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Discuss the Potential for H5N1 influenza viruses to cause a human pandemic

Tutor – Dr Benjamin Hale

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Discuss the potential for H5N1 influenza viruses to cause a human pandemic

Influenza viruses have long been the cause of pandemics throughout human history, from the Middle

Ages to the 21st century, persisting due to its constant genetic assortment and high mutation rate.

Recent pandemics include the H1N1 Spanish flu of 1918 which claimed over 40 million lives, the

1957 H2N2 Asian flu with a death toll of around 2 million and the 1968 H3N2 Hong Kong flu

causing 1 million deaths worldwide. With all previous pandemics having some segments of avian

origins in combination with the resulting high mortality rate, fears have arisen that the HPAI H5N1

avian influenza will have similar consequences (Horimoto and Kawaoka 2005) The WHO defines a

pandemic as the worldwide spread of a disease, with three conditions which must be met; 1) there

must be a disease new to a population - or at least a disease that had not surfaced for a long time. 2)

This disease must be caused by disease-causing agents that infect humans, causing serious illness. 3)

The agents must spread easily and sustainably among humans. These conditions are met to some

degree by HPAI H5N1 virus. It is a new influenza virus strain to humans and has the potential to

infect humans and cause serious illness. The main barrier presently which prevents a pandemic is that

H5N1 is not easily transmissible in humans. Here we will discuss the potential for H5N1 virus to

become pandemic among humans by addressing host adaptation factors, epidemiology, transmission,

pathogenesis and molecular biology of this virus.

H5N1 Structure

Annual epidemics are a result of Influenza viruses A, B or C whereas large pandemics are usually

associated with Influenza A. Influenza A viruses are enveloped RNA viruses consisting of an eight-

segmented, negative sense, single stranded genome. The genome encodes 10 proteins which are the

nucleocapsid, Neuraminidase (NA), Haemagglutinin (HA), Non Structural (NS) proteins NS1 and

NS2, matrix proteins M1 and M2, three polymerases, PB1, PB2 and PA (Figure 1). PB1-F2 protein

present in some influenza viruses is a recent addition to the known encoded proteins (Bruns et al

2006)

Figure 1. Structure of Influenza A virus Virion (Horimoto and Kawaoka 2005)

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Influenza A viruses are identified by surface proteins HA and NA where a different iteration of the

surface proteins is coded for by a number for example H1N1. Genetic diversity in Influenza is

generated through mutations and genetic re-assortment. Mutation in HA and NA genes lead to

antigenic drift over time which explains the recurrent influenza epidemics. The segmented genome

allows genetic re-assortment of HA and NA which leads to generation of novel viruses. These viruses

will go on to cause a pandemic due to the fact that the population is immunologically naïve to the

antigens. The 1918 H1N1 virus is thought to have arisen via direct adaptation of avian flu to humans

through mutation. Whereas the 1957 strain was caused by H1N1 acquiring novel H2, N2 and PB1.

Similarly the 1968 strain was a result of re-assortment of the previous strain with H3 and a novel PB1

gene. (Horimoto and Kawaoka 2005)

Pathogenesis

H5N1 is a potentially fatal disease causing infection of the respiratory system. It may spread to other

vital organs and causes dysregulation of cytokines and chemokines. Symptoms include high fever,

malaise, cough, sore throat, abdominal and chest pain, diarrhoea, ARDS and possible

neurologicalchanges. (WHO, 2013). Usually transmitted from animal to human, there are reports of

human to human transmission. Lungs have been observed with diffuse alveolar damage and positive

stranded viral mRNA, a sign of viral replication, has been detected in the trachea and lungs. Damage

has been observed in other organs including the spleen, lymph nodes, intestinal tissues, brain and

placenta. Positive stranded mRNA has been detected in the brain and intestines although this may be

attributed to viraemia. H5N1 has been shown to induce higher expression of cytokines and

chemokines than human influenza. (Korteweg and Gu 2008). Up regulation of TNF alpha and TRAIL,

important molecules involved in cell death signalling and apoptosis, has been shown in macrophages

(Zhou et al 2006). HA of H5N1 has also been known to suppress perforin expression in cytotoxic T

cells. The pathogenesis of H5N1 is summarised in Figure 2.

