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Approximately all documented viruses that cause influenza in avians belong to the speciesÂ influenza A virus. Almost all the subtypes of Influenza A virus cause illness in avians but besure not all the strains of all subtypes are disease causing agents in Avians,therefore Avian Flu is normally considered as Influenza A Virus.
(Where as "A" doÂ notÂ stand for "avian").
The adaptations are not constrained. Adaptation towards some specific species do not completely limitize the adaptations, or partial adaptations, in infecting different species. Numerous species have adapted to Strains of influenza viruses , that might show compatability of adaptation to specific host. As for example, influenza pandemicsÂ causing viruses are adapted to both humans and avians. ModernÂ influenza researchÂ into the genes of theÂ Spanish fluÂ virus shows it to have genes adapted to both humans and avians , with more of its genes from avians than less lethal later pandemic strains.
The "Influenza Viruses" are longitudinally or spherically shaped envelop particles, single stranded RNA genome of negative polarity and with an up to eight- fold segmented. The "Influenza Viruses" belong to in Orthomyxoviridae family and classified into A, B and C based on antigenic differences of their nucleo- and matrix proteins. The "Avian Influenza Virus" (AIV) belong to type A. On the structure and replication strategy of Influenza Viruses special reviews have been published recently (2004, Sidoronko and Reichl).
The antigenic determinants of Influenza A virus are the Haemagglutinin (H or HA) and Neuraminidase (N or NA) transmembrane glycoproteins can cause subtype specific and immune responses and protection.
Influenza A viruses are classified into sixteen H ( H1 - H16 ) and nine N ( N1 - N9 ) subtypes based on the antigenicity of these glycoprotein,. When phylogenetically analyzed the above said clusters are substantiated the nucleotide and deduced amino acid sequences of the HA and NA genes . ( Fouchier, 2005).
Nomenclature of Influenza Virus isolates requires implication of the Influenza Virus type, the host species (omitted in the case of human origin), the geographical site, serial number, and year of isolation.
Since 2003, its most highly pathogenic strain Avian Influenza (H5N1) had been spreading throughout Asia. In 2005, the Avian Influenza reach Europe, and the Middle East, and Africa, the following year.Â On January 22, 2012, China reported its second avian flu death in a month following other fatalities in Vietnam and Cambodia.
1.2. Genetic Factors:
Genetically the Avian Influenza Virus and Avian Influenza Viruses are differentiated as:
(RNA polymerase) PB2:Â Amino acidÂ (orÂ residue) position 627 in the PB2 protein encoded by the RNAÂ gene PB2Â . H5N1 and all known Avian Influenza viruses had aÂ GluÂ at position 627, while all human influenza viruses had aÂ Lys.
Hemagglutinin (HA): The Avian Influenza HA viruses bind alpha 2-3Â sialic acidÂ receptors, while human influenza HA viruses bind alpha 2-6 sialic acid receptors.Â Swine influenzaÂ viruses have the capability to bind both types of sialic acid receptors. (HA) Hemagglutinin is the major antigen of the virus against Avian Influenza which produced neutralizing antibodies, and influenza virus epidemics are related with changes in its antigenic structure. The virus isolates from the pigs are referred to as pig virus
At present there are many subtypes of Avian Influenza viruses, but only some strains of four subtypes have been highly pathogenic in humans. These types include H5N1, H9N2, H7N7, and H7N3.
1.4. The Spreading/ Contraction of Avian Influenza:
The majority human contractions of the avian flu are a result of either handling dead infected avians or from contact with infected fluids. While wild avians normally have only a mild form of the H5N1 strain, once domesticated avians such as chickens or turkeys are infected, it could become much more deadly because the avians are often within close contact with one another. Due to low hygienic conditions and close quarters, a large threat of Avian influenza H5N1 subtypes in Asia with infected poultry. Though it is easy for humans to become infected from avians, it's much more difficult to do so from human to human without close and lasting contact.
H5N1 strain spread from Asia to Europe and is much more likely the cause of both illegal and legal poultry trades than dispersing through wild avian migrations, being that in recent studies, there were no secondary rises in infection in Asia when wild avians migrate South again from their breeding grounds. The infection patterns followed transportation such as rail roads, roads, and country borders, suggesting poultry trade as being much more likely. Whereas there have been strains of avian flu to exist in the United States, such as Texas in 2004, they have been extinguish and have not been known to infect humans.
The Examples of Avian Influenza A virus strains:
Avian InfluenzaA Viruses
A/fowl plague virus/Dutch/27(H7N7)
A/fowl plague virus/Rostock/34(H7N1)
1.5. The Influenza Pandemics:
The Pandemic flu viruses have some avian flu virus genes and usually someÂ human fluÂ virus genes. TheÂ H2N2Â andÂ H3N2Â pandemic strains contained genes from avian influenza viruses. New subtypes arose in pigs co-infected with avian and human viruses, and were soon transferred to humans. The swine were considered the original "intermediate host" for influenza, because they supported reassortment of divergent subtypes. Though, other hosts appear capable of similar co infection (e.g., many poultry species), and direct transmission of avian viruses to humans is possible.Â Spanish fluÂ virus strain may have been transmitted directly from avians to humans.Â Because of their pandemic connection, avian influenza viruses are noninfectious for most species. They are usually asymptomatic even when they are infectious, therefore the carrier does not have any disease from it. Consequently, while infected with an avian flu virus, the animal does not have a "flu". Usually, when infection (called "flu") from an avian flu virusÂ doesÂ occur, it is the result of an avian flu virus strain adapted to one species spreading to another species (usually from oneÂ avianÂ species to another avian species). Therefore, the most common result of this is an illness so minor as to be not worth noticing (and thus little studied). Whereas with the domestication of chickens and turkeys, humans have created species subtypes (domesticated poultry) that can catch an avian flu virus adapted to waterfowl and have it rapidly mutate into a form that kills over 90% of an entire flock in days, can spread to other flocks and kill 90% ofÂ them, and can only be stopped by killing every domestic avian in the area. In the 1990s untilÂ H5N1Â infected humans, this was the only reason avian flu was considered important. From the time then, avian flu viruses have been intensively studied; resulting in changes in what is believed about flu pandemics, changes in poultry farming, changes in flu vaccination research, and changes in flu pandemic planning.
