Tweet
Avian Influenza virus program - Virginia-Maryland Veterinary Medicine
<TABLE height=2161 cellSpacing=0 cellPadding=0 width=700 bgColor=white border=0><TBODY><TR height=875><TD vAlign=top width=759 height=875>
Our mission at the Avian Influenza Virus Program at the Virginia-Maryland Regional College of Veterinary Medicine is to determine the molecular basis for interspecies transmission and pathogenesis of avian influenza viruses.
Our Program is located in the Department of Veterinary Medicine at the Avrum Gudelsky Bldg.,College of Agriculture and Natural Resorces, University of Maryland, College Park Campus.
Research Interests
The molecular basis of interspecies transmission, pathogenesis, and cross-protection of influenza A viruses.
Aquatic birds are the natural reservoir of influenza A viruses. Occasionally, some of these viruses cross to other animal species, in which they can cause severe disease outbreaks that include pandemics. In the 20th century, humans experienced three major pandemics. The most devastating was the 1918 "Spanish flu" that killed between 20 and 40 million people. The emergence of another influenza pandemic is considered imminent. Unfortunately, little is known about the molecular features that allow an influenza A virus to cause pandemic disease, although multiple genes, encoding both the surface and internal proteins of the virus, are thought to be involved. We have recently developed a plasmid-based reverse genetics system for influenza A viruses that allows the complete manipulation of the viral genome. We are currently using this system to map specific amino acid residues involved in interspecies transmission of H9, H2 and H7 influenza viruses, which are believed, along with H5 influenza viruses, to have the greatest potential to become pandemic viruses. Our studies are elucidating the most important factors in the spread and pathogenesis of influenza viruses from the aquatic bird reservoir to land-based birds and mammals.
Want to know more? Check the background information below and our publications. Click on the icons on the left for more information about resources and our program.
The information below has been extracted from the book "Biodefense: Principles and Pathogens" edited by M.S. Bronze and R.A. Greenfield, chapter 21 "Miscellaneous threats: Highly pathogenic avian influenza, and novel bio-engineered organisms." by D.R. Perez, S.H. Nazarian, G. McFadden, and M.S. Gilmore. Copyright ? 2005, Horizon Bioscience, Norfolk, England.
Influenza infections have been recognized in humans since ancient times. The name influenza refers to the ancient belief that it was caused by a malign and supernatural influence (Latin influentia). Year-to-year epidemics in humans, recurrent epidemics in swine and horses, sporadic cases in minks, seals and whales, and devastating outbreaks in poultry constantly remind us that influenza is a non-eradicable disease.
Highly Pathogenic Avian Influenza
Avian influenza was defined as ?fowl plague? in 1878 as a disease causing high mortality in chickens in Italy (Perroncito, 1878). It was not until 1955 that it was demonstrated that fowl plague was an avian influenza virus whose genomic composition was virtually identical to the one found in the human influenza virus (Schafer, 1955). By then it was already shown that human influenza viruses, identified as a virus in 1933, (Smith et al., 1933) shared with their avian counterparts many biological properties including the ability to grow in chick embryos and agglutinate red blood cells (Hirst, 1941). The terminology ?highly pathogenic avian influenza? was officially adopted in 1981 at the First International Symposium on Avian Influenza to designate the highly virulent forms of avian influenza. The Office International des Epizooties (OIE) that codifies sanitary and health standards, and is affiliated to the World Trade Organization (WTO), has included HPAI as a List A reportable disease (OIE, 2003). Countries with representation in the WTO are obliged to report outbreaks of HPAI. Any AI virus that fits into either one of the following criteria is considered an HPAI virus
1) Lethal for six, seven, or eight 4 to 6 week-old chickens within 10 days following intravenous inoculation with 0.2 ml of a 1:10 dilution of virus in a bacteria-free, allantoic fluid.
2) It has a polybasic amino acid region at the hemagglutinin cleavage site and is of the H5 or H7 subtype (see below).
3) It is not an H5 or H7 virus but kills one to five chickens and grows in cell culture in the absence of trypsin.
