Influenza virus A (H5N1): a pandemic risk? · NEW MICROBIOLOGICA, 30, 65-77, 2007 Influenza virus A...

NEW MICROBIOLOGICA, 30, 65-77, 2007

Influenza virus A (H5N1): a pandemic risk?

Muhammed Babakir-Mina1, Emanuela Balestra1, Carlo Federico Perno1, Stefano Aquaro1,2

1Department of Experimental Medicine and Biochemical Science, University of Roma Tor Vergata, Rome;2Department of Pharmaco-Biology, University of Calabria, Rende (CS), Italy

Influenza A subtype H5N1 has represented a growing alarm since its recent identification in Asia. Previously thoughtto infect only wild birds and poultry, H5N1 has now infected humans, cats, pigs and other mammals in an ongo-ing outbreak, often with a fatal outcome. In order to evaluate the risk factors for human infection with influenzavirus H5N1, here we summarize 53 case patients confirmed with H5N1 infection during 2006. The review also com-pares the mortality rate among human cases from late 2003 until 15 June 2006 in different countries. Neither howthese viruses are transmitted to humans nor the most effective way to reduce the risk for infection is fully under-stood. The association between household contact with diseased poultry in human infection has been demonstrated.This association could possibly operate by 2 mechanisms. First, transmission may be by inhalation or conjuncti-val deposition of large infectious droplets which may travel only in short distances. Second, having infected poul-try in the home and preparation of infected poultry for consumption may result in exposure to higher virus con-centrations than other types of exposure. There is so far no significant evidence for repeated human to humantransmission, yet some cases of human to human transmission among the family relatives in Indonesia, Azerbaijan,Iraq and Turkey have been described. Recent outbreaks of highly pathogenic avian influenza A virus (H5N1 sub-type) infections in poultry and humans (through direct contact with infected birds) have raised concerns that anew influenza pandemic might occur in the near future.

KEY WORDS: Avian Influenza, Pandemic, Transmission, Pathogenicity

SUMMARY

Received February 27, 2007 Accepted March 25, 2007

INTRODUCTION

The term influenza refers to illness caused byinfluenza virus. This is commonly called flu, butmany different illnesses cause flu-like systemicand respiratory symptoms such as fever, chills,aches and pains, cough, and sore throat. In addi-tion, Influenza itself causes many different ill-ness patterns, ranging from mild common coldsymptoms to typical flu to life-threateningpneumonia and other complications, including

secondary bacterial infections particularly in eld-erly persons with underlying chronic diseases.Influenza viruses constitute the genusOrthomyxovirus, which consists of three speciesA, B, and C (Stephenson and Democratis,2006). The differences among these viruses arebased upon the antigenic characteristics of theM protein of the virus envelope and the nucle-oprotein within the viral particles. Influenza epi-demics have been recorded throughout history.In temperate climates, the epidemics typicallyoccur in the winter and cause considerable mor-bidity in all age groups. The likelihood of ahuman influenza pandemic has increased overthe past few years with the emergence and out-break of a highly virulent avian influenza virus,including influenza virus A(H5N1) that causesa contagious disease. Usually, influenza virusesattack the respiratory tract in humans, like throat

Corresponding authorProf. Carlo-Federico Perno, MD, PhDDepartment of Experimental Medicineand Biochemical ScienceUniversity of Roma Tor VergataVia Montpellier, 1 - 00133 Rome, ItalyE-mail: [email protected]

0000_Micro2_02_BABAKIR:0479_06_Micro6_02_Fiorentini 23-05-2007 17:22 Pagina 65

and lungs. The presence of certain receptors inthese target regions are very important in the fluvirus infectivity and pathogenicity. In the past,the disease was termed fowl plague, describedin 1878 as a serious disease of chickens in Italy(Kamps et al., 2006). All influenza virusesaffecting domestic animals (equine, swine,avian) belong to Type A which is the most com-mon type producing serious epidemics inhumans. Other influenza types, B and C, do notaffect domestic animals, Influenza C infects mostpeople when they are young and rarely causesserious illness. Type B occasionally causes localoutbreaks of influenza and is usually confinedto youngsters. Despite the WHO efforts to con-trol the avian influenza pandemic, sporadic casesof human influenza A H5N1 infection are stillbeing reported from time to time in the endem-ic countries and the disease was high in severi-ty in the year 2006. The failure to control avianinfluenza infections in the domestic birds andanimals make the virus probably reassortant,then increase the ability of the virus to infecthumans that improves human to human trans-missibility. The WHO has advised all nations onvigilance for an influenza pandemic in view ofthe growing threat of H5N1.

