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Avian influenza is a highly contagious infectious disease of birds caused by type A influenza viruses. The disease was identified first in Italy in1878 WHO, 2005. Wild waterfowl, shorebirds and gulls constitute the natural reservoirs of type A influenza viruses (Slemons et al., 1974; Webster et al., 1992; Olsen et al., 2006; Slemons and Easterday, 1977). The infection from infected migratory birds such as seagulls spreads to healthy poultry flocks through contaminated materials. The infection causes high degree of mortality and morbidity in infected poultry and is a major ground for potential economic loss (Lupiani and Reddy, 2009).
The first human transmission of avian influenza was reported in Hong Kong in 1997 (WHO, 2008). After 1997, frequent and severe human transmissions were reported in many parts of the world indicated the potential health risk associated with HPAI viruses. These outbreaks also resulted in the loss of hundreds of millions of poultry flocks either directly through infection or by culling. High degree of variation in the severity of the virus is often associated with the rate of mutation in the genome through genetic reassortment resulting in the emergence of a highly pathogenic avian influenza strains. According to Meltzer et al., 2001 the next influenza pandemic will result in approximately 734,000 hospitalizations and 207,000 deaths in the United States alone. Avian influenza, especially the high pathogenic H5N1, is the infectious agent that is most likely to cause the recent pandemic, leading to massive fatalities worldwide and hence considered one of the major threats to both public health and poultry industry (J.S. Malik Peiris et al., 2007).
Biology of the virus
Influenza viruses belonging to the family Orthomyxoviridae are enveloped viruses (Lamb and Krug, 2001). The virus particles are spherical or filamentous in form. The lipid bilayer is derived from the host cell plasma membrane in the process of virus release by budding and is known to contain cholesterol-enriched lipid rafts and non-raft lipids [Scheiffele et al., 1999; Zhang et al., 2000; Nayak et al., 2009]. The virion consists of eight-segmented single-stranded, negative-sense RNA genome (Mjaaland et al., 1997). The eight segmented fragments constitute for a total length of 13.6 kilobases (kb) and consists of eleven open reading frames namely HA, NA, M1, M2, PB1, PB2, PB-F2, NP, PA, NS1 and NS2 (Fields et al., 2007; Webster et al., 1992; Lamb and Krug, 2001).
The HA codes for haemagglutinin protein which is the major structural glycoprotein found on the surface of the influenza viruses (Ruigrok, 1998). The HA protein facilitates the binding of the virus to the cells via different types of sialic acid receptors depending on the cell type. Mutations in HA proteins can affect its binding affinity to particular type of sialic acid residue and also results in change of host specificity (Skehel and Wiley, 2002). HA is the most abundant structural antigen and approximately 500 copies (80%) of HA is needed to make a single virion. The NA gene encodes neuraminidase protein which is the second major surface protein of avian influenza viruses and approximately 100 copies (17%) of NA antigen makes up one virion. The NA protein found on the surface of the influenza viruses, facilitate the virus release after the replication by cleaving the bond between the HA and the sialic acid residue in the target cells. The NA also prevents the virus aggregation and helps in spreading the virus (Lamb and Krug, 2001).
The M gene codes for two matrix proteins namely M1 and M2. The M1 and M2 are encoded from the same RNA segment using two different open reading frames. The matrix protein M1 along with the two major surface proteins HA and NA make up the capsid of the virus. The M2 is an ion channel transmembrane protein which plays a major role in virus uncoating and thereby releasing the viral RNA into the cytoplasm of the host cell at the time of infection. The M2 is a minor structural antigen and only 16-20 molecules are present per virion (Holsinger and Lamb, 1991; Pinto et al., 1992).
The RNA segments are surrounded by the nucleoprotein which is coded by the NP segment. The NS segment codes for two non-structural proteins namely NS1 and NS2. The NS2 was also called as nuclear export protein or NEP. The non-structural proteins are known to play a role in RNA transport, splicing and translation. The non-structural antigens also involves in enhanced proinflammatory cytokine response, especially TNFa. The PA, PB1 and PB2 segments code for the viral RNA polymerase. The PB1-F2 protein is encoded by PB1 RNA segment using an alternative open reading frame and plays a role in apoptosis. The PB1-F2 is believed to play a major role in pathogenicity of pandemic avian influenza viruses.