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Figure 2. Pathogenesis of H5N1 in Humans (Korteweg and Gu 2008)

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H5N1 virus was first detected in Guangdong Province of China in 1996, it was found to be prevalent

in geese. In the following year the first instance of a purely avian flu virus causing disease and death

in humans occurred, with 18 cases and 6 deaths. This 1997 strain of virus was a re-assortment of the

previous 1996 strain with H9N2 and H6N1 genes. Due to the crackdown on poultry farms and the

slaughter of 1.5 million poultry in Hong Kong this strain has not returned. The 2005 outbreak of

H5N1 in migratory waterfowl was a major factor in facilitating transmission across Asia (Peiris et al

2007). As a result, following this incident, the geographic distribution widened, outbreaks occurred in

Croatia, Turkey, Russia, Egypt and Nigeria (WHO 2013).

Since then various other H5N1 re-assort ants have been detected. Previously the strains were

classified A, B, C, D, E, V, W, X0-3, Y, Z and Z+ 2 according to variability in re-assortments of the

genotype (Figure 3) Around 2003 genotype Z began to emerge as the dominant genotype and from

this V, W and G genotypes also emerged. Genotype V became endemic in Japan and South Korea

whereas in Vietnam, Cambodia, Indonesia, Thailand and southern China genotype Z became

dominant.

With increasing variability and difference a new classification method was put forward by the WHO.

This current phylogenetic classification method consists of clades 0-9 with further sub clades

(Appendix 1). Clades 2.2 and 2.3 have been the dominant circulating strains during 2011 and 2012

(FAO, 2013)

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Figure 3. Evolution of H5N1 from 1999 to 2005 (Peiris et al 2007)

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2003 brought the second incidence of H5N1 in humans. It presented in a father and son returning

from holiday in Fujian province. Subsequently further human cases have occurred across Asia and the

Middle East with 615 cases in total, 364 of which have been fatal (WHO 2013).

The ability for H5N1 to cause a future human pandemic depends on several factors. Effective host

adaptation is required for human infection. Genetic reassortment and mutation of the virus has led to

efficient transmission in avian sources however human to human transmission is still not prevalent but

recent studies have revealed the potential for it.

Transmission of H5N1 in Humans

Host Receptor Adaptation

For effective transmission from bird to humans, H5N1 requires affinity for human host receptors.

Factors which determine viral tropism are not very well understood and is thought to be mainly

determined by HA and NA as well as other genes including PB2 (Neumann and Kawaoka, 2006).

Avian influenza viruses have an affinity for alpha 2-3 linkage sialic acid linked galactose. Human

influenza viruses bind to alpha 2-6 sialic acid linked galactose usually found in the upper respiratory

tract (Rogers and Paulson, 1983). Considering this, the lower respiratory tract, namely the terminal

bronchioles and alveolar epithelial cells have been shown to possess the alpha 2-3 and alpha 2-6

receptors. (Kyoko et al 2006)

Other cells which have been shown to have alpha 2-3 receptors include pancreatic and bile duct

epithelial cells, endothelial cells throughout body, intestinal mucosa epithelia, T cells and Kupffer

cells (Ulloa and Real, 2001, Yao et al, 2008). H5N1 was shown to infect and replicate in ex vivo

cultures of lung fragments (Nicholls et al, 2007). Additionally a small number of H5N1 strains have

accumulated mutations in the HA gene at positions 192 and 182 which allows them to bind to alpha 2-

6 receptors. Although these mutations do not allow efficient human to human transmission (Yamada

et al 2006). Related to this is the fact that quails possess alpha 2-6 receptors which bind human

influenza. This may be a potential re-assort ant vessel for H5N1 in adapting to human receptors. (Wan

and Perez, 2006). Surprisingly ex vivo cultures of the upper respiratory tract also seem to be receptive

to the virus although they seem to be lacking in alpha 2-3 receptors (Nicholls et al, 2007). This along

with the absence of infection of cells possessing alpha 2-3 receptors throws doubt on the validity of

this receptor as the sole determinant in host binding, therefore further study is required.

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Haemagglutinin and Neuraminidase

H5N1 like most Influenza A viruses come in two formals which are Low Pathogenic Avian Influenza

(LPAI) and Highly Pathogenic Avian Influenza (HPAI) (http://www.cdc.gov/flu/avian/gen-info/flu-

viruses.htm). LPAI strains have the ability to evolve into HPAI which is the source of concern for the

current H5N1 strains. The HPAI H5N1 strain is largely determined by mutations in the HA protein.