HISTORY OF AVIAN INFLUENZA VIRUSAND ITS GEOGRAPHIC DISTRIBUTION

On the basis of their ability to cause disease,influenza A viruses infecting poultry can be divid-ed into two distinct groups: highly pathogenicavian influenza (HPAI) and low pathogenic avianinfluenza (LPAI). Highly pathogenic avian influen-za (HPAI) viruses have periodically occurred inrecent years in Australia (H7), England (H7), Italy(H5), South Africa (H5), Scotland (H5), Ireland(H5), Mexico (H5), Pakistan (H7), and the UnitedStates (H5). Because laboratory facilities are notreadily available in some parts of the world to cor-rectly diagnose HPAI, the actual incidence of HPAIin the world’s poultry flocks is difficult to define.It can occur in any country, regardless of diseasecontrol measures, probably because of its preva-lence in wild migratory waterfowl, sea birds andshore birds. Outbreaks of less virulent avianinfluenza (AI) have frequently been described indomestic ducks in many areas of the world. The

AI viruses are often recovered from apparentlyhealthy migratory waterfowl, shore birds, and seabirds worldwide. The epidemiologic significanceof these isolations relative to outbreaks in domes-tic poultry has led to the generally accepted beliefthat waterfowl serve as the reservoir of influen-za viruses.

Main influenza pandemicsInfluenza pandemics can occur when a novelstrain of virus causes an epidemic that spreadsover a wide geographic area and affects an excep-tionally high proportion of the population. Thenovel strain will be produced when there is a cer-tain genetic change, antigenic shift, in the cir-culating strain of influenza (Figure 1). At pres-ent influenza A virus causes the most severe dis-ease in humans, and has good chance of triggeringa pandemic. Three main pandemics occurred inthe 20th century. The pandemic influenza of 1918-1919, termed Spanish flu, was the biggest disas-ter in flu history, with more than 20 million deaths(Mills et al., 2004); most of them were childrenand young adults (see below). As expected, manyof the deaths in 1918 were from pneumoniacaused by secondary bacterial infections. TheSpanish flu also caused a form of primary viralpneumonia, with extensive hemorrhaging of thelungs. The second pandemic in 1957, Asian flu,was on the whole a milder illness than that of1918, yet the global death toll was estimated tobe about 1 million. At that time, the causativeagent was quickly identified, due to advances inscientific technology and the vaccine was avail-able in limited supply by August 1957. An anti-genic shift occurred between an avian virus(H2N2) and the causative agent of the 1918 pan-demic (H1N1) (Belshe, 2005). After eleven years,another pandemic appeared, known as HongKong flu (H3N2), by which around 1 million per-sons died. Earlier infections by the Asian flu virusmight have provided some immunity against theHong Kong flu virus that may have helped toreduce the severity of illness. Instead of peakingin September or October, as had in the previoustwo pandemics (Spanish and Asian flu), the HongKong flu pandemic did not occur until near theschool holidays in December. Since children wereat home and did not infect one another at school,the rate of influenza illness among schoolchild-ren and their families was blocked. Also,

66 M. Babakir-Mina, E. Balestra, C.F. Perno, S. Aquaro


improved medical care and antibiotics that aremore effective for secondary bacterial infections,were available for those who became ill. Thereis no evidence that the 1957 or 1968 pandemicviruses originated in pigs (Neumann andKawaoka, 2006) even though the flu viruses canbecome reassortant in this intermediate host.There are three notable flu outbreaks, but theyare not categorized as true pandemics, whichinclude a pseudopandemic in 1947 with low sever-ity and death rates, an epidemic in 1977 that wasa pandemic in children, and a potential epidemicof swine influenza in 1976 that was termedabortive pandemic (Kilbourne, 2006). There is noexact information which type of influenza will bethe next pandemic strain, but influenza virusA(H5N1) is the first candidate (Figure 5B).