The influenza virus infection cycle can be divided into six major stages. The binding and entry of viruses into the host cell; entry of vRNPs into the nucleus; transcription and replication of the viral genome; export of the vRNPs from the nucleus; and assembly and budding at the host cell plasma membrane (Tasleem Samji, 2009).
The infection of avian influenza virus begins when the HA antigen on the virus particle docks the virus onto the cells via the sialic acid receptors (Skehel and Wiley, 2000). After the specific binding of the virus onto the cell surface, the cell surface folds inwards and wrap up the virus in cell membrane. This helps the virus particles to sink deep enough into the cell forming the membranous "bubble" (or vesicle). The external scaffold of the vesicle is formed by the protein clathrin, a protein that forms an external scaffold that causes the cell membrane to invaginate and finally form the vesicle. The clathrin coat is subsequently lost and the engulfed virus then forms an endosome (Skehel and Wiley, 2000; Huang et al., 2003). The internal environment of the endosome is more acidic and the M2 protein present on the membrane of the virus enables the entry of more hydrogen ions into the virus and thus lowering the pH inside the virus. This causes the matrix protein to disassociate from the ribonucleoprotein (RNP) complex and releases the viral contents into the cytoplasm. At this stage, the nucleocapsid segments migrate to the nucleus and move into the nucleus via the nuclear pore complex. This process delivers the viral genome into the nucleus.
In the nucleus, the viral negative sense genetic material produces positive sense viral messenger RNAs of various kinds with the help of RNA polymerase complex. The messenger RNA travels to the cytoplasm through the nuclear pore complex and begins the translation of viral proteins. At the same time, the negative sense RNA is made inside the nucleus from initially synthesized positive sense templates. The mRNA which was transported into the cytoplasm makes enough structural and non-structural and non-structural proteins in the cytoplasm of the infected cells. The M1 and the NS2 proteins are transported back into the nucleus through the nuclear pore complex and along with the newly synthesized negative sense RNA and forms the progeny viral RNAs. At the same time, the surface antigen M2, HA and the NA proteins are processed through different route. These proteins are produced in the rough endoplasmic reticulum and proceeds through the Golgi apparatus for maturation. These proteins are finally discharged onto the cell surface. The NA and HA proteins are transferred to the infected cell surface which will latter become the membrane envelope of the mature virions. The newly formed progeny RNPs are transported through the nuclear pore complex and reaches the cytosol. In the cytosol the newly transported RNPs undergo further assembly with the M1 protein. This process covers the RNPs with the layer of matrix proteins on which the M2 also assembles to form the ion channel. The provirions reaches the cell membrane where the HA and NA already formed a layer. The virus particles now begin to take a shape and gets ready to bud from the infected cells by taking a layer of cell membrane which is covered by HA and NA proteins (Murphy and Bang, 1952; Compans and Dimmons, 1969; Nayak et la., 2004; Palese et al., 1974; Burleigh et al., 2005). The mature virus particles released are now ready to undergo further infections.