The H5N1 strainÂ has evolved into a flu virus strain that infects more species than any previously known strain, is deadlier than any previously known strain, and continues to evolve, becoming both more widespread and more deadly. The fact initiatedÂ Robert G. Webster,an American scientist a leading expert on avian flu, to publish an article titled "The world is teetering on the edge of a pandemic that could kill a large fraction of the human population". He suggested for adequate resources to fight what he sees as a major world threat to possibly billions of lives.Â Till the time, the article was written, the world community has spent billions of dollars fighting this threat with limited success.
1.6. The Influenza A virus subtype H5N1Â andÂ Transmission and infection of H5N1:
Highly pathogenic influenza A virus subtypeÂ H5N1Â is an emerging Avian Influenza virus that has been causing global concern as a potentialÂ pandemicÂ threat. This is often referred to simply as "Avian Flu" or "Avian Influenza", even though it is only one subtype of Avian Influenza causing virus.
The subtype H5N1 has killed millions of poultry in a growing number of countries throughout Asia, Europe and Africa. The health experts are concerned that the coexistence of human flu viruses and avian flu viruses (especially H5N1) will provide an opportunity for genetic material to be exchanged between species-specific viruses, possibly creating a new virulent influenza strain that is easily transmissible and lethal to humans.Â Mortality rate for humans with H5N1 is about 60%.
Ever since the first H5N1 outbreak occurred in 1987, there has been an increasing number of Highly Pathogenic Avian Influenza (HPAI) H5N1 avian-to-human transmissions, leading to clinically severe and fatal human infections. As a significant species barrier exists between avians and humans, though, the virus does not easily cross over to humans, though some cases of infection are being researched to detect whether human to human transmission is occurring.Â To understand the pathogenesis and epidemiology of the H5N1 virus in humans further research is necessary. Disease transmission characteristics, the exposure routes and others, such as genetic and immunological factors that may increase the likelihood of infection, are not clearly understood.
Though millions of avians have become infected with the virus since its discovery, 306 humans have died from the H5N1 in twelve countries according toÂ WHOÂ data as of February 2, 2011.
Avian flu claimed at least 300 humans in Azerbaijan, Cambodia, China, Egypt, Indonesia, Iraq, Laos, Nigeria, Pakistan, Turkey, and Vietnam. The epidemiologists are afraid the next time such virus mutates, it could pass from human to human; however, the current A/H5N1 virus does not transmit easily from human to human. But in future if this form of transmission occurs, another pandemic could result. Therefore, the disease-control centers around the world are making avian flu a top priority. The organizations encourage poultry-related operations to develop a preemptive plan to prevent the spread of H5N1 and its potentially pandemic strains. Their recommended plans center on providing protective clothing for workers and isolating flocks to prevent the spread of the virus.
Thailand outbreak of avian Influenza caused massive economic losses, especially among poultry workers. The Infected avians were culled and sacrificed. Thus the public lost confidence with the poultry products, thus decreasing the consumption of chicken products. That also elicited a ban from importing countries. It included the factors which aggravated the spread of the virus, including avian migration, cool temperature (increases virus survival) and several festivals at that time.
1.7. The Infection of Avians/ poultry with Influenza Viruses:
The Influenza viruses have shown to infect a great variety of avians of which migratory waterfowl are recognized as a natural reservoir. Infrequently these viruses also infect some mammalian species, such as humans, pigs, horses, cats, minks and marine animals.
In poultry Influenza causes two distinct clinical forms:
(HPAI) Highly Pathogenic Avian Influenza
(LPAI) Low Pathogenic Avian Influenza
Highly Pathogenic Avian Influenza (HP Avian Influenza) cause serious disease in almost all poultry species resulting in very high mortality. The adults duck may not always show clinical signs, HP Avian Influenza produce disease only by some viruses of H5 and H7 subtypes which exhibit certain molecular characteristics that are at the basis of the systemic infection and reason for the severity of the disease.
LP Avian Influenza viruses belong to all H subtypes, but the lack of molecular traits, Avian Influenza of HP Avian Influenza viruses, thus causing only localized infection. It is generally recognized that LP Avian Influenza viruses of the H5 and H7 subtype represent the progenitors of HP Avian Influenza viruses which may mutate and acquire the molecular traits of HP Avian Influenza subsequent introduction into poultry.
1.8. THE POULTRY FARMING IN PAKISTAN:
Poultry farming has become one of the most vibrant associated parts of agriculture throughout the world. In South Asian countries the poultry culture is growing rapidly and the rate of growth of commercial layer and broiler (meat producing) farms is phenomenal to combat the ever increasing demand for proteins through poultry meet and eggs. The poultry industry IN Pakistan, had produce considerable contribution to food production and plays a vital role in the economy of the country. In Pakistan, raising of poultry has in effect proven a money-making enterprise as it is the best source of cheap, palatable and nutritious food protein. The poultry farming describes that Poultry science is the study of principles and practices involved in the production and marketing of poultry and its products. The poultry farming is rasing chickens, turkeys, ducks and other fowl for meat or eggs. The poultry farms can be:
1. Layer Farms where they produce eggs.
2. Breeding Farms where they raise poultry for meat,
1.8.1. History Of Poultry Farming In Pakistan:
Poultry production started in Pakistan in 1963. The poultry as on a commercial scale in the private sector started due to new effect made by Pakistan International Avian Influenza (PIA) in 1965, when the first modern hatchery unit in Karachi was established. Afterward , the investors have been taken keen interest in this sector, as was result of which considerable development has taken place towards growth of this industry. Commercial poultry farming emerged through the combined efforts and foresight of the government and the private enterprises. At the present commercial poultry production is concentrated around the large urban centers in the provinces of Sindh and Punjab and initially at Karachi, Lahore with the passage of time, it is now fairly well extend all over the country. The poultry avians (particularly Chicken) were mostly maintained in the backyard of the houses in villages on a small scale, with very little investment by the villagers in terms of money and material. The profit rate to those avians was too little, and there was no attraction for investors to operate the same on large scales.