Recent scares of flu pandemic in humans1) 1997: In Hong Kong, avian influenza A

(H5N1) infected both chickens and humans.This was the first time an avian influenzavirus had ever been found to transmit direct-ly from birds to humans. During that out-break, 18 people were hospitalized and 6 ofthem died (Zhou et al., 1999).

2) 1999: In Hong Kong, cases of avian influen-za A (H9N2) were confirmed in children, but

this infection resulted in only mild, self-lim-iting illnesses. The evidence suggested thatpoultry was the source of infection and themain mode of transmission was from bird tohuman (Hien et al., 2004).

3) 2003: Two cases of avian influenza A (H5N1)infection occurred among members of a HongKong family that had traveled to China(Enders et al., 2005), from whom one personrecovered and the other died. How or wherethese 2 family members were infected was notdetermined. Also in the same year anotherfamily member died of a respiratory illnessin China, but no testing was done. No addi-tional cases were reported.

4) 2003: Avian influenza A (H7N7) infectionsamong poultry workers and their familieswere confirmed in the Netherlands during anoutbreak of avian flu among poultry. Morecases of H7N7 illness were reported: the symp-toms were mostly confined to eye infections,yet some respiratory symptoms occurred: 1patient died (Fouchier et al., 2004). There wasevidence of some human to human trans-mission. In the same year another 2 mild clin-ical cases in Hong Kong were reported withH9N2 subtype virus (Chotpitayasunondh etal., 2005).

Influenza virus A (H5N1): a pandemic risk? 67

FIGURE 1 - Themain 3 pandemicsof the 20th centuryand 2 expectationsfor the next pan-demic. The figureshows how anti-genic shift in theinfluenza virusgenome makes thevirus able to causepandemics.


5) 2004: In an outbreak in Vietnam and Thailandmany birds and humans were infected withH5N1; among humans 32 fatal cases havebeen documented (Ungchusak et al., 2005).

6) In the year 2005, 42 fatal cases were confirmedin Cambodia, China, Indonesia, Thailand andVietnam. Also in the year 2006, from Januaryto 15th June, 53 fatal cases were confirmedin Azerbaijan, Cambodia, China, Egypt,Indonesia, Iraq and Turkey.

GENETIC VARIATION AND PANDEMICALERT

The Spanish flu pandemic virus that caused thehighest number of known flu deaths did not orig-inate through a reassortment event. Theresearchers indeed identified that the 1918-1919pandemic evolved by mutational adaptation of theavian strain (Belshe, 2005). Also, genetic studiesshowed that all eight segments of the virus aremore closely related to avian influenza virusesthan any other influenza virus species(Taubenberger et al., 2005). Genetic variationmakes the virus more virulent. For example, oneof the five amino acid changes in the polymerasegene (PB2) of the 1918 virus made the virus infecthumans (Belshe, 2005). This type of mutation wasseen in several human isolates of the 1997 HongKong H5N1 virus and 2004 Vietnam H5N1 virus.So, if H5 viruses do persist they will likely con-tinue to evolve and then be more easily transmittedamong people (Chotpitayasunondh et al., 2005).Influenza viruses have a high level of recombi-nation between genes because of the segmentednature of their genomes. As a consequence of this,gene reassortment through dual infection withhuman and avian strains of the Asian influenzaand Hong Kong influenza may occur (Belshe,2005). In recent years, there is no large geneticvariation like the past pandemics. However, anti-genic analysis of the new avian isolates showsreactivity pattern differences from the H5N1 virus-es isolated in 1997 and 2001 (Sturm-Ramirez etal., 2004). On the other hand, the genomicsequence for the virus (A/Beijing/ 01/2003) whichwas isolated from a 24-year-old Chinese man sug-gests that the virus may be a mixed virus becauseits gene segments are closely related to some otherspecies (Qin et al., 2006). In the 2 human deaths