Precursor HA (HA0) is postranslationally cleaved into HA1 and HA2 by host proteases. These two

subunits are connected by a peptide chain. The dual nature of HA mediates the fusion of viral

envelope and host membrane using trypsin like proteases which are restricted to the airways

(Bottcher-Friebertshauser et al, 2010). Mutations in the connecting peptide between HA1 and HA2

increase the sensitivity to proteolytic activity so that it has high cleavability permitting greater virus

replication and dissemination (Horimoto and Kawaoka, 2001).

NA stalk deletions correlate with expanded host range indicating that this mutation is associated with

interspecies transmission (Wang et al, 2006). NA sialidase activity is important for effective virus

replication and must be balanced with HA activity. NA stalk deletion results in reduced enzymatic

activity which balances with the weaker H5 HA activity, hence restoring the functional balance. The

NA stalk deletion has been shown to contribute to increased virulence and pathogenicity. This

mutation was found in all 173 human cases of H5N1 from 2004-2007 which may indicate an

association with gradual transmission to humans (Zhou et al 2009).

Two of eight Vietnamese patients died from H5N1 infection. Isolates of the virus were taken and a

tyrosine to histidine substitution at position 274 in the H5N1 NA gene was found to confer resistance

to the influenza drug oseltamivir. A major concern, this may lead to increased persistence of H5N1 in

humans therefore a greater risk of transmission (de Jong et al, 2005).

Polymerase and Matrix Proteins

PB1, PB2 and PA make up the polymerase proteins which are involved in influenza RNA synthesis.

Several mutations have been shown to increase pathogenicity in H5N1. A substitution at position 627

in the PB2 gene from glutamic acid to lysine was detected in human strains isolated in Vietnam and

Thailand. Strains containing this substitution replicated efficiently in the lungs and at a lower

temperature of 33 degrees celcius, facilitating greater dissemination and transmission of the virus. In

the context of other mutations in receptor specificity it provides a platform for efficient human to

human transmission. (Hatta et al, 2007)

PB1-F2 and M2 genes were found to be the only genes under positive natural selection in human

strains (Smith et al 2006). PB1-F2 is involved in increased host sensitivity to apoptic stimuli

(Conenello et al 2007). M2 has been shown to be involved in interspecies transmission. Also a

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substitution at residue 31 of the M2 protein from serine to asparagine is associated with amantadine

resistance. Although only a few clade 2 viruses possess this mutation, it may be possible that in future

re-assortants it will become prevalent. (Smith et al, 2006).

Non-structural Proteins

Non-structural proteins are involved in viral replication of influenza. NS1 is vital in evading host

immune system via inhibition of type I interferon response. A glutamic acid present on NS1 at

position 92 was found to confer resistance to TNF alpha. The resulting H5N1 virus was unaffected by

the antiviral effects of TNF alpha in porcine epithelial cells. (Seo et al, 2002). This may cause an

increased risk of virus transmission, due to the persistence of the virus, in the case of a human

infection.

Airborne Transmission

Recently it was found that four amino acid substitutions in the HA gene and one in the polymerase

complex protein PB2 allowed H5N1 to become airborne transmitted it ferrets. These were

consistently present throughout strains which were airborne transmissible. The four mutations in HA

were Q222L, G224S, H103Y and T156A. In PB1 the mutation was E267K. The mutations in HA

changed the binding preference of H5N1 from avian alpha 2-3 SA to human alpha 2-6 SA receptors.

Q222L and G224S are also associated with receptor binding specificity in H2 and H3 viruses which

makes these the main suspects in H5N1 receptor specificity change. The role of the E267K mutation

in PB2 was unclear (Herfst et al, 2012).

Another study also identified a reassortant H5N1 which preferentially recognized human type

receptors and spread efficiently through respiratory droplets (Imai et al, 2012).

H5N1 Epidemiology in Humans

Bird to Human

The prevalence of H5N1 in avian sources and other animal sources creates a risk of re assortment

and mutation to occur within these species which will allow the virus to adapt to interspecies

transmission especially humans. Cracking down on avian and animal reservoirs of infection is a vital

preventative measure in the introduction of a possible human transmissible H5N1.