1.8.2. THE POULTRY STATES IN PAKISTAN:
The poultry produced in Pakistan is being established through two management systems that is rural poultry farming and commercial farming. Commercial poultry farming sector is a classic example of private sector enterprise with a phenomenal growth of 20 to 25 % per annum. The attainment of this rapid growth was made to the incentives granted by the government time by time through income tax, liberal financing etc.
In the provinces of Sindh, there are farms in Mirpurkhas, Sukhar, Nawabshah as well as in small cities. Similarly, in the province of the Punjab, poultry farms have been well established in the areas of Rawalpindi, Faisalabad, Multan, Lahore, Bhawalpur, Rahimyar Khan and Mianwali.
The large scale investment and proper incentives have resulted in the establishment of infrastructure comprising of 252 hatcheries with capacity to produce 346 million a day- old chicks per annum, 141 feed mills with the capacity to produce 2540 thousand tons of compounds feed per annum and 13154 poultry farms with the capacity to produce 98 million broilers. Present total poultry population is estimated to be 319 million, out of which 137 million is rural (44%), 160 million broilers (50%), 17 million layers (5%) and 5 million breeding (2%) stock annually.
1.8.3. WEIGHT AGAINST PRODUCTION:
On average a laying avian produces an average of 250 eggs per year and the average live weight of the broiler is recommended as 1200 grams ( or 1.2 kg), at 8 weeks of age. The Pakistan produces 6.5 thousand million eggs and 340 thousand tons poultry meat. Consumption of poultry feed increased with their ages. It attained its peak (135g/avian/ day) at 25 weeks age. After that it declined slightly reaching 124g / avian/ day at 33 weeks of age. Some of the local breeds, are highly productive and are more adaptive to the local conditions. The important poultry breeds of fowls are cross bred with each other to produce. The product is accumulated across the large urban centers in the provinces of Punjab, Sindh and NWFP. Majority of such farmers are usually in and around the cities like Karachi, Lahore, Peshawar, and Faisalabad. Capacity of the layers farm varies from 2000 to 20000 avians and for the broilers farms from 103 to 403 avians per annum. The elevated cost of poultry feed is primarily due to the increase in the price of the various ingredients particularly fishmeal in their poultry feed. At present the prices of poultry are very high due to the cost of ingredients needed for the manufacturer of the feed meal.
1.8.4. The Types of Poultry Farms:
The different types of poultry farms supply to different segments of the market. The breeding farms are established for the sole purpose of hatching and raising poultry for sale to other farmers. Farmers who buy poultry from these farms set up broiler farms.
Broiler The chickens are grown just for their meat. The normal procedure is to procure one day old chicks, keep them for six weeks and market them. For the duration of the time that they are in the farm, their feeding, supplements, etc. is closely checked.
second type of poultry farm is
Layer farm, where chicks are grown for the purpose of egg production.
1.8.5. The Types Of Pakistani Poultry Chickens:
Asil (or Aseel):
Asil or Aseel is a breed of chicken originating from South Punjab/ Sindh area of Pakistan . Similarly fowl are found throughout Southeast Asia and have names like Shamo, Avian Influenza wan, etc. the Asils were first used for cock fighting. The Aseel is noted for its pugnacity. These chicks often fight when they are just a few weeks old and mature roosters will fight to the death. The Hens of this breed are also be very aggressive towards each other.
The Sadal (called Malay in Europe). This is a very large breed of chicken from Pakistan . This breed have longer legs with thin thighs and little wattles with pea-combs.
Afghan game fowl Pakistani Rooster
2.1. Background of Avian Influenza Virus
2.1.1. History of Avian Influenza Virus:
A contagious disease of poultry associated with high mortality in relation to the early description of Avian Influenza (AI) dates back to 1878 in Northern Italy, a contagious disease of poultry associated with high mortality was when Perroncito [Perroncito E. Epizoozia tifoide nei gallinacei. Annali Accad Agri Torino 1878; 21:87-126]. The ailment, called "fowl plague", was initially perplexed with acute septicemic type of fowl cholera. Whereas, in 1880, after its first depiction, Rivolta and Delprato [as reported by Stubs EL. Fowl pest, In: Biester HE, Devries L, editors. Diseases of poultry 1st ed. Ames, IO: Iowa State College Press; 1943. p. 493-502] described it to be dissimilar from fowl cholera, and called it Typhus Exudatious Gallinarum based on clinical and pathological properties,. In 1901, Centanni and Savunzzi [Centanni E, Savonuzzi E, La peste aviaria I & II, Communicazione fatta all'accademia delle scienze mediche e naturali de Ferrara, 1901] determined that fowl plague was caused by a filterable virus; however, it was not until 1955 that the classical fowl plague virus was shown to be a type A influenza virus based on the presence of type A influenza virus type-specific ribonucleoprotein [Schäfer W. Vergleichender sero-immunologische Untersuchungen über die Viren der Influenza und klassischen Geflügelpest. Z Naturf 1955;10b:81-91]. The term fowl plague was substituted by the more appropriate term highly pathogenic avian influenza (HPAI) at the First International Symposium on Avian Influenza [Proceedings of the First International Symposium on Avian Influenza. Beltsville, MD. 1981, Avian Dis 47 (Special Issue) 2003.] and will be used throughout this review when referring to any previously described fowl plague virus.