Etiology
The influenza A viruses belong to the family Orthomyxoviridae (Lamb, 1989). Other members of the family include influenza viruses type B and C, the insect viruses Togotho and Dhori, and Salmon Anemia virus. Influenza A virions are 80-120 nm in diameter. The influenza viruses are relatively unstable in the environment. Heat, extreme changes of pH, or nonisotonic conditions and dryness can readily inactivate the influenza viruses. The virus has an envelope with a host-derived lipid bilayer and covered with about 500 projecting glycoprotein spikes with hemagglutinating and neuraminidase activities. These activities correspond to the two major surface viral glycoproteins: the hemagglutinin (HA) and neuraminidase (NA), present as homotrimers and homotetramers, respectively. Within the envelope, a matrix protein (M1) and a nucleocapsid (NP) protein protect the viral RNA (Lamb, 1989). The type designation (A, B, or C) is based upon the antigenic features of the M1 and NP proteins. Eight segments of single stranded RNA of negative polarity, totaling approximately 14 kilobases, encode for at least 10 viral proteins (McGeoch et al., 1976). Approximately half of the total genome encodes for the three viral polymerase proteins (segments 1, 2 and 3; (Palese et al., 1977). Segment 5 encodes the NP protein. The three-polymerase subunits, the NP and the vRNA are associated in virions and infected cells in the form of viral ribonucleoprotein particles (vRNPs). Segments 4 and 6 encode for the HA and NA genes, respectively. The two smallest segments (7 and 8) encode two genes each with overlapping reading frames, which are generated by splicing of the co-linear mRNA molecules (Lamb and Lai, 1980; Lamb and Lai, 1984; Lamb et al., 1981). In addition to M1, segment 7 encodes for the M2 protein, which has ion channel activity and is embedded in the viral envelope. Segment 8 encodes for NS1, a nonstructural protein that blocks the host?s antiviral response, and NS2 or NEP that participates in the assembly of virus particles.
Brief Overview of the Influenza Life Cycle
Influenza virus attachment to the susceptible cell is mediated by the interaction between the viral hemagglutinin and sialic acid receptors present on glycolipids and glycoproteins on the cell surface (Lamb, 1989). At this stage, the sialidase activity of the neuraminidase prevents binding of the HA to sialic acids present in mucopolysaccharadies, which would otherwise interfere with the virus binding to the adequate cellular receptors. The virus is internalized by endocytosis and, upon acidification of the endosome, conformational changes on the hemagglutinin lead to the fusion between the viral and the endosomal membranes (Lamb, 1989). Acidification of the endosomal lumen also activates the ion channel activity of the viral membrane protein M2 (Pinto et al., 1992). Activation of M2 generates an inward current of protons into the virion?s interior that triggers the disassembly of M1 from the vRNPs, which are transported to the nucleus, the site of influenza virus transcription and replication (Holsinger et al., 1994; Martin and Helenius, 1991a). A minimal set of four viral proteins is essential for influenza virus transcription and replication: PB1, PB2, PA - referred to as P-proteins-, and the NP protein (Huang, Palese, and Krystal, 1990). Two different populations of positive sense RNAs are synthesized from vRNA templates: messenger RNAs (mRNAs) and complementary RNAs (cRNAs). Viral mRNAs are primed by 5? capped (m7GpppNm-containing) fragments derived from newly synthesized host-cell RNA polymerase II transcripts (Beaton and Krug, 1986; Krug et al., 1989; Plotch et al., 1981; Ulmanen et al., 1983)Viral mRNAs are polyadenylated by a stuttering mechanism involving the viral polymerase and a stretch of uridines, which are located 17-22 nucleotides before the 5? end of the vRNAs (Hay et al., 1977; Robertson et al., 1981); Synthesis of cRNA is the first step in influenza virus replication. Transcription of cRNAs occurs in the absence of primer or polyadenylation and they represent full-length copies of vRNAs (McGeoch et al., 1976). The second step in viral replication is the synthesis of progeny vRNA molecules from cRNAs templates (McGeoch et al., 1976). Towards the end of the infection cycle and once enough molecules of M1 and NEP have been produced, the newly synthesized vRNPs are exported out of the nucleus and assembled into full virus particles. The final assembly steps occur at the plasma membrane exposing the newly synthesized hemagglutinin, neuraminidase proteins, and M2 (Helenius, 1992.) Once the final assembly events are completed, new virus particles bud from the plasma membrane. The activity of the neuraminidase becomes again important by disrupting viral aggregates and thus releasing viral particles that can start a new cycle of infection.