The 1918 influenza pandemic among humans killed more people than the bubonic plague. Though many influenza outbreaks have been reported even after, the origin of the causative agent was not clearly understood until when the influenza virus was isolated in 1995. The virus responsible for the pandemic in 1968 consisted mixture of components from viruses which caused previous infection in humans and avian species. The highly pathogenic avian influenza viruses were isolated in 1996 from infected geese in the province of Guangdong, located in the southern part of china. The 1997 Hong Kong outbreak was clear enough to demonstrate the potential danger of highly pathogenic avian influenza viruses. Though there were many influenza infection in avian species have been reported in many parts of the globe, after 1997, there were no reported cases of human infection until 2003. In 2003, there were two new cases of human infections were reported in Hong Kong. At the end of 2003, another outbreak of HPAI infection was identified in chicken in South Korea, lasting until September of 2004. From 2004, the pandemic began to affect other countries such as Japan, Thailand, Indonesia, Cambodia, Laos and Malaysia. In the following year, new cases of avian influenza infections were identified in Russia, Mongolia, Kazakhstan, Kuwait and Turkey. Subsequently, the infection has started spreading to outside Asian countries including European countries such as England, Croatia, Romania and the Ukraine. In 2006, the pandemic began to spread to other parts of Asia and the Middle East including Iran, Iraq, Pakistan, India, Israel, Jordan and Afghanistan. The infection has spread to many European countries such as Bulgaria, Slovenia, Italy, Greece, Germany, France, Albania, Austria, Bosnia, Slovakia, Azerbaijan, Hungary, Serbia, Georgia and Montenegro, Switzerland, Denmark, Sweden, Poland, the Czech Republic and Spain. In Africa, the infection has been reported to have occurred in Nigeria, Egypt, Sudan, Djibouti and Cameroon. In 2007, Bangladesh, Ghana, Saudi Arabia, Myanmar and Benin also reported new cases in domestic poultry. Infection with HAPI H5N1 viruses in chicken is already considered endemic in China, Thailand, Cambodia and Laos. It is estimated that it will take several years to control the disease in these regions (WHO, 2008).
For many years, India was free from HPAI viruses till January 2006. In February 2006, the High Security Animal Disease Laboratory (HSADL) Bhopal reported to have diagnosed two H5N1 outbreaks in chickens, the first outbreak in the districts of Nandurbar and the second outbreak in Jalgaon of Maharashtra. Both the outbreaks showed high mortality among infected chicken population and lasted for a period of 12 days. In the first outbreak, the H5N1 virus was isolated from the cloacal swab of chickens while the virus was isolated from dead chickens in case of second outbreak. Both the isolates were identified to be of highly pathogenic avian influenza virus belonging to the H5N1 virus group. From the study, it was clearly evident that the H5N1 virus that caused outbreak in Jalgaon where the first outbreak occurred was from the HPAI virus which caused outbreak in Maharashtra. Thus, both the viruses may have introduced at two different times into populations and outbreak in Maharashtra did not originate from the Nandurbar outbreak (WHO, 2012).
Infections by low pathogenic avian influenza viruses are common among ducks and geese (Olsen et al., 2006). These infections usually results in milder to no clinical severity. On the other hand, the domestic poultry such as chicken and turkey have high rates of susceptibility to epidemics of HPAI with the mortality rate ranging from 90% to 100% (Alexander et al., 1978; Alexander et al., 1986; Narayan et al., 1969; Westbury et al., 1981; Westbury et al., 1979). The influenza viruses belonging to the subtype of H2, H5, H6, H7, H8, H9, and H10 are most likely to infect humans. H1N1 and H3N2 subtypes have caused milder to severe outbreaks in pigs while H7N7 and H3N8 in horses. The human influenza infections are routinely caused by subtypes belonging H3N2, H2N2, H1N1, and H1N2. The subtype H1N1 was responsible for the Spanish influenza pandemic of 1918 that killed 20 million people (Gary Adam Zeitlin and Melanie Jane Maslow, 2005).
The months for seasonal risk of influenza are November-April for the northern hemisphere, April-November for the southern hemisphere and year-round for the middle hemisphere. The influenza infections reach peak prevalence in winter which may be because the Northern and Southern Hemispheres have winter at different times of the year.
A major reason why outbreaks of the influenza occur seasonally rather than uniformly throughout the year is may be because the people stay close and indoor during the winter. This may help in promoting transmission from person to person. Another possible factor is that at low temperatures with relatively lower humidity, the virus harboring mucus gets dehydrated and thus preventing the body from effectively expelling virus particles. The lower air humidity in winter indeed seems to be the important cause of seasonal influenza transmission in temperate regions. However, in some countries the peaks of infection are often seen during the raining season. The HPAI H5N1 viruses exhibit seasonality in both birds and humans. An alternative hypothesis describes that reduced levels of vitamin D during winter may play a role on reduced immunity to the virus and hence increases the rate of disease transmission.