First detected in domestic geese in China there is reason to believe that a major method of

transmission of H5N1 is through the poultry trade. In Thailand and Vietnam human infection

through direct contact with backyard flocks and poultry have been In one study of human cases, 12

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confirmed and 21 suspected cases were associated with abnormal chicken deaths including direct

contact and possession of backyard flocks have been reported (Chotpitayasunondh et al, 2005 Dinh

et al, 2006). Use of faeces as fertilizer is widespread and is another potential risk factor (WHO

Writing Committee, 2005). Waterfowl such as ducks have been widely investigated and shown to be

the “Trojan Horse” in H5N1 generation, maintenance and transmission (Li et al). H5N1 from infected

ducks remain asymptomatic with effective transmission and possess a long virus shedding period

allowing the virus to persist. Free-range ducks drink from common water sources, swim in them and

mix with other species (Sturm-Ramirez et al, 2005). Humans which utilise this water source are put

at a risk of acquiring infection (WHO Writing Committee, 2005). Mixed flocks can be implicated in

the re-assortment of H5N1. Much of the Asian population keep backyard flocks and some alongside

pigs. (Ly et al, 2007)

The H5N1 outbreak in migratory waterfowl in Qinghai Lake, China highlighted the possibility of wild

migratory birds contributing to viral transmission (Liu et al, 2005). A study done comparing the

phylogenetic relationships of virus isolates with the migratory movements of wild birds proved that

spread to most countries in Europe was likely through migratory birds. H5N1 could transmit through

wild birds migrating from Siberia to the USA across the Bering Sea (Kilpatrick et al, 2006).

Human to Human

Cases of human to human transmission are few and far between as of yet. One case of possible

person to person transmission was described in Thailand and resulted in one death (Ungchusak et al

2005). Another group of cases in Indonesia suggested that person to person contact may have been

responsible for H5N1 transmission. The patients were related and lived in small enclosed spaces with

lots of close contact (Kandun et al 2006). Another person to person case was reported in China

where a father and son were infected. Isolates of the virus from each of the patients were

genetically identical apart from one nucleotide substitution (Wang et al 2008). These cases

demonstrate the ability of H5N1 to become transmissible in humans and although these events are

relatively rare, with the possibility of acquiring the mutations described above it could become more

prevalent.

Prevention

For effective control procedures there must be a combination of several measures. An obvious factor

is early detection of the virus so that control measures can be taken and prevent further

transmission of the virus. Quarantine of infected patients would be vital to prevent further spread

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and vaccination of unaffected is required to prevent potential infection. In preventing a H5N1

introduction in humans the primary options for control of avian sources which have proved to work

are stamping out, culling of poultry, movement controls, vaccination and education (Sims et al,

2005). Predictive modelling and epidemic simulation have been shown to be effective tools in the

control of influenza. Situations can be predicted in advance and possible control strategies

developed. From using these tools strategies have been developed for the control of a pandemic.

Treatment of cases with antivirals is effective if they are given within a day of symptoms emerging.

Drugs for at least 50% of the population combined with school closures can reduce attack rates by

40-50%. Case isolation and household quarantine can also have a significant impact (Ferguson et al

2006). The main lessons to be learnt from previous avian outbreaks are also applicable to possible

human pandemics, these are that; intensive surveillance should be implemented to allow for early

detection, to have contingency plans in the case a rapid response is required, preventative measures

should be taken.

Treatment

Antivirals provide another option to prevent further transmission. The main ones consist of

amantadine, rimantadine, oseltamivir and zanamivir. Adamantines were effacious on H5N1 strains in

the 1997 Hong Kong outbreak. H3N2 have now acquired resistance to these and they are no longer

recommended (Bright et al, 2005). This is a danger in that a reassortment could provide H5N1 with

both oseltamivir and adamantine resistance. Effective administration strategies must be developed

such as combination therapy and intravenous dosing in severely ill patients where the digestive

system is compromised. Rising resistance calls for further research to be done into novel antivirals.