in Turkey (Figure 5B), two different mutationswere observed. The first was at position 223 (seetable 1) of the haemagglutinin receptor proteinwhich increases the ability of the virus to bindthe human receptors more than avian receptors(Butler, 2006). The same mutation was observedtwice in a father and son in Hong Kong in 2003,and in one fatal case in Vietnam in 2005. The sec-ond mutation observed in the Turkish deaths(Glu627Lys) is in the polymerase protein. Thismutation was seen in other flu sequences fromEurasian poultry throughout 2005 in fatal caseswho died during an outbreak of H7N7 in theNetherlands in 2003 and in a few people inVietnam and Thailand. Another new finding forgenetic variation in the genome of the influenzavirus A (H5N1) is that of those 32 mutations thatwere found in the isolates of the family clustersin Indonesia, the 21 mutations that occurred inthe isolate of a 32-year-old man (Butler, 2006).Now, it can be suggested that genetic variationin the certain flu genome may increase the affin-ity of the virus for humans. Also, all these recentmutations increase the likelihood for human tohuman transmission (Figure 5B). Some specialmutations in the influenza virus genome mayincrease viral resistance for antiviral agents. Manystudies have been carried out to understand theeffects of the mutations on anti-influenza virusdrugs. The H274Y mutation confers resistance tooseltamivir, which is a neuraminidase inhibitorused in patients with H5N1 infection (Moscona,2005). Also, an in vitro study for adamantines sug-gests that resistance to adamantines is a commonfeature of H5 isolates of 2004 (Chotpitayasunondhet al., 2005). Additionally, another scientificstudy shows that a mutation in the non-structuralprotein NS1 (Asp92Glu) makes the virus resist-ant to interferon and special tumor necrosis fac-tor (Writing Committee of the World HealthOrganization WHO Consultation on HumanInfluenza A/H5. Avian Influenza A (H5N1)Infection in Humans, 2005).

EPIDEMIOLOGY AND TRANSMISSION OF THE VIRUS

Waterfowl is the natural reservoir of all influen-za A viruses, usually carrying the infection withno sign of disease (Sturm-Ramirez et al., 2005).



Also migratory birds are considered to have anepidemiological role during flu outbreaks(Hlinak et al., 2006). The infection of humanswith an H5 avian influenza virus in Hong Kongin 1997 resulted in a reconsideration of the roleof the avian species in the epidemiology of humaninfluenza (Buxton Bridges et al., 2002). The infect-ed birds excrete virus in high concentrations intheir faeces, nasal and ocular discharges. The dis-ease generally spreads rapidly in a flock by directcontact, but on occasions spread is erratic.Airborne transmission may occur if birds are inclose proximity and with appropriate air move-ment. Once introduced into a flock, the virus isspread from flock to flock by the usual methodsinvolving the movement of infected birds, con-taminated equipment, egg flats, feed trucks, andservice crews. Usually, infection by H5N1 virusoccurs in humans when there is a direct contactwith infected birds. An epidemiological study inVietnam showed that 9 of 10 patients had a clearhistory of direct contact with poultry (Hien et al.,2005). Normally, typical influenza is primarilytransmitted from person to person via dropletsfrom the nose and throat. Thus, close contactsare in general required for transmission. Thetransmission may also occur through indirectcontact with respiratory secretions (for exampletouching contaminated surfaces then touching

the eyes, nose or mouth). The in vivo studies illus-trated that influenza viruses replicate to higherlevels in the trachea than in the cloacae (Sturm-Ramirez et al., 2005), meaning that the chanceof influenza virus transmission is greater via res-piration than faeces. The conformation of the sial-ic acid linkage has been established to controltropism of the influenza viruses (Victor et al.,2004). Avian influenza viruses bind to cell-sur-face glycoproteins containing sialyl-galactosylresidues linked by a 2-3-linkage. By contrast,human influenza viruses bind to receptors thatcontain terminal 2-6-linked sialyl-galactosylmoieties, since alpha 2-3 sialic acid receptors arefound mainly in the lower respiratory regions.Thus, avian flu viruses can replicate efficientlyonly in cells in the lower region of the respira-tory tract where specific receptor is more preva-lent (Shinya et al., 2006). Also, the findings implythat specimens from the lower respiratory tract,such as sputum or bronchoalveolar lavage, havea higher sensitivity for viral detection than anupper respiratory specimen like nasopharyngealaspirates or throat swab specimens. Moreover,the absence of viral antigen in the trachea indi-cated that the upper airway is probably not anactive site of avian viral replication (Uiprasertkulet al., 2005). This restriction may be the causeof limited human-to-human transmission of