Epidemic and pandemic spread
Although the frequency of influenza infections can vary extensively between years, 36,000 deaths and more than 200,000 hospitalizations are associated with influenza each year in the United States. The genetic reassortment and minor mutations in the hemagglutinin and neuraminidase antigens causes minor to major changes on the surface of the virus resulting the evolution of HPAI influenza viruses with altered virulence. These genetic reassortments may result in either antigenic drift or antigenic shift which can result in novel and highly pathogenic strains of influenza viruses with modified host specificity, virulence and pathogenicity. These new variants often causes epidemic in avian and human population. However, the population having immune response for particular subtype can offer low to high level protection against new strains of avian influenza viruses depending upon the antigenic similarities. But, if the newer strain is completely new, the whole set of population will be susceptible and the strain will eventually spread uncontrollably, causing a pandemic. An alternative approach has been proposed in contrast to this model of pandemics based on antigenic shift and drift, where the sporadic pandemics are produced by exchange of a set of viral strains among the population with a persistently changing immunity levels against different strains.
Host range and specificity
Influenza A viruses have been isolated from a wide variety of animals, including seals, whales, pigs, horses, minks, different avian species as well as humans (Webster et al., 1992). Though the interspecies transmission occurs very rarely, few cases have already been reported. For instance, the H1N1 avian subtype (Scholtissek et al., 1983) and H3N2 human subtype (Tumova et al., 1980; Ottis et al., 1982; Mancini et al., 1985; Haesebrouck; Pensaert, 1988 and Wibberley et al., 1988) viruses have been reported to have transmitted to pigs. Similarly, the H1N1 swine viruses to humans (Rota et al., 1989) and the avian viruses to horses (Guo et al., 1992), seals (Webster et al., 1981; Hinshaw et al., 1984), whales (Hinshaw et al., 1986), and mink (Klingeborn et al., 1985) have also been documented. Experiments on transmission of avian influenza viruses to humans (Beare and Webster, 1991) and other primates (Murphy et al., 1982) have failed.
Similarly, the human influenza viruses did not replicate efficiently in waterfowl when introduced by natural routes (Hinshaw et al., 1983). On the other hand, the avian influenza viruses can be directly transmitted to humans, as evidenced by the recent occurrence in Hong Kong (Claas et al., 1998; Subbarao et al., 1998). But the probability of establishing such transmission is however lower. This indicates that the opportunities for the generation of human-avian reassortant viruses are limited. In 1985, Scholtissek et al., proposed that pigs may serve as `mixing vessels' for the development of reassortant influenza viruses. It was demonstrated by Hinshaw et al., in 1981 and Kida et al., in 1994 that upon experimental infection the avian and human influenza viruses can replicate efficiently in pigs.
The host range and species specificity are mainly influenced by the viral surface glycoproteins such as HA and NA. However, the internal proteins such as RNA polymerase (PB2, PB1, PA), nucleoprotein (NP), matrix proteins M1, M2 and the non-structural proteins (NS1, NS2/NEP) also contributes to the host range. Different avian influenza haemagglutinin proteins show different receptor binding preferences and hence the changes in HA protein contributes mainly for the host range and specificity. Human influenza viruses preferentially recognize SAÎ±2, 6Gal containing sialyloligosacchrides as avian viruses preferentially recognize SAÎ±2, 3Gal sialic acids (Rogers and Paulson, 1983; Rogers et al., 1983). Interestingly, the tracheal epithelial cells of pig contain both types of sialic acids and both (SAÎ±2, 6Gal SAÎ±2, 3Gal) types of linkages (Ito et al., 1998). This explains the high susceptibility of these animals to both human and avian influenza viruses and help the reassortment between human and avian viruses which eventually becomes the source of pandemic strains, however, there are no clear evidence exists that the 1957 or 1968 pandemic viruses originated in pigs (Kida et al., 1994). In addition, the HA cleavability also seems to have a role in pathogenicity and disease progression in animals.