One potential target is the highly virulent polymerase complex genes (Salomon et al, 2006). In the

meanwhile, one strategy which should be immediately implemented is to reduce the abuse of

antivirals. With reports of antiviral resistance in H5N1 viruses due to excessive administration, the

use of such antivirals should be limited to extreme cases and even then they should be administered

with restraint. One point to note is that the strains found to be effective in human to human

airborne transmission remain susceptible to oseltamivir (Herfst et al 2012).

Vaccination

Vaccines are the focus of investigation currently. Vaccines have been used as preventative measures

in H5N1 outbreaks such as in Hong Kong (Ellis et al, 2004). Vaccination even if it is of low efficacy can

significantly reduce attack rates if stockpiled in advance (Ferguson et al 2006). A pandemic vaccine

may be a useful tool in prophylaxis as demonstrated by Lin et al in a phase I trial of a H5N1

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inactivated whole virus vaccine (Lin et al, 2006). A cold adapted, live attenuated vaccine has been

shown to provide broad cross protection against antigenically diverse H5N1 strains. Pre-emptive

vaccination may be a risk due to the possibility of reassortment. This ceases to be an issue in the

case of a pandemic (Suguitan et al 2006). The Holy Grail lies in a universal influenza vaccine although

this is far from reality just yet. The M2 protein possesses antigenically conserved epitopes across

different subtypes and opens up the possibility of a universal vaccine (Neirynck et al 1999)

Conclusion

In conclusion the risks of H5N1 are clear and reason to believe that it has the ability to cause a

pandemic is well grounded. Although effective human to human transmission is not yet possible

with H5N1, it possesses the capacity for it. The main risk of infection currently is through direct

contact with poultry which limits infection to a certain portion of the world population. A pandemic

of poultry to human infection is unlikely to occur. There is much to be discovered regarding H5N1

receptor specificity. Current knowledge is restricted to what we know of sialic acid receptors and

studies have shown that it cannot be the determining factor in human infection. There may be other

host factors at play here. A Cambodian incident of H5N1 occurred in a village where many of the

villagers were in direct contact with infected poultry yet only a single person acquired infection

(Vong et al, 2006). This calls for further research to be done into the host factors which make a

person susceptible to infection. Control measures have been shown to be effective in reducing viral

transmission. At present the antivirals are becoming obsolete as H5N1 increasingly becomes

resistant to them. Further targets have been identified and now it is a case of developing the tools.

Vaccination is still in preliminary stages but greater understanding of the flu virus should lead to

development of an effective pandemic vaccine. Currently there are many biological barriers in the

way of H5N1 preventing it from becoming truly dangerous among humans, but the potential for it to

overcome these through re-assortment and mutation exists, and that creates a very real risk of a

future H5N1 pandemic.

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Appendiz 1

WHO | FAQs: H5N1 influenza. (n.d.). WHO.

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Brief overview of role of ducks in H5N1 persistence

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in the Apical Membrane of Cultured Epithelial Cells and Tissues. J Histochem Cytochem 49,

501–509.

Evidence for human host distribution of H5N1 receptors

Vong, S., Coghlan, B., Mardy, S., Holl, D., Seng, H., Ly, S., Miller, M. J., Buchy, P.,

Froehlich, Y. & other authors. (2006). Low Frequency of Poultry-to-Human H5N1

Transmission, Southern Cambodia, 2005. Emerg Infect Dis 12, 1542–1547.

Highlights important issue of looking into other host factors involved

in H5N1 infection

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Wang, Q., Long, J., Hu, S., Wu, Y. & Liu, X. (2006). [Biological significance of amino acids

deletion in NA stalk of H5N1 avian influenza virus]. Wei Sheng Wu Xue Bao 46, 542–546.

Understanding of NA stalk deletion and its effects

WHO | FAQs: H5N1 influenza. (n.d.). WHO.

Statistics of H5N1 epidemiology

WHO | Influenza at the Human-Animal Interface (HAI). (n.d.). WHO.

Statistics regarding bird to human H5N1 epidemiology

WHO Writing Committee. (2005). Avian Influenza A (H5N1) Infection in Humans. New

England Journal of Medicine 353, 1374–1385.

Provides global understanding of previous H5N1 outbreaks

Yamada, S., Suzuki, Y., Suzuki, T., Le, M. Q., Nidom, C. A., Sakai-Tagawa, Y., Muramoto,

Y., Ito, M., Kiso, M. & other authors. (2006). Haemagglutinin mutations responsible for

the binding of H5N1 influenza A viruses to human-type receptors. Nature 444, 378–382.