FIGURE 2 -Transmission ofinfluenza A virusesto wild birds,domestic birds, andintermediate hostslike pigs. The figureshows how thestrains circulateamong their hosts.


avian flu including H5N1 viruses. To be easilytransmitted among humans, avian viruses musthave the ability to bind cells that display the 2-6 receptors, so that it can enter the cells and repli-cate in them. Transmission of influenza virusesis illustrated in (Figure 2).

PATHOGENICITY AND INFLUENZA VIRUSVIRULENCE

HPAI are virulent viruses that cause fowl plague.Mortality rate may be as high as 100 %. Theseviruses have been restricted to subtypes H5 andH7, but not all viruses of these subtypes are high-ly pathogenic. Haemagglutinin subtypes can bedifferentiated by developed multiplex reversetranscriptase polymerase chain reaction (Xie etal., 2006). This differentiation is important dur-ing any flu outbreak. Also, a discriminationamong H5 subtypes must be made, because theyare not always from HPAI strains (Payungpornet al., 2006). The hemagglutinin glycoprotein forinfluenza viruses is produced as a precursor, HA0,that cleaves to HA1 and HA2 by host proteases.These precursor derived subunits are a prereq-uisite for fusion of the viral and endosomal mem-branes and, therefore, for viral infectivity(Neumann and Kawaoka, 2006). Pathogenicavian influenza viruses present a single Arginineresidue at the cleavage site recognized by extra-cellular trypsin-like proteases which are thoughtto be secreted only by cells of the respiratory andintestinal tract and consequently limit infectionsto these organs. In contrast, highly pathogenicavian viruses possess multiple basic amino acidsat the cleavage site that are recognized by anyintracellular subtilisin-like proteases that thustrigger systemic infection. The HA cleavabilityis affected by the absence or presence of a car-bohydrate side chain near the cleavage site thatmay interfere with the facility of host proteasesto the cleavage site. When the human airwayepithelial cells are exposed to the avian influen-za A (H5N1), virus haemagglutinin (HA) can rec-ognize its receptors on the surface of the airwayepithelial cells (Yuen and Won, 2005). After thetarget recognition, the viruses enter the cells,replicate and rapidly (hours) spread throughoutthe human respiratory tract, damaging vitalorgans and tissues. Genetic variation in the H5N1

genome may increase the pathogenic propertiesof the virus in humans and animals. For exam-ple, certain mutations in PB2 (E627K) affect thevirulence of the virus in mice (Fouchier et al.,2004). Also, studies of the isolates of avianinfluenza virus A (H5N1) from patients in 1997revealed that the virulence of the virus increas-es with the highly cleavable hemagglutinin, spe-cific substitution in the polymerase basic proteinPB2 (Glu627Lys), and by substitution in non-structural protein NS1 (Asp92Glu) (WritingCommittee of the World Health OrganizationWHO Consultation on Human Influenza A/H5.Avian Influenza A(H5N1) Infection in Humans,2005). For more information on the effect ofgenetic variation on influenza virus virulence seeTable 1.

CASE CONFIRMATION AND LABORATORYPROCEDURE

Avian influenza virus is most commonly isolat-ed by inoculation of swab material or tissuehomogenates into embryonated chicken eggs bythe allantoic sac route (Mahy, 1985). Theembryos may or may not die, but in any case thepresence of the virus can be detected by haemag-glutination tests on harvested allantoic. Then itsidentity is confirmed by agar gel diffusion orhaemagglutination inhibition tests using specificantiserum. Also, rapid diagnosis can be made bythe detection of viral antigen in tissue impres-sion smears using immunofluorescence, or byantigen detection enzyme-linked immunosorbentassay (ELISA) on tissue homogenates.Cytological tests (including those done at autop-sy) can be performed for virus detection. Lungmay be the candidate organ because it wasdemonstrated that the pneumocytes are themajor site for H5N1 virus replication(Uiprasertkul et al., 2005). Searching for H5N1in plasma may be necessary, since a clear-cutviremia can be found (Chutinimitkul et al., 2006).Furthermore, the virus can be serotyped to deter-mine its haemagglutinin and neuraminidase sub-types. In vitro tests use plaque assay for viralquantification (Mastrosovich et al., 2006).However, this assay depends on the ability of thevirus to produce plaques in cell cultures. Also,polymerase chain reaction and gene sequencing



