant viruses [1]. The US reported around 92% resistance among H3N2 viruses by the year 2005. A recent study based on the frequency and distribution of M2 gene mutations in influenza virus variants that circulated between 1902 and 2013 showed that 45.2% of all resistant influenza A viruses (H1-H17) circulating globally had S31 N mutations [91].

Similarly the NA mutations causing resistance to neuraminidase inhibitors (NAI) has lots of variations. The most common mutation observed is the H275Y that confers high resistance to oseltamivir [92]. A study showed that the amino acid changes at residue 223 (I → R/V) conferred reduced inhibition to oseltamivir and zanamivir [93]. The N2 subtype has been associated with oseltamivir resistance due to mutation at E119V and R292K. The R292K has also been linked to zanamivir resistance [94]. Studies have demonstrated that the most frequent mutation conferring the oseltamivir resistance in NA of the H1N1 and H5N1 subtypes was H274Y, while the E119V and R292K mutations were more common among the H3N2 and H7N9 subtypes [95]. Another study showed that R292K mutation in NA protein in the H7N9 virus strains were detected in patients after drug treatment. This substitution promoted resistance against oseltamivir [96]. Similarly oseltamivir resistance was associated with the H274Y NA mutation in H5N1 influenza viruses detected in patients during treatment or prophylaxis [97]. Few other studies have reported that the Egyptian H5N1avian influenza isolates from humans had N294S NA mutation [98]. Boltz et al. reported that H5N1 viruses of clade 2.3.2 isolated from the Republic of Laos in 2006–2008 had V116A, I222L, and S246 N mutations in NA [99]. The ongoing concerns about influenza A viruses and increasing antiviral resistance needs immediate attention, better antiviral surveillance for better management and control of future influenza pandemics.

## **7. Infection control, advances in vaccines and therapeutics**

are considered more important [83]. Nevertheless, successful application of influenza vac-

Antiviral resistance in influenza viruses is a global concern and the number of resistant mutants is increasing year after year. The antiviral drugs have been formulated mainly against the M2 ion channel (amantadine and rimantadine) and the neuraminidase proteins (oseltamivir and zanamivir) of influenza viruses. These FDA approved drugs are currently used for prophylaxis and treatment of influenza A infections and are effective against the HPAI H5N1 viruses [84]. The effectiveness of these drugs ranges from 80 to 90% if the treatment had begun within 48 hours of infection [85]. The antiviral resistance in influenza may develop during disease treatment and occasionally spreads widely to replace the susceptible strains in the absence of drug pressure. An example of this event is the global spread of adamantaneresistant H3N2 viruses in the year 2003, oseltamivir-resistant seasonal H1N1 viruses since 2007 and more recently the adamantane-resistant pandemic A (H1N1) viruses in 2009. Such events show the highly unpredictable nature of influenza viruses and increase the challenge of its management. Sometimes a single reassortment event or mutations leads to emergence of variant influenza viruses such as the pandemic 2009 or seasonal A (H1N1) viruses that becomes completely unresponsive to most antiviral drugs. The amantadine resistance was soon observed after the discovery of the drug in early 1960s and studies subsequently reported that a single point mutation in the M2 protein lead to the emergence of high-level resistant mutant viruses showing resistance to both amantadine and rimantadine [86]. Other studies also suggested that resistance to M2 blockers (amantadine/rimantadine) can be achieved by only a few substitutions in the codon L26, L27, A30, A31 and G34 of the M2 gene [87] and these mutants retain the virulence and are transmissible between humans [88]. A study showed that adamantane resistance emerged in about 30% of patients post few days of treatment [89]. Another study has shown the synergistic antiviral effects of amantadine-oseltamivir combination chemotherapy [90]. The adamantanes were very effective for almost 4 decades after which the frequency of adamantine resistance among influenza A H3N2 viruses started to increase. The global resistance among H3N2 virus was as low as 0.8% between the periods 1991 to 1995. The adamantine resistance has now been reported for human H1N1, H3N2 and H5N1 avian influenza viruses. The frequency of resistance further increased to 28% during 2004–2005 and to 72% in 2005–2006 for H1N1 variant viruses [1]. The US reported around 92% resistance among H3N2 viruses by the year 2005. A recent study based on the frequency and distribution of M2 gene mutations in influenza virus variants that circulated between 1902 and 2013 showed that 45.2% of all resistant influenza A viruses (H1-H17) circulating globally

Similarly the NA mutations causing resistance to neuraminidase inhibitors (NAI) has lots of variations. The most common mutation observed is the H275Y that confers high resistance

cines in animals may contribute in reducing zoonotic transmission.

**6. Antiviral resistance mutants**

44 Influenza - Therapeutics and Challenges

had S31 N mutations [91].

Generally, people infected with the flu are advised to stay home and rest, both to recover and to avoid infecting others. In severe cases, or for individuals at high risk of complications, physicians may prescribe antiviral medication. The antiviral drugs currently available against influenza viruses are adamantane derivatives (amantadine and rimantadine) and neuraminidase (NA) inhibitors (zanamivir, oseltamivir and peramivir). A viral infection can be inhibited at several crucial steps, such as entry, signaling, assembly, and egress [1].