Transmission of the disease
Wild waterfowl are the core natural reservoir of different strains of avian influenza A viruses. Most of the infected wild birds do not show any symptoms specific to influenza viruses but excrete large amounts of infectious virus. Hence, in most of the cases they act as "silent" reservoirs of the virus. The infected wild birds often become the source of transmission of influenza A viruses. Domestic waterfowl such as ducks acts as a common intermediary between wild waterfowl and domestic poultry in the transmission of avian influenza. Although generally the wild birds transmit low pathogenic avian influenza to domestic poultry, the virus may undergo one or more mutations during replication in the new host and become a highly pathogenic virus.
Infected birds shed large quantities of virus in their body fluids such as saliva, nasal secretions as well as in their faeces in the first two weeks of infection. The chances of infected, symptomatic or asymptomatic waterfowl and migratory birds to spread the infections through the secretions are huge because, these birds often share common water sources. The contaminated water bodies are the main source of disease transmission from the wild and migratory to domestic birds.
Transmission of avian influenza A viruses from animals to humans are usually rare and occurs either directly from exposure to infected domestic birds or indirectly through an intermediate host, such as a pig. Humans acquire infection most often through close and frequent contact with domestic birds while handling infected birds and cleaning their shed without proper personal safety (Berton and Teixeira, 2005; Brankston et al., 2007). However, any material belonging to the infected animal containing infectious virus can also become potential source for transmission (Cunha, 2004; Brankston et al., 2007). So far, there are no reported cases of a human avian influenza infection being transmitted by a mammal. The adaptation of avian influenza infection in humans is believed to happen through an intermediary species which is often pig (Cunha, 2004). However, it is also believed that the H5N1 virus adapts to humans through the genetic reassortment between the avian and human strains of the virus when a person is infected with both (Cunha, 2004; Taubenberger et al., 2005).
Pathogenesis of H5N1 virus
The site of avian influenza replication does not always begin at the route of entry of a pathogen. The site of influenza virus replication in mammals may vary and both viral and host factors are believed to influence tissue tropism (16). According to one study, more number of influenza viruses was isolated from the cloacae than from the trachea of domestic poultry. This indicates that the gastrointestinal tract may be the primary site of viral replication (2,17). But another study in ducks involved 23 different isolates from Asia found to have higher levels of influenza virus replication in the trachea than in the cloaca of both inoculated and contact birds which is suggesting that the trachea may be the primary site of H5N1 influenza virus replication. In humans, influenza virus replicates primarily in the respiratory tract. Diarrhea is one of the major symptoms in infected human population and the viral RNA was frequently isolated from faecal samples of infected humans suggesting that the H5N1 virus gastrointestinal tract (8, 18, 19) may also be the site for viral replication in humans.
Infection in lungs usually shows diffuse alveolar damage. Though, hyperplasia of type II pneumocytes was demonstrated in many of the autopsy samples, predominant appearance of macrophage cells within the alveoli, scattered histiocytes with hemophagocytic activity have also been observed in the lungs. In addition to desquamation of epithelial cells into alveolar spaces, cystically dilated air spaces, bronchiolitis, pleuritis, hemorrhage, apoptosis in alveolar epithelial cells and leukocytes and features of interstitial pneumonitis have also been documented. On the other hand, it may be difficult to differentiate the histopatholoigical features caused severe acute respiratory syndrome (SARS), coronavirus (SARS-CoV) as the above features are not unique to avian influenza infections. In such cases, specific tests such as reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and virus isolation assay are required to confirm H5N1 infection.