</TD></TR><TR height=875><TD vAlign=top width=759 height=875>
Natural Reservoir of Avian Influenza The antigenic diversity of influenza A viruses occurs primarily at the surface glycoproteins. Differences of up to 80% have been found in these proteins (Wharton et al., 1989). The viral glycoproteins are classified into different subtypes on the basis of their reaction with hyperimmune sera using the letters H and N with the appropriate numbers in the virus designation e.g., H5N1. Among the influenza A viruses, 15 hemagglutinin and 9 neuraminidase subtypes have been described. (Wharton et al., 1989). Wild aquatic birds (Orders Anseriformes and Charadriiformes) are the major natural reservoir of influenza A and from which viruses corresponding to all15 HA and 9 NA subtypes have been isolated, reviewed in (Alexander, 2000). Given the segmented nature of their genome, influenza A viruses easily exchange genomic information, a process known as genetic reassortment. Thus, when a single cell is infected by two strains of influenza A with different antigenic subtypes reassortment can occur, further increasing the diversity of these viruses. Influenza A viruses in many different combinations of HA and NA subtypes have been isolated from wild and domestic birds supporting the notion that reassortment occurs freely in nature (Hinshaw et al., 1979). The intestine is the primary site of replication of influenza A virus in waterfowl, where the infection is usually asymptomatic and large quantities of viral particles are excreted with the feces into the water, perpetuating a cycle of oral-fecal transmission (Slemons and Easterday, 1977; Slemons and Easterday, 1978).
Genesis of HPAI
It is commonly accepted that migratory waterfowl, including ducks, sea birds, or shorebirds are responsible for introducing AI into domestic poultry (Alexander, 2000). Some of these viruses can establish stable lineages in terrestrial birds (Order Galliformes) and a limited number of mammalian species (Scholtissek, 1994; Scholtissek, 1995; Scholtissek, 1997; Webster, 1997). All highly pathogenic avian influenza viruses (HPAI) identified to date differ from their low pathogenic (LPAI) counterparts in the susceptibility of the HA to host proteases. HPAI are characterized by HAs that are highly susceptible to cleavage by numerous cellular proteases, which are ubiquitous in many cell compartments and organ systems. In contrast, the LPAI HA requires specific active extra-cellular proteases ?such as trypsin- for cleavage and activation of infectivity. Trypsin-like proteases are restricted to the lumen of the respiratory and intestinal sites.
Theoretically, viruses of all HA subtypes have the potential to cause disease. HPAI viruses have been historically restricted to only two HA subtypes, H5 and H7; although not all viruses that belong to these two subtypes are highly pathogenic. Viruses with any other subtype can cause a mild, primarily respiratory disease in poultry, which may be exacerbated by other infections or environmental conditions. An H5 or H7 virus can start out by presenting itself as a low pathogenic virus but can mutate without warning to become highly pathogenic. Unlike other HA subtypes, the highly virulent H5 and H7 viruses possess multiple basic amino acids at the cleavage site of the HA, that increase the spectrum of proteases that recognize the site (Swayne and Suarez, 2000). The cleavage of HA can also be modulated by the presence of additional glycosylation sites spatially located nearby. For example, a loss of a glycosylation site at amino acid 13 and the presence of the polybasic amino acid region in the HA are pathotypic markers of the H5N2 virus that caused the 1983-1984 outbreak in Pennsylvania (Banks and Plowright, 2003; Kawaoka et al., 1984). The exact contributing factors that lead to the accumulation of the polybasic amino acid region at the HA cleavage site remain largely unknown, although it is postulated that viral adaptation to terrestrial birds plays a major role.