procedures can be used for rapid determinationof the pathogenic potential of an AI virus. Thereal-time RT-PCR assay is a rapid, specific, anda relatively sensitive method for directly detect-ing influenza A subtype H5 virus and may be use-ful in routine diagnostic testing (Enders et al.,2005). All cases described in this paper have beenobtained from the WHO and the centre for infec-tious disease research and policy (CIDRAP). Atfirst, cases were confirmed by the ministries of

health of the countries in which the outbreakoccurred. The ministry of agriculture had alsogiven the epidemiological informations to theinvestigators. Then the samples were sent to theWHO centres for reconfirmation that are theUnited States Naval Medical Research Unit 3(NAMRU-3) in Cairo, WHO H5 reference labo-ratories in Hong Kong and the USA, WHO col-laborating laboratory in the United Kingdom,Pasteur Institute in Cambodia, and CollaboratingCentre for Influenza at the National Institute for


TABLE 1 - Mutations in the Influenza genome favouring viral pathogenicity.

Type of the mutations Type of the mutant genes Effect of the mutations Reference

R152K NA gene Associated with Kamps B.S., Hoffmann C.,oseltamivir-resistant Preiser W., 2006

R292K, N294S, NA gene Associated with Moscona A. 2005H274Y and E119V oseltamivir-resistant

Dual mutation motif M2 gene Associated with Cheung C-L. et al., 2006Leu26Ile-Ser31Asn Amantadine- resistance

V27A, A30S and S31N M2 gene Associated with Ilyushina N.A. et al., 2005Amantadine resistance

D126N and K152N HA gene Reducing virulence Kaverin N.V. et al., 2002and lethality in mice

Lys88Arg, Asp701Asn, NS2 gene, PB2 gene, It is suggested that they Brown E.G. et al., 2001Phe103Leu NS1 gene respectively may operate in a certain

virus to mediate itsvirulence

Glu627Lys and PB2 gene Both mutations that Smith G.J.D. et al., 2006Asp92Glu have been associated

with increased virulenceof influenza viruses

S129L HA gene Effect atomic contactwith cellular sialosidesreceptors

The World Health OrganizationA156T HA gene Reduce viral affinity Global Influenza Program

for sialosides Surveillance Network,

S223N HA gene Facilitate bindingOctober 2005

of sialosides

E119A, E119G NA gene Associated withzanamivir-resistant

Mishin V.P. et al., 2005E119D NA gene Associated with zanamivir

and peramivir resistance

Some of these mutations make the virus resistant to anti-influenza drugs and others increase its virulence.


Medical Research (NIMR). In these centres, theRT-PCR assay, haemagglutination inhibitiontest, virus isolation in embryonated eggs andMDCK cells, and gene sequencing have been per-formed to detect avian influenza virus A (H5N1).

SPREAD OF AVIAN INFLUENZA VIRUS IN HUMANS

We retrieved the epidemiological data for thisreview from the daily news of the WHO, CDC,and CIDRAP. According to the report of WHO,from 2003 to 15th June 2006, 230 persons result-ed infected with H5N1 virus; 57% died (Figure4). In 2003, there were 4 confirmed human cases:3 in Vietnam and 1 in China. All of them werefatal (Figure 3). In 2004, 17 cases in Thailand and29 in Vietnam were reported; about 70% of themdied (Figure 3). In 2005, avian flu was presentin Cambodia, China, Indonesia, Thailand andVietnam. The confirmed human cases in thesecountries are 4, 8, 19, 5 and 61, respectively, ofwhom 43% died (Figure 3). Avian influenza casescaused by H5N1 were detected in numerouscountries in 2006 compared with the years 2003,2004, 2005. In the year 2006 there were 10 coun-