The low levels of T lymphocyte counts in infected animals can be attributed to prolonged H5N1 viral replication in pharyngeal region. Very high levels of chemokines and cytokines are often found to be present in peripheral blood of critically ill patients . The viral RNA was isolated up to 15-16 days from respiratory specimens [19, 37] and up to 27 days from trachea and lung autopsy specimens . The clinical specimens of humans infected with H5N1 viruses were found to have elevated levels of proinflammatory cytokines in primary alveolar and bronchial epithelial cells, and in macrophages [73-75]. Experimentally infected non-human primates showed severe lower respiratory disease with H5N1 viruses targeting type II pneumocytes [77, 78] and macrophages . But in macaques, alveolitis and severe necrotizing bronchiolitis were observed within 24 hours. In addition, interleukin (IL)-6 cytokinemia and disruption of the cell mediated antiviral response was also observed . It is already reported that elevated levels of cytokine induction by H5N1 virus often damages lung tissues. In three fatal cases of influenza virus infection, over-expression of cyclo-oxygenase (COX)-2 was found in pneumocytes and bronchial epithelial cells, but, not in alveolar macrophages . Inactivated H5N1 virus was reported to induce acute lung injury mouse alveolar macrophages. In addition, the inactivated virus induced respiratory oxidative stress was also observed in human peripheral blood monocytes . In a murine model, activated Toll-like receptor 4 and stimulated IL-6 production leading to inflammation and alveolar damage  was also documented with inactivated H5N1 virus. Elevated levels of oxidized phospholipids were found in inflammatory exudates that lined the injured air spaces and alveolar macrophages of lungs from H5N1 virus-infected patients with acute respiratory distress syndrome . Mice deficient in IL- 6, TNF-a, or the chemokine CCL2 or mice treated with corticosteroids died when infected with H5N1 virus . These findings in the pathogenesis of acute lung injury implicate prolonged viral replication and the induction of high levels of proinflammatory cytokines.
The complications of influenza infection are not restricted to but also extend to outside of the respiratory tract. Although in most of the autopsy samples the appearance of histiocytes has been found to be less prominent, reactive histiocytes with hemophagocytic activity have been identified in the spleen, bone marrow, lymph node, lungs, and liver. In case of severe influenza infection, the spleen displays white pulp atrophy and congestion with depletion of lymphoid cells. In addition to hemophagocytosis the lymph nodes show loss of germinal centers and focal necrosis in few cases. However, acute tubular necrosis has been reported in many cases. Lymphocytes undergoing apoptosis have been detected in both intestinal and spleen tissues. Tissue necrosis, cholestasis, activated Kupffer cells and fatty acid changes have been observed in liver tissue specimens. Brain edema without any significant histopathological changes have been observes in several cases, but, demyelinated areas, reactive histiocytes and foci of necrosis have also been reported in two cases. No remarkable histological changes have been observed in other organs.
The viral RNA was isolated from blood, plasma and serum samples of prolonged and severely infected patients and such cases are often associated with 100% mortality. In contrary to non-fatal seasonal influenza infections, few fatal cases of H5N1 virus infection had higher pharyngeal viral loads and viral RNA was also detected in blood samples . The presence of influenza virus in cerebrospinal fluid specimen with encephalitis was associated with fatal infections . H5N1 viral antigen and RNA, or segments of nucleic acid were detected in extrapulmonary tissue specimens such as cerebral neurons, lymph node T cells, bone marrow, astrocytes, the small and large intestines [19, 84].
A number of immunohistochemistry studies with monoclonal antibodies to hemagglutinin (HA) and nucleocapsid protein (NP) and/or in situ hybridization with sense and anti-sense probes to HA and NP were undertaken to detect genomic sequences and viral antigens in various organs. In addition, to investigate virus tissue tropism, strand-specific RT-PCR, RT-PCR, nucleic acid sequence-based amplification and H5 detection assays have also been performed. Early studies on H5N1 infection appeared to be restricted only to the lungs recent studies however, indicate that the virus disseminates beyond the respiratory tract.
The influenza genomic sequences and few viral antigens have been found in epithelial cells of alveoli and both ciliated and nonciliated cells of trachea. The alveolar cells were identified with antibodies to surfactant protein while tracheal epithelial cells were identified with antibodies to tubulin. Positive-stranded RNA has been detected by RT-PCR and nucleic acid detection based on H5 detection assays in tissue samples of both the trachea and lungs. These findings suggest active viral replication at trachea and lungs.