Manifestations of HPAI
The most virulent forms of HPAI are characterized by a highly fatal systemic infection that spreads to most organ systems including the cardiovascular and nervous systems (Acland et al., 1984; Gross et al., 1986; Perkins and Swayne, 2002a; Perkins and Swayne, 2002b; Perkins and Swayne, 2003; Slemons et al., 1991). Morbidity and mortality can be as high as 100%, particularly in gallinaceous species. The incubation period is usually between 3 to 7 days depending on the virus isolate, age of the bird and bird species. Death may occur within 24 to 48 hr after the onset of symptoms but it may be delayed for as long as one or two weeks. In chickens, the initial stages of HPAI infections are usually accompanied by marked depression with ruffled feathers, loss of appetite, excessive thirst, watery bright green diarrhea, and markedly lower egg production. Adult chickens frequently have swollen combs, wattles, and edema surrounding the eyes. Cyanosis is often observed at the tips of the combs and it is not uncommon to observe plasma or blood vesicles on the surface with dark areas of hemorrhage and necrotic foci. Laid eggs are frequently without shells. Edema of the head and the neck are usually observed. The conjunctivae are congested and swollen with occasional hemorrhage. Areas of diffuse hemorrhage may be observed on the legs, between the hocks and feet. Respiratory syndrome and mucus accumulation can be significant characteristics of the disease. Some severely affected hens may occasionally recover, although neurological sequelae may be observed, such as torticolis and ataxia. The disease in turkeys, ducks, geese, and quail is similar to that seen in chickens, but it may last 2 to 3 days longer and is occasionally accompanied by swollen sinuses. Mortality rates may be lower in ducks and geese than those observed for chickens or turkeys, although younger birds may exhibit neurologic sequelae (Swayne and Halvorson, 2003).
Histologic lesions may not be apparent in birds that died soon after the onset of the infection other than severe congestion of the musculature and dehydration. However, in birds that survive the acute form of the disease, significant gross lesions are observed, although they may resemble those observed with velogenic viscerotropic Newcastle disease (NDV), and thus, they cannot be used for differential diagnosis of HPAI. During post-mortem examination, fluid and mucous exudates from the nares, oral cavity, the trachea, and subcutaneous edema of the head and neck may be observed. The conjunctivae are severely congested, often presenting petechia. Pinpoint petechiae are frequently observed on the inside of the keel, the abdominal fat, serosal surfaces, and peritoneum. Kidney function is usually severely compromised, with tubules that are plugged with white deposits of urate. In layers, the ovary may be hemorrhagic or degenerated with darkened areas of necrosis. In layers that survive longer than a week, the ova are ruptured and the yolk fills the peritoneal cavity causing severe airsacculitis and peritonitis. Hemorrhages may be present on the mucosal surface of the proventriculus and underneath the gizzard. The intestinal mucosa may have hemorrhagic areas, especially in the cecal tonsils and other lymphoid foci (Swayne and Halvorson, 2003).
Transmission and Control of HPAI
Since ancient times, humans have altered the natural ecosystem and created new ones for many animal species including avian species. By domestication and captivity of many bird species, humans have exposed themselves to the potential biohazards carried and/or transmitted by these animals. Such is the case of diseases like Psittacosis (parrot fever), Campylobacteriosis, Salmonellosis, and Avian Tuberculosis, to name a few. Humans have created niches where disease agents have the chance to jump from natural to non-natural hosts. Such is the case of AI in its many virulent forms. As mentioned earlier, live bird markets have played a major role in the emergence of HPAI. The live bird markets in the East Coast of the U.S, in Hong Kong, and Italy, to name a few, have been the originators of very important outbreaks of HPAI that extended beyond their borders and were brought into vast regions of their respective countries involving many small and large poultry production facilities and farms. Once introduced into a flock, it is very easy to spread the virus from flock to flock if not adequate biosecurity measures are taken. Movement of infected birds, excretions from infected birds, if carried in contaminated equipment, clothing, hands, shoes, egg flats, feed trucks, water, and food are all major contributors in the spread of the virus. GIS and retrospective studies have shown how HPAI viruses can be transmitted from farm to farm simply by the movement of trucks and personnel that work in different farms (Ehlers et al., 2003; Valleron and Vidal, 2002). Airborne transmission may occur but it is rather limited to birds that are in close proximity.
Proper sanitation and biosecurity cannot be over emphasized; they are the first line of defense against AI. Thus, all methods for preventing and controlling the spread of AI are related to controlling the contamination of equipment and personnel. Most HPAI viruses start out from non-virulent AI forms, so efforts should be made to control AI before it is too late. Since wild birds are considered the major source of infection for domestic poultry, particularly those in live bird markets and those raised on open range, it is essential to reduce the contact between these two groups. Appropriate education programs, in different languages and/or dialects, are needed to help people of different ethnic groups become aware of the risk factors involved in having flocks infected with AI. At the industrial level the incorporation of education programs about AI and its risks, monitoring, reporting and ?responsible response? initiatives as they were implemented in