tries with virus outbreak (Figure 3): Iraq, Egypt,Azerbaijan, Djibouti and Turkey are the new ones.From January to 15th June 2006, 82 cases wereconfirmed in all countries (except Thailand andVietnam); 65% were fatal (Figure 3). From the53 confirmed deaths in 2006, 12 patients died inJanuary, 8 in February, 12 in March, 6 in April,14 in May and 1 in June (Figure 5A). The largestnumber of deaths was reported in Indonesia (27cases). In 2006, the disease predominantlyaffected children and young adults. The age distribution is shown in Figure 6: 81%of the deaths are between (5-34) years, 7.54%between (34-55) and 1.88% between (55-80); 9.4%are less than 5 years old. The total median ageis 20.36 years. The causes of this prevalence inyoung people are not fully defined, but mostprobably nearly all people are immunologicallynaive for the virus (Bartlett, 2006). This age dis-tribution of deaths in 2006 is compatible with1918 pandemics, in which the ages most affect-ed by the virus were between 15 and 35 years.Another study in Thailand shows that the fatal-ity rate is very high in children younger than 15years (Areechokchai et al., 2006). Not surprisingly,a recent epidemiological study in the area ofRome, Italy, showed that influenza viruses cir-


FIGURE 3 - a) Thegeographic distri-bution of thehuman cases anddeaths by influenzavirus A (H5N1) inthe year 2006 (fromJanuary to 15th

June). b) The geo-graphic distribu-tion of the humancases and deathsby influenza virus A(H5N1) from late2003 until 2005.The box shows thetotal death percent-ages in the years(2003, 2004, 2005and 2006).

a

b



FIGURE 5 - a)comparison of thehuman death ratefrom January until15th June 2006.The highest numberof the deaths wasdocumented inMay. b) the relativehuman deaths byH5N1 in the year2006 (until 15thJune) in the fourdesignated coun-tries (Azerbaijan,Iraq, Indonesia andTurkey). The agesand dates of the rel-ative deaths areshown. Each sym-bol represents acluster.

FIGURE 45 - Thetotal human casesand deaths withinfluenza virus A(H5N1) in late 2003until 15th June2006; 10 countries(Azerbaijan, Cam -bodia, China, Egy -pt, Indonesia, Iraq,Thailand, Turkey,Djibouti and Viet -nam) are indicatedwith virus out-break.

a

b



FIGURA 7 - a) thepercentage of bothmales and femalesamong all 53deaths in the year2006 (from Janua -ry until 15th June).b) the days betweenonset of symptomsto hospitalization(left columns) andthe days betweenhospitalization anddeath (right colum -ns) of 17 patients.

a

b

FIGURE 6 - a) agedistribution of all53 deaths in theyear 2006 (fromJanuary until 15thJune). b) medianage of all 53 deathsin the year 2006(from January until15th June) in eachcountry. The figureshows that childrenand young adultsare at high risk dur-ing influenza virusoutbreaks.

a

b


culates far more in younger patients. There wasno documented case in persons over 65 years(Rezza et al., 2006).The rates of male/female deaths due to the avianflu change over the world. As shown in Figiure 7a, the death rates amongfemales in Azerbaijan, Egypt, China and Turkeyare more prevalent when compared to othercountries. This difference depends on the tradi-tions, customs and economy of these countries.In Azerbaijan 80% of the deaths were femalebecause in this community defeathering of thebirds is a task usually undertaken by adolescentand young women (Gilsdorf et al., 2006).Avian flu causes death in humans a few days afterthe onset of symptoms. The period between onsetof symptoms, hospitalization and death isshown in Figure 7b for 17 deaths in the year 2006(for the others no information was available). Allevidence indicates that no sustained human tohuman transmission has occurred, and direct orindirect contact with infected or dead birdsremains the principal source of infection.