A Highly Pathogenic Avian Influenza Virus H5N1 subtype, in 1997 infected the humans for the first poultry outbreak in Hong Knog SAR, China. In 2003 and 2004, the virus re-emerged and widely spread from asian countries to Europe and African countries and has become ingrained in poultry in some of these countries, which resulted in hundreds and millions of poultry ailments and infections, quite a lot of human cases, and numerous human deaths. The financial system economy and international trading and business in affected countries during the outbreaks in poultry and have seriously impacted livelihoods. Continuing transmission of H5N1 viruses in poultry birds, particularly when endemic continues to induce threats to public health, this is because these viruses have equally the potential to cause serious disease in human beings and strong enough to transform or adapted into a form that is more contagious and transferable amongst human beings. Other subtypes of influenza viruses also travel in poultry and other birds and animals,which become the constant threats for the public health.
Starting in 1997, when highly pathogenic avian influenza (HPAI) H5N1 was discovered in humans in Hong Kong, the virus (also known as avian flu) has received unprecedented publicity. Avian influenza outbreak has been spread to 15 countries in Asia, Africa, and the Middle East, till 1997 and, lateron till December, 2011, it has infected 573 people. Out of these 573 people, 336 people died because of this fatal virus.
Millions of poultry birds have been died or been culled to prevent further spread of the virus. Even though the large number of humans infected with avian influenza are relatively few as compared to seasonal human Flu, the percentage of people who die from HPAI H5N1 is very high (approximately 60%).
The public health officials are concerned that Highly Pathogenic Avian Influenza (HPAI) H5N1 possibly will mutate into a form that could spread from human to human. Recently , human outbreaks of Avian Influenza particularly HPAI and casualities have resulted mainly from direct contact with infected avians.
Although many different influenza viruses infect avians and have for many years, the history of the avian influenzaÂ H5N1 virus in humans is relatively brief, because the first cases noted occurred in 2003 in China and Vietnam, according to the World Health Organization (WHO). The WHO reported only the confirmed cases, in which the presence of Highly Pathogenic Avian Influenza H5N1 microbes have been identified by blood tests or swabs of the infected person's nose or throat.
Undomesticated avians carry the viruses, but they are might be unaffected by them. However, in domestic avians (chickens, ducks, parrots and turkeys) this virus can cause illness and even it can be fatal. The symptoms include mildly ruffled feathers and less egg productions and severe symptoms include spread of virus to the multiple organs and fatality rate sometimes reached to 90-100% within shorter span of time i.e. 36-48 hours. It is this supposed that the level of differences in avian influenza symptoms is directly related to the strain of the influenza virus affecting the avians. The Highly Pathogenic Avian Influenza strain H5N1 microbes can cause severe signs and symptoms in poultry animals and in many cases complete flocks have to be ruined to block the spread of disease.
Infectivity with the avian influenza micro-organisms amongst humans is very unusual and normally occurs in persons who are in direct contact with infected birds of poultry and most strains, producing only mild illnesses. The olden times avian influenza H5N1 virus has shown that this strain can be fatal to humans. Since 2003, the confirmed cases had been about 253 that results in 148 casualities. This high percentage of deaths of about 58% because of infection with avian influenza micro-organisms create the situation which cause the scientists and officers of public health sciences through out the world to get worried.
Over a period of time the virus particales mutate at very slow rate therefore the human immune system recognize them easily because these viruses are similar copies of the previous existing viruses and thus they spontaneously respond them. Very rarely in the past times, viruses show changes which are, referred to as "antigenic shift", which cause severe disease, number human fatalities and global epidemics. Every so often these virus particles did not formerly infected humans, but had infected other animals, such as avians. They had not been highly infectious amongst humans, as with the H5N1 strain, but abruptly mutate and become easily transmitted among human beings. The history of the avian influenza H5N1 shows that the virus has revealed that it can infect human beings , researchers believe that it may possibly become highly contagious amid them, causing pandemics or international epidemics. The researchers believe that simply two proteins in the H5N1 avian influenza micro-organisms would need to change in order for it to become as easily transmitted among human beings as the seasonal influenza.
Presently, if an individual do not have contact with wild birds or domestic poultry in areas where H5N1 have recognized, then you run no danger of contracting the disease. That there will be epidemics of influenza every year is a virtual certainty. It is also noted that the influenza starts generally in late winters and ends up in one or two months. (Brammeret al., 2002). Therefore, outside of those general rules, estimating the time, degree and severity of influenza epidemic is a alarming public health challenge. Influenza A viruses exists in human beings largely and epidemiologically spread in numerous distinctive ways: as localized outbreaks, as yearly area wise epidemics and, rarely, as worldwide pandemics. Influenza cause the hospitalization of about 1,00,000 and kills over 30,000 people in year (SimonsenÂ et al., 2000;Â ThompsonÂ et al., 2003) in USA. After every 2 to 3â€…years, influenza epidemics increase the annual number of casualities, causing about 10,000-15,000 additional deaths. Infrequently, and unpredictably, influenza cleans the world, and infect 22 to 40â€Š% of the general public in one year. In those pandemic years, which have occurred every 15 to 50â€…years for at least several decades, the number of deaths are increasingly above average (Beveridge, 1977;Â Cox & Subbarao, 2000;Â Wright & Webster, 2001). It is concluded that the influenza will come back in pandemic form. In recent times, it was determined in USA alone that the next influenza pandemic may result in about 200â€Š000 deaths, 735â€Š000 hospitalizations, about 42 million outpatient visits and about 48 million extra illnesses (MeltzerÂ et al., 1999). The economic loses ranged between 70-180 billion dollars which did not include damages to the society. Presently, it is not possible to estimate the timings and severity of the subsequent pandemic outbreak, whereas study of the genetical and epidemiological characteristics of historic pandemics may suggest where observations and investigations would suggested best (LayneÂ et al., 2001; Taubenberger & Layne, 2001).