Oseltamivir, works by blocking neuraminidase that enables newly made influenza virus to escape from an infected cell. Zanamivir (inhaled), peramivir (intravenous), and inavir (inhaled) operate in a similar way. Baloxavir, discovered in Osaka, received preliminary approval in Japan in January 2018 and will be filed for regulatory review in the US and Europe thereafter. Baloxavir requires a single dose, unlike oseltamivir which is taken twice a day for 5 days [100].

Efforts to improve currently available vaccines have been explored over the last 2 decades such as: increasing the antigen dose, intradermal route of administration to activate other arms of the immune system, and adding immunostimulating compounds such as adjuvants [78]. The main areas of research and development in flu vaccines involve:


**3.** Development of a universal vaccine that protects against influenza regardless of what influenza viruses are circulating. These includes vaccine targeting the HA stalk domain [102, 103], and the use of influenza-virus-like particles as vaccines [104].

**9. Future perspectives**

**Abbreviations**

**Disclosure of potential conflicts of interest**

CDC centers for disease control and prevention

FDA Food and Drug Administration

HPAI highly pathogenic avian influenza

IIV inactivated influenza vaccine

LAIV live-attenuated influenza vaccine LPAI low pathogenic avian influenza

PMO phosphorodiamidate morpholino oligomer

HA hemagglutinin

IAV influenza A virus

mAb monoclonal antibody

NAI neuraminidase inhibitors

QIV quadrivalent influenza vaccine

NA neuraminidase

RNA ribo nucleic acid

SIV swine influenza virus

WHO World Health Organization

ECDC European Centre for Disease Prevention and Control

GISRS global influenza surveillance and response system

The public health threats from influenza viruses have always been a global concern. They are not only responsible for annual epidemics throughout the world, but also affect quality of life and have negative impacts on the economy due to frequent school and work place absenteeism. The frequencies of influenza infections have further increased due to co-mingling in shared humananimal environment. The virus is known to acquire antigenic shift and drifts and thus pose challenges in control measures and management. Advancements in vaccination strategies, discovery of novel drugs and antiviral therapeutics along with development of a universal influenza vaccine are promising approaches toward the management of future epidemics and pandemics.

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All authors declared that they have no conflict of interest (financial or non-financial).

In addition to antiviral drugs and vaccines, several novel therapeutic alternatives may prove to be beneficial in the near future. The long-acting inhaled neuraminidase inhibitor CS-8958 (also known as R-118958) has shown promising results in murine models of influenza treatment while a polymerase inhibitor, T-705 (Toyama Chemical), that inhibits viral RNA polymerase has been found to be effective against all three influenza virus types (A, B and C) and to some extent against other RNA viruses, including hemorrhagic fever viruses. The drug, DAS181, a fusion construct that includes the sialidase from *Actinomyces viscosus*, affects the viral attachment process during the early stages of influenza replication. Another study demonstrated the antiviral properties of chlorogenic acid (CHA) and its inhibitory effect on A/PuertoRico/8/1934 (H1N1) and oseltamivir-resistant strains in the late stage of the infectious cycle. Other novel antiviral drugs under clinical development include AVI-7100, a 20-mer phosphorodiamidate morpholino oligomer (PMO) IV formulation that hinders translation and splicing of mRNA from the matrix gene. EV-077, a dual thromboxane receptor antagonist and thromboxane synthase inhibitor, prevents virus replication by inhibiting prostanoids associated with influenza infections. Aureonitol, a compound obtained from fungi, has shown inhibitory effects against both influenza A and B virus replication by impairing virus adsorption. Monoclonal antibodies, CR6261 and CR8020, bind to the conserved stalk region of HA and inhibit the entry and fusion stages. A broad spectrum human monoclonal antibody (mAb- MEDI8852), which unlike other stem-reactive antibodies, used a rare heavy chain VH (VH6-1) gene, was found to be effective in mice and ferrets and better than oseltamivir [1]. These novel approaches will potentially become effective tools for managing seasonal, zoonotic and pandemic influenza virus infections.

#### **8. Conclusions**

Influenza viruses have a silent reservoir in the aquatic avian species and continuously pose threat to human population. The avian, swine and other zoonotic influenza infections may range from a mild upper respiratory tract infection to a more severe pneumonia, acute respiratory distress syndrome and even death. Humans can be infected with a wide range of avian [subtypes A(H5N1), A(H7N9), and A(H9N2)] and swine [subtypes A(H1N1), A(H1N2) and A(H3N2)] influenza viruses. Although sustained human to human transmission is lacking, these viruses can be transmitted when there is a direct contact with infected animals or contaminated environments. The virus shows a tremendous potential to mutate, re-assort and give rise novel variants to evade host immunity and vaccination strategies. The emergence of antiviral mutants has further worsened the worldwide control measures. Although management of influenza has been a challenging task owing to its large reservoir and ability to mutate rapidly, the disease can be controlled in the animal source to decrease the risk to human population. With advancements in modern diagnostic methods, vaccination and antiviral strategies, the annual epidemics and occasional pandemics can be managed efficiently.