DISCUSSION

Human cases of avian influenza virus A (H5N1)infection have remained rare and sporadic, butthe disease is very severe and the case fatality ishigh. Genetic variation, including reassortment,is the main factor producing a flu pandemic(Figure 1). However, the development of the 1918pandemic virus cannot be related to gene reas-sortment, since simple adaptive mutations(drift) were sufficient to generate a strain ableto kill millions of people (Taubenberger et al.,2005). The recent genetic sequencing for the iso-lates of the 2 cases in Turkey (Figure 5B) demon-strates that the strains contain two mutationswhich probably make the virus easier to adaptto humans (Butler, 2006). The first mutation is in the pol gene at position627 (compatible to the mutation that occurredin the 1918 pandemic). The other mutationoccurred in HA at position 223 (detected in HongKong in 2003 and in one fatal case in Vietnamlast year). Both mutations found in Turkey casesshow the importance of keeping the level of alerthigh, since a few mutations may confer pandemicpotential on avian viruses that can efficiently

replicate in humans and cause human to humantransmission (Shinya et al., 2006). Indeed, in 2004a possible human to human transmission wasreported in a family cluster of the disease inThailand (Ungchusak et al., 2005). A 10 year oldboy was infected by a slightly mutated virus. Hepassed the mutated virus to this father, closelyinvolved in caring for his son; this contact is con-sidered a possible source of infection (http://www.cidrap. umn.edu/cidrap/content/ influenza/avianflu/news/jun2306cluster.html). Thus, furtherstudies are needed to shed more light on the epi-demiology of the disease. For 17 of the 53 deaths in 2006 the time inter-vals between onset of symptoms and hospital-ization was about 5 days; between hospitaliza-tion and death it was about 3 days (Figure 7b).These data illustrate that the virus is very severewhen it infects humans. For this reason the min-istry of Public Health in each country mustinform people about viral severity. Also infect-ed persons should go directly to the health cen-ters after onset of flu symptoms. Avian influen-za is more severe in children, who should avoidhandling dead and sick poultry during flu out-break.

CONCLUSIONS

The conclusions that arise from these viro-epi-demiological data are:1) Infection of humans with the virus (H5N1)

is very severe: only a few days are needed fromonset of symptoms to death.

2) The disease is more severe in children andyoung adults. Also, female deaths are pre-dominant when compared with males.

3) The mortality rate in the years 2004 and 2006were nearly equal (69.5% and 64.6% respec-tively). However, there is a difference in thenumber of countries with flu outbreak; thismay mean that the affinity and ability of thevirus to infect human may increase on a day-by-day basis.

4) In the next possible pandemic, influenza virusA (H5N1) may be the first candidate, since themutations in the virus genome make the virusmore adapted to infect humans. Today thereis no evidence of repeated events of humanto human transmission. So, long-term pub-



lic health surveillance and control measuresare needed to monitor for person-to-persontransmission and the emergence of a poten-tially pandemic H5N1 influenza virus.

5) Handling of dead or sick poultry in an H5N1infected area is the risk factor for humans.Close contact with them is the primary sourcefor transmission of the virus to humans.

6) Influenza virus A (H5N1) can replicate effi-ciently only in cells in the lower regions of therespiratory tract where the avian virus recep-tor is prevalent. This is the main cause of inef-ficient transmission of the viruses amonghumans (Shinya et al., 2006), yet, if the viruscontinues to circulate widely among poultry,it has a greater potential to infect humans andother animals. Genetic reassortment may thustake place and create a new pandemic strain.In a recent report of H5N1 virus infection ina domestic cat infected by eating a pigeon car-cass, the virus isolated from the pigeon andthe cat shows the same cluster as the virus-es obtained during the outbreak in Thailand(Kuiken et al., 2004). This affinity of the virusto infect other animals (as well as humans andbirds) may cause genetic variation, that in turnmay anticipate the next avian influenza out-break.

7) Vaccines against the next flu pandemics arenot available, because there is not enoughinstruction on which type of influenza viruswill be the cause. Available vaccines for birdsand humans may be useful for reducing therisk of pandemics, by decreasing the circu-lation and replication of common flu virus-es (avian and humans), that in turn may lowerthe risk of viral reassortment and mutations.For instance, no significant flu outbreak wasdetected in Thailand and Vietnam in the year2006 (from January until 15th June), becauseof the good vaccination process and surveil-lance performed in the previous year.

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