Influenza pandemic follow the classic outline of influenza epidemiology in many different ways in 1918. After the previous pandemic of 1890, it occurred again after 28â€…years and emerged internationally suddenly in September 1918 immediately after a limited signal earlier in the year. The morbidity rate ranged between25-40â€Š% whereas the majority cases were self limiting. The age related morbidity was also alike to other pandemics, with the kids under 15â€…years of age experiene the highest rates of infection (Jordan, 1927). On clinical basis, the pandemic of 1918 shows the same signs and symptoms and shows similarities as influenza of other years and, on pathological basis, the disease was similar to other pandemics which showed the clinical signs and symptoms including respiratory tract (Wolbach, 1919;Â WinternitzÂ et al., 1920). the The pandemic of 1918 is different from other pandemics in a few aspects. First of all, the clinical course in the majority of cases was mild, a considerably high percentage of cases showed severe pneumonia and its complications. These effected cases showed the mortality rate of about 2.5% in USA which is many fold greater than comtemporary average. During the pandemic of 1918 the death rate was gathered in adolescent age grouped people (Linder & Grove, 1943;Â Marks & Beatty, 1976;Â Rosenau & Last, 1980). The mortality rate was as high as 99% among the people between 60- 65 years old because of influenza virus in 1918. In 1957 and 1968 influenza outbreaks, the mortality rate was 36 - 48 % of increased in population under 65 years of age, (SimonsenÂ et al., 1998). During the pendamic of 1918, the people of 20-40 years old were the main effecties and this age group showed 50% of casualities of total deaths caused by influenza virus.
Recently, the pandemic strain of 1918 was not available for study purposes,and this is because the influenza virus was not isolated and cultured uptill 1930's.After that because of circulation of virus for about 15 years among the human beings resulted in considerable change in antigenicity of the circulating influenza virus at H1 haemagglutinin units as tested by serological analysis. (Shope, 1936;TaubenbergerÂ et al., 2001), therefore the strain of 1918 can be analysed on limited basis by some indirect method. Now a days, the frozen lung and tissue sample can be used for virus identification and sequencing of viral genome of the strain of 1918 pandemic of Influenza virus. (TaubenbergerÂ et al., 1997). From these frozen samples four genes from total of eight gene segments had sequenced (ReidÂ et al., 1999,Â 2000,Â 2002;Â BaslerÂ et al., 2001). They study of the strain of 1918 pandemic has two main purposes, first of all this study described the virulence factor of the 1918 influenza virus and also the origin of this virus which caused the pendamic. The study and understanding of the structure, genome and virulence factor of the virus provide the basis for the development of prevention and treatment guide lines of influenza virus.
The disease called Avian Influenza (AI) which is caused by influenza A viruses is an ailment of many kinds of poultry birds, natural and caged birds recognized by the considerable by distinct variation in mortality, morbidity, signs and lesions. Furthurmore, the infection causes periodical epidemics in variety of avians including humans, seals and others. (Swayne and Halvorson, 2003). All the Avian Influenza (AI) viruses are members of Orthomyxoviridae family. Within the family there are three types of influenza: A, B and C affect only humans (Scholtissek et al.,1983). The RNA virus is enveloped, sensitive to organic solvents and different chemical disinfectants. On the basis of antigenic relations among the surface glycoprotein's, Influenza A virus is grouped into two subtypes that are Haemagglutinin (HA) and Neuraminidase (NA). There are sixteen H subtypes and nine N subtypes. The virus genome is single stranded, and the progeny viruses are produced by the reassortment of parental genes which is derived from different viruses. The recent studies showed that 256 of progeny viruses originated from the parent viruses. (Murphy and Webster, 1996; Anon,2000; Suarez,2000).
The most virulent form of the Avian Influenza was designated as fowl plague in poultry. The term "highly" virulent influenza virus now replaced the primitive term fowl plaque (1918). The term Highly pathogenic Avian Influenza (HPAI) is described by the surface antigen and pathogenicity is recommended. (Anon,2000). Presently, the H5 and H7 subtypes have caused the Highly pathogenic Avian Influenza whereas all H5 and H7 subtypes are not virulent. But it has been proved by researchers that the the Highly Pathogenic Avian Influenza strain i.e. H5 and H7 subtypes are originated from the Low Pathogenic Avian Influenza progenitors. (Garcia et al.1996; Perdue et al 1997; Villarreal and Flores, 1998). Also, in 2004, highly pathogenic Avian Influenza was detected in Commercial and backyard flocks in Thailand. Recently several outbreaks of Avian Influenza H5N1 have been confirmed among poultry in Cambodia, China, Hong Kong, Indonesia, Japan, Laos, South Korea, Vietnam and Thailand.
2.1.2. Taxonomy of Avian Influenza Virus:
The Influenza A virus is an Orthomyxovirus, and its receptor binding complex is comprised of two primary structural proteins, Hemagglutinin (HA) and Neuraminidase (NA). It has been determined that Hemagglutinin is the primary protein responsible for binding to receptor sites on the cell membrane, allowing the virion to enter the cell (Subbarao 2000). Hemagglutinin is species specific binding protein that binds only to matched sialic acid receptors in host cells (Subbarao 2000). The molecule under study here is the HA protein extracted from the H1-human influenza strain.