Provides evidence for H5N1 adaptation to humans

Yao, L., Korteweg, C., Hsueh, W. & Gu, J. (2008). Avian influenza receptor expression in

H5N1-infected and noninfected human tissues. FASEB J 22, 733–740.

Human host receptor distribution

Zhou, H., Yu, Z., Hu, Y., Tu, J., Zou, W., Peng, Y., Zhu, J., Li, Y., Zhang, A. & other

authors. (2009). The Special Neuraminidase Stalk-Motif Responsible for Increased

Virulence and Pathogenesis of H5N1 Influenza A Virus. PLoS ONE 4, e6277.

Inidence of NA stalk deletion

Zhou, J., Law, H. K. W., Cheung, C. Y., Ng, I. H. Y., Peiris, J. S. M. & Lau, Y. L. (2006).

Functional tumor necrosis factor-related apoptosis-inducing ligand production by avian

influenza virus-infected macrophages. J Infect Dis 193, 945–953.

Pathogenesis of H5N1

Horimoto, T. & Kawaoka, Y. (2005). Influenza: lessons from past pandemics, warnings from

current incidents. Nature Reviews Microbiology 3, 591–600.

Provides basic understanding of H5N1 genetics and structure as well

as diagram of virus particle

Korteweg, C. & Gu, J. (2008). Pathology, molecular biology, and pathogenesis of avian

influenza A (H5N1) infection in humans. Am J Pathol 172, 1155–1170.

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Review of pathogenesis of H5N1 throughout human body on a

cellular and anatomic level

Peiris, J. S. M., Jong, M. D. de & Guan, Y. (2007). Avian Influenza Virus (H5N1): a Threat to

Human Health. Clin Microbiol Rev 20, 243–267.

Understanding of the factors involved in for a potential pandemic

Bruns, K., Studtrucker, N., Sharma, A., Fossen, T., Mitzner, D., Eissmann, A., Tessmer, U.,

Röder, R., Henklein, P. & other authors. (2007). Structural characterization and

oligomerization of PB1-F2, a proapoptotic influenza A virus protein. J Biol Chem 282, 353–

363.

Primary source for understanding of PB1-F2 protein

Ungchusak, K., Auewarakul, P., Dowell, S. F., Kitphati, R., Auwanit, W., Puthavathana, P.,

Uiprasertkul, M., Boonnak, K., Pittayawonganon, C. & other authors. (2005). Probable

Person-to-Person Transmission of Avian Influenza A (H5N1). New England Journal of

Medicine 352, 333–340.

Case of human to human H5N1 transmission

Kandun, I. N., Wibisono, H., Sedyaningsih, E. R., Yusharmen, Hadisoedarsuno, W., Purba,

W., Santoso, H., Septiawati, C., Tresnaningsih, E. & other authors. (2006). Three

Indonesian Clusters of H5N1 Virus Infection in 2005. New England Journal of Medicine 355,

2186–2194.

Another case of H5N1 human to human transmission

Wang, H., Feng, Z., Shu, Y., Yu, H., Zhou, L., Zu, R., Huai, Y., Dong, J., Bao, C. & other

authors. (2008). Probable limited person-to-person transmission of highly pathogenic avian

influenza A (H5N1) virus in China. Lancet 371, 1427–1434.

And another case of human to human H5N1 transmission

Ferguson, N. M., Cummings, D. A. T., Fraser, C., Cajka, J. C., Cooley, P. C. & Burke, D. S.

(2006). Strategies for mitigating an influenza pandemic. Nature 442, 448–452.

Control strategies proposed by carrying out a pandemic simulation

Suguitan, A. L., McAuliffe, J., Mills, K. L., Jin, H., Duke, G., Lu, B., Luke, C. J., Murphy,

B., Swayne, D. E. & other authors. (2006). Live, Attenuated Influenza A H5N1 Candidate

Vaccines Provide Broad Cross-Protection in Mice and Ferrets. PLoS Med 3, e360.

Possible influenza vaccine in the making

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Neirynck, S., Deroo, T., Saelens, X., Vanlandschoot, P., Jou, W. M. & Fiers, W. (1999). A

universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med

5, 1157–1163.

Proposal for a universal influenza vaccine

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