Gene segments belonging to the influenza virus and viral antigens have been found in neurons of the brain. The H5N1 virus has been isolated from cerebrospinal fluid of infected humans. From brain tissue samples, negative- and positive-stranded RNA of influenza virus have been isolated by RT-PCR. Based on studies in mouse model, the virus was found to disseminate from lungs to the central nervous system via blood or via the afferent fibers of the olfactory, vagal, trigeminal, and sympathetic.
The in situ hybridization revealed the presence of viral genomic sequences in epithelial cells of the intestine. Both negative- and positive-stranded RNA were detected in the intestines by RT-PCR. These findings are consistent with frequently observed clinical symptoms related to the gastrointestinal tract. However, viral antigens could not be detected in samples from the intestines.
In the placenta of a female infected with influenza virus, viral antigens and gene segments have been found in Hofbauer cells and cytotrophoblasts, but not in syncytiotrophoblasts. In addition to RT-PCR, in situ hybridization, immunohistochemistry and real-time RT-PCR also confirmed influenza infection of the fetus, demonstrating that the virus is vertically transmissible from mother to fetus. Vertical transmission may take place either via transcytosis across syncytiotrophoblasts to cytotrophoblasts in chorionic villi or via invasive cytotrophoblasts within the uterine wall after contact with maternal blood. These cells would subsequently transmit the virus via the cell columns to the anchoring chorionic villi and may then be transmitted to Hofbauer cells of the fetus.
Influenza viral gene fragments and antigens have been detected in Hofbauer cells as well as in lymphocytes in lymph node tissue, Kupffer cells and mononuclear cells in the intestinal mucosa and are consistent with in vitro experiments demonstrating infection of macrophages by the influenza virus and with ex vivo experiments showing virus attachment to and infection of alveolar macrophages in human lung tissue. Viremia was evidenced by virus isolation from serum and plasma samples and extra-pulmonary dissemination may be the result of viremia or of infected immune cells transporting the virus to other organs.
Host immune responses
Both mucosal and systemic immunity contributes to protection against influenza infection and disease. The secretory IgA antibodies in the upper respiratory tract are a key factor involved in protection of the upper respiratory tract and serum IgG contributes to protection of the lower respiratory tract during natural infection. The immune response induced either by infection or by vaccination protects against re-infection with the same virus or an antigenically similar viral strain. Influenza viruses undergo frequent and unpredictable changes. Therefore, the effective period of protection against one strain of influenza virus provided by the host's immunity may help only for similar strains for a limited period of time or till the next infection by newer strains of influenza viruses. The humoral immune response, including both the mucosal and systemic immunity, plays a major role in immunity to influenza infection while the cell-mediated immune response helps particularly in clearing virus-infected cells.
During influenza infection, the animal develops antibody response to almost all viral proteins. Antibodies to major surface glycoproteins, HA and NA are contributes resistance to infection, while antibodies to the conserved internal antigens such as M and NP are not protective. The structural antigens HA and NA primarily induces humoral immune response and the M and NP proteins induce cytotoxic T-cell response. Although the T cell response against M and NP does not confer protection against infection, it is appeared to be important for the clearance of virus and help recovery from illness. Neutralizing antibodies against major structural antigen HA plays vital role in protection against the infection whereas antibodies to structural antigen NA do not confer protection in animals but helps in controlling the disease progression.
Apart from innate immunity, the mucosal immune system offers the first line of protection against infection. Secretory IgA and, to some extent, IgM particularly to influenza HA and NA are the major antibodies helps in inhibiting viral replication. It was observed during primary infection that, the IgG and IgM are predominant whereas the IgA and IgG are the dominant Ig classes in secondary infection. Local IgA response can be found in case of primary and secondary infection. However, protection in animals is always correlated with the presence of neutralizing antibodies against HA and NA antigens in serum and is mainly belonging to IgG classes. The amount of these antibodies is often measured to correlate the protection against influenza. In humans, the level of serum neutralizing antibody to HA and NA can be correlated with resistance to disease following experimental and natural infection. During primary infection, IgA and IgM levels peak after 2 weeks and then begin to decline, whereas the level of IgG peaks at 4-6 weeks.