The structure of Hemagglutinin is very similar between strains, and differs in only a few structural difference. What makes this strain differ from the H5N1 influenza now posing a pandemic threat is the speficity of its binding regions, which will be the basis of this study. What makes this strain of influenza so dangerous is both its rapid ability to evolve and the fact that mammals, specifically humans contain to immune defenses against this avian strain (Suarez 2000). In 1918, a strain of influenza A virus killed 20-40million people worldwide and the World Health Organization estimates nearly 7 million dead and 1 billion ill from an outbreak of the current H5N1 strain (Horimoto 2001).
Researchers have been able to determine that Hemagglutinin (HA) is a species specific binding protein that allows for the virus to bind to the cell membrane of host respiratory cells and propagate through cellular processes. By examining this process, medical researchers hope to determine a vaccine that may prevent this binding from occurring, thus preventing host infection.
I. General Structure:
Hemagglutinin is a trimer protein composed of aÂ globular domainÂ and astem domain, divided along the longitudinal axis of the proteinÂ Â . As was stated, HA protein is made up of three monomers;Â HA1,Â HA2, and HA3Â domainsÂ Â . Each of these monomers is comprised to two subdomains, in the stem domain the two helix are bonded atÂ Phe-88Â toÂ Phe-63Â a clear difference between influenza strains, in the avian H5N1, it displays a inward facing Phenylalanine ring, while H1- human strains display an outward facing ring shown here. Along the longitudinal axis, the protein is comprised of structuralÂ alpha-helices andÂ beta sheetsÂ Â are seen especially in forming the "bonding depression" in the globular region.
TheÂ beta-sheetsÂ are primarily present in the globular headÂ where the binding region to sialic acid resides and theÂ alpha helicesÂ make up the stem region of the HA monomers .
II. Receptor Binding Sites
The hemagglutinin protein, as was previously stated, is a trinomer protein, primarily responsible for the binding of the Influenza A virion to cell surface receptors, membrane fusion and intracellular infection, which is the first stage of viral infection. Hemaggluntin recognizes sialic acid components of cell-surface glycoproteins and glycolipids (Gamblinet al., 2004; HaÂ et al., 2000). Hemagglutinin contains a shallow depression at itsÂ "head"Â Â which allows the sialic acid sequence to move into like aÂ "lock and key."Â According to HaÂ et al.Â (2000), species specific sailic acid receptor analogs do not have a binding affect on the orientation of the sialic acid domain into the base of the hemagglutinin depression. One side of the sialic acid's pyranose ring faces the base of the binding depression. The axial carboxylate, acetamido nitrogen, and the 8- and 9-hydroxyl groups face the site and form bonds.
The binding depression surrounds the sialic acid domain with three primary regions of the hemagglutinin structure. This region is comprised of aÂ loop-helix-loopÂ structure, which surrounds the sialic acid. TheÂ 130-loop,Â 190-helixÂ andÂ 220-loopÂ structures form the triangular opening into the beta-sheet depression.Â Â These three structures from the primary binding to the sialic acid and specific regions in their domains (Gamblinet al.,Â 2004). The exact binding geometry differs between species specific hemagglutinin and cell surface proteins, however
there are primary cites on the loop-helix-loop complexes that form specific integrations allowing for the binding of the hemagglutinin and the subsequent cell infection. In human-avian receptor complexes theÂ Glu-190Â Â residue on theÂ 190-helixÂ forms a hydrogen bond to theÂ 9-hydrxyl group.Â Thr-136Â Â as well as amino-acids at residuesÂ 135Â Â andÂ 137Â on theÂ 130-loopÂ form hydrogen bonds to the sialic acid'sÂ carboxylate. Also,Â Lys-222Â Â andÂ Gln-226Â Â of theÂ 220-loopÂ form bonds withÂ 8-hydroxyl groupÂ of the sialic acid (GamblinÂ et al.,Â 2004) (Figure 1). Together this binding forms around the sialic acid domain of the cell surface glycoprotein or glycolipid in the HA depression, connecting each monomer to the sialic acid on the cell, initiating viral infectionÂ Â .(Figure 1,2)
III. Species Specific Binding
The HAs of viruses recognize different linkage if the HA strain is avian or human. Avian strains have receptor-binding specificity for sialic acid receptors in alpha-2,3 linkage while human strains have specificity for alpha-2,6 linkage. Avian H5 HA hydrogen bonds through Gln-226 to the glycosidic oxygen that is exposed in a trans coformation in the alpha ;2,3 sialic acid-to-galactose link. Alpha-2,6-Linked sialosides bind in a cis conformation, exposing the glycosidic oxygen to solution and nonpolar atoms of the receptor to Leu-226, a human-specific residue. Although each strain contains the both types of alpha linkages, the Gln-226/Gly-228 of the avian strain prefer the alpha-2,6 linkage while the Leu-226/Ser-228 of the human strain prefers the alpha-2,3 linkage. The two can geometrically distinguished because the human strain is much wider between the 226 and 228 positions while the avian strain is much more closed.
2.1.3. SURFACE ENVELOPE GLYCOPROTEIN
Segment 4 (1,698 nt ). Denoted as HA (or H) is surface glycoprotein that is responsible for viral binding and entry into host epithelial cells. HA contains two domains (HA1 and HA2) and several importantÂ epitopesÂ - parts of the viral protein that are recognized by either cellular or humoral arms of the immune system, and that undergo constant changes for virus to be able to breach host's immune defenses. H is the principal component of any influenza vaccine. For virus to be infectious, the HA must undergo post-translational cleavage into two peptides, HA1Â andÂ HA2, by a tripsin-like protease. Modifications in cleavage mechanism and/or site can lead to drastic changes in the virus pathogenicity. For example, influenza A viruses subtypes H5 and H7, usually asymptomatic in wild waterfowl, may become highly pathogenic once introduced into domestic poultry. Acquisition of extra amino acids at the cleavage site transforms the virus from one in which cleavage activation of the HA is restricted to the respiratory and intestinal tract, where there are tripsin-like proteases, into one that can be activated by cell-associated proteases that are found throughout the body, resulting in so-called viralÂ pantropism.
Segment 5 (1,407 nucleotides). The NA constitutes 20-25% of the surface of the viral particle. The NA is an enzyme named for its ability to cleave neuraminic or sialic acid from complex carbohydrates such as mucin. NA is responsible for the release of influenza particles from the infected cells, and thus, is important as a spreading factor, and is targeted by the current antiviral drugsÂ oseltamivir (Tamiflu)Â andÂ zanamivir (Relenza).Â The NA antigen is second most important component of anti-flu vaccine. (Figure 3,4)
2.1.4. Types of genetic variations in influenza viruses
Constitutes minor incremental changes in viral surface proteins (antigens). In the course of continual evasion of host's immune responses, viruses accumulate mutations in the HA an NA surface glycoproteins thus modifying their antigenic makeup. The antigenic drifts within single species usually result in mild outbreaks. However, antigenic drift that lead to adaptation of zoonotic virus to the human host, might result in a major pandemic.
b) Re-assortmentÂ (also can be referred as anÂ antigenic shift)
is a form of recombination in which two or more influenza viruses, of same or of different subtypes, co-infect a single cell and exchange their RNA segments to form genetically novel viruses. Antigenic shifts frequently result in pandemics. The novelty of 2009/A/S-OIV constitutes a majorÂ antigenic shift. Pigs have been hypothesized to serve as a mixing vessel in which avian and human influenza A viruses reassort. Swine are very susceptible to infection with avian flu viruses, which further facilitated by proximity to natural water reservoirs inhabited by wild fowl. This has been attributed to the fact that swine tracheal epithelium expresses both Î± 2,3- and Î±-N-acetylneuraminic acid-galactose linked receptors. The former,
found in waterfowl enteric tract, bind avian influenza A viruses, and the latter, located in the human respiratory tract, bind human host enabled influenza A viruses, thus facilitating occurrence of co-infections and making re-assortment possible.
To cause a pandemic among humans the zoonotic influenza virus should undergo genetic modifications that would lead to aÂ host switch. The host switch means that the virus not only can pass from avians or animals to humans, but also acquires human-to-human transmitability. The host switch can occur as a result of incremental changes in the process ofÂ adaptation to the foreign host (antigenic drift) as well as a result of accidental re-assortment between two or more viral subtypes (antigenic shift). For example, the 1918 virus may have arisen wholly from an avian influenza virus by adaptive mutations, whereas, 1957 and 1968 pandemic viruses resulted from re-assortments.
2.1.5. Morphology and genetic structure of the virus
The morphology of influenza A (H5N1) is basically that of an orthomyxovirus as it is a subtype of the type A influenza virus. The typical virion is enveloped, spherical (100 nm), with a nucleocapsid of helical symmetry surrounding a minus sense single stranded 8 segmented RNAÂ . The envelope is internally lined by a matrix protein (M) and externally with glycoprotein peplomers-rod shaped haemagglutinin (HA) which are homotrimers of class I membrane glycoproteins and mushroom shaped neuraminidase (NA) molecules which are tetramers of a
class II membrane protein.
Based on the variation on HA and NA molecules there exists 15 HA and 9 NA subtypes of influenza A virus. The avian strains differ from human strains in that they have all the 15 subtypes of HA in contrast to only three in case of humans.
The virulent avian influenza H5N1 strains differ from other avian strains in that, there lies a link between HA cleavage and degree of virulence. In virulent strains the HAs contain multiple basic aminoacids at the cleavage site, which are cleaved intracellularly by endogenous proteases. In contrast, in case of avirulent avian strains as well as non-avian influenza A viruses, the HAs lack the basic aminoacid residues, hence not subjected to cleavage by such proteases. Moreover, all types of influenza A viruses are antigenically labile, well adapted to evade host defences and lack mechanisms for "proof reading"; hence constant, permanent and small changes in antigenic composition are very common, which is known as antigenic drift. Another important characteristic of great public health concern is antigenic shift which results from reassortment of genetic material from different species resulting in variability of HA spikes, keeping the basic structure of the virus constant.
Viruses of this family contain 6 to 8 segments of linearÂ negative-senseÂ single stranded RNA.
The total genome length is 12000-15000Â nucleotidesÂ (nt). The largest segment 2300-2500 nt; of second largest 2300-2500 nt; of third 2200-2300 nt; of fourth 1700-1800 nt; of fifth 1500-1600 nt; of sixth 1400-1500 nt; of seventh 1000-1100 nt; of eighth 800-900 nt. Genome sequence has terminal repeated sequences; repeated at both ends. Terminal repeats at the 5'-end 12-13 nucleotides long. Nucleotide sequences of 3'-terminus identical; the same in genera of same family; most on RNA (segments), or on all RNA species. Terminal repeats at the 3'-end 9-11 nucleotides long. Encapsulated nucleic acid is solely genomic. Each virion may contain defective interfering copies.