The cell-mediated immunity does not seem to play a significant role in protection against infection but appear to plays a major role in recovery from influenza infection. The secretions of the lower respiratory tract and blood found to have influenza-specific lymphocytes during infection. In infected or vaccinated individuals, the primary cytotoxic response associated with the infection is detectable in blood after 6-14 days and disappears by day 21. The cytotoxic T lymphocytes specific to Influenza do not exhibit cross reactive specificities and hence they lyse cells infected with the same type of influenza but not with other types. The cytotoxic T lymphocytes specific to the Matrix protein and the internal proteins such as NP and PB2 have significant variations in the reactivity pattern between subjects.
The memory cells of cytotoxic T lymphocytes exhibit a cross reactivity pattern similar to primary response and reaches peak levels at day 14 and return to baseline after six months. Following experimental influenza infection it was understood that the amount of memory cells to influenza does not correlate with susceptibility to infection or illness, but it does correlate with the rate of viral clearance from the respiratory tract. Influenza infection also induces a strong T-helper response, which plays an important role in stimulating antibody production against the viral antigens. The CTL response is cross-reactive between influenza A strains and plays an important role in minimizing viral spread in combination with neutralizing antibody.
Laboratory diagnosis of influenza H5N1
Virus isolation from secretions of infected animal not only indispensable for virus characterization but also remains the gold standard of diagnosis. However, rapid confirmation of suspected influenza infection in routine, diagnostic laboratories is usually targets the detection of influenza virus antigens by immunochromatographic, immunofluorescent and viral nucleic acids by RT-PCR detection in respiratory specimens. In addition, commercial ELISA kits based on the detection of antibodies to conserved nucleoprotein antigens are available. Further subtyping of influenza antigens or detection of their specific antibodies, in the absence of circulating avian influenza strains in the population, are usually not performed at routine diagnostic laboratories, but are analyzed only at reference laboratories involved in epidemiological studies. However, in addition to diagnosis, the subtyping of influenza virus should also be performed at the routine diagnostic laboratories since immediate knowledge about the infecting influenza subtype is essential for implementation of quicker control strategies and for epidemiological investigations. Most of the diagnostic reference laboratories are situated in Southeast Asian countries and hence that may potentially results in unacceptable delays and hampers timely recognition of outbreaks and delays the implementation of adequate control measures (Hien et al., 2004). However, many affected countries are often not sufficiently equipped for virological diagnostics.
Virus isolation from samples of infected animals is a superior diagnostic methodology. The usual method for avian influenza virus isolation is performed by infection in embryonated eggs or in cell culture, using Madin Darby canine kidney (MDCK) cells or rhesus monkey kidney (LLC-MK2) cells from which the virus could be readily isolated. Unlike low pathogenic avian influenza viruses, the highly pathogenic avian viruses do not require the addition of exogenous trypsin for efficient replication in cell culture and they use cellular proteases for the cleavage of HA. Hence, the isolation of highly pathogenic avian influenza virus requires biosafety level 3 or higher. However, the virus isolation requires further characterization for the effective identification the subtype. The cytopathic effects of influenza viruses in cell culture are non-specific and hence, initial identification of influenza virus can be performed by immunostaining with fluorescent labeled monoclonal antibodies against the nucleoprotein. In addition, the subtyping of HA and NA antigens using haemagglutination and neuraminidase inhibition assays using a panel of reference antisera against various subtypes and subtype-specific RT-PCRs of culture supernatant can help identify the subtype. In case of human infections, the viruses have mostly been isolated from conjunctival swabs and respiratory specimens such as throat and washings or nasal secretions (Fouchier et al., 2004; Tran et al., 2004; Yuen et al., 1998). In case of one reported H5N1 infection, the virus was also isolated from cerebrospinal fluid, serum and a rectal swab (de Jong et al., 2005).