2.2. AVIAN INFLUENZA VIRUS
2.2.1. Epidemiology of Highly Pathogenic Avian Influenza of H5 and H7 Subtypes:
Upto the end of 2003, Highly Pathogenic Avian Influenza (HPAI) was thought to be a rare disease in poultry animals . Globally only 24 primary outbreaks were reported till 1959. Amongst them most of the outbreaks occurred in America and Europe. Most outbreaks were geographically limited, with only five resulting in significant spread to numerous farms, and there was only one which spread worldwide. Among the all outbreaks, the size of Asian outbreak in 2004 was largest. (WHO 2004/03/02). It is also noted that among all the outbreaks uptill now the biggest and extremely pathogenic Influenza A virus is of subtype H7 and H5. In 1997, the original H5N1 virus was discovered and it was also researched that this virus camed from domestic geese (A/goose/Guangdong/1/96, donating the HA) and a H6N1 virus, probably from teals (A/teal/ HongKong /W3 12/97, donating the NA and segments for the internal proteins), which underwent many more cycles of reassortation with other unknown avian influenza virus (Xu,1999, Hoffmann, 2000, Guan, 2002b). Several different genotypes of the H5N1 lineage have been described (Cauthen, 2000, Guan, 2002, 2003). The so-called genotype "Z" has dominated the outbreaks since December 2003 (Li, 2004). A new dimension of HPAI outbreaks became evident late in 2003. From mid-December 2003 through to early Feburary 2004, outbreaks in poultry caused by the Asian lineage HPAI H5N1 virus were reported in Republic of Korea, Vietnam, Japan, Thailand, Cambodia, Leo People's Democratic Republic, Indonesia and China. The simultaneous occurrence in several countries of large epidemics of highly pathogenic H5N1 influenza in domestic poultry is unprecedented. All efforts aimed at the containment of the disease have failed so far. Despite the culling and the pre-emptive destruction of some 150 million avians, H5N1 is now considered epidemic in many parts of Indonesia and Vietnam and in some parts of Cambodia, China, Thailand and possibly also the Laos.
In January 2004, an outbreak of highly pathogenic Avian Influenza outbreak occurred in Thailand. HPAI was detected in the layer farms, Bangplama district, Suphanburi Province, central region of Thailand. The peak of the infection rate and seasonal influenza outbreaks was during the end of rainy season and winter season, it occurred in commercial poultry and backyard flocks.
2.2.2. Mode Of Infection And Transmission
Domesticated avians may become infected with avian influenza virus through direct contact with infected waterfowl or other infected poultry, or through contact with surfaces (such as dirt or cages) or materials (such as water or feed) that have been contaminated with virus. People, vehicles, and other inanimate objects such as cages can be vectors for the spread of influenza virus from one farm to another. When this happens, avian influenza outbreaks can occur among poultry.
Infected avians shed the virus in fecal and oculo-nasal discharges. Even though recovered flocks shed less virus than clinically ill flocks, recovered flocks will intermittently shed and should be considered infected for life.
Waterfowl (wild and domesticated) are the primary natural reservoir of influenza viruses. Wild waterfowl usually do not show clinical signs, but they can excrete the virus for long periods of time. In addition, waterfowl can be infected with more than one type of influenza virus. Detection is further complicated by the fact that they often do not develop a detectable antibody response after exposure to the virus.
Influenza virus has been recovered from water and organic material from lakes and ponds utilized by infected ducks. Co-mingling of these avians with range-reared flocks is a factor in some outbreaks.
The avian influenza virus can remain viable for long periods of time at moderate temperatures, and can survive indefinitely in frozen material. As a result, the disease can be spread through improper disposal of infected carcasses, manure, or poultry by-products.
The disease also can be easily spread by people and equipment contaminated with avian influenza virus. Avian influenza viruses can be transmitted on contaminated shoes, clothing, crates, egg flats, egg cases, vehicles, and other equipment. Any object located on an infected poultry farm must be considered contaminated and should be completely cleaned and disinfected before it is moved from that premises. Clothing worn on an infected farm should be laundered.
Insects and rodents may mechanically carry the virus from infected to susceptible poultry.
Influenza virus has been isolated from turkey eggs suggesting vertical transmission, although typically the virus kills the embryo. There is little or no evidence of egg-borne infection of poults. However, eggshell surfaces can be contaminated with the influenza virus, and thus are a means of transmission.
Avian influenza viruses have frequently been isolated from clinically normal, imported exotic avians. These infected avians are a potential threat to cage avians, wild avians, and poultry.
Live-avian markets are reservoir of infection. Such markets serve as a focal point for gathering and housing many species of avian. These facilities are rarely cleaned or disinfected.
Incidences of avian influenza among poultry occur worldwide from time to time. Since 1997, for example, and based on the World Organization for Animal Health (OIE) reporting criteria for Notifiable Avian Influenza in commercial poultry, the United States has experienced 17 incidences of H5 and H7 low pathogenic avian influenza (LPAI), and one incidence of highly pathogenic avian influenza (HPAI) that was restricted to one poultry farm.
Low pathogenic forms of avian influenza viruses are responsible for most avian influenza outbreaks in poultry. Such outbreaks usually result in either no illness or mild illness (e.g., chickens producing fewer or no eggs), or low levels of mortality.
When highly pathogenic influenza H5 or H7 viruses cause outbreaks, between 90% and 100% of poultry can die from infection. Animal health officials carefully monitor avian influenza outbreaks in domestic avians for several reasons:
the potential for low pathogenic H5 and H7 viruses to evolve into highly pathogenic forms
the potential for rapid spread and significant illness and death among poultry during outbreaks of highly pathogenic avian influenza
the economic impact and trade restrictions from a highly pathogenic avian influenza outbreak.
the possibility that avian influenza could be transmitted to humans when avian influenza outbreaks occur in poultry, quarantine and depopulation (or culling) and surveillance around affected flocks is the preferred control and eradication option.
2.2.3. Clinical Signs and Gross Leisions
The severity of the disease ranges from in noticeable (mild) to rapidly lethal. Fatal strains of the virus can attack rapidly , specially to the young chickens, and this cause sudden death without showing any clinical signs and symptoms.
Avian influenza viruses of low to moderate pathogenicity are identified regularly in the United States in the domestic poultry populations. Avian influenza virus is reintroduced into domestic poultry by migratory waterfowl, which are carriers of the influenza virus.
Clinical signs vary greatly and depend on many factors including the age and species of poultry affected, husbandry practices, and the inherent pathogenicity of the influenza virus strain. Clinical signs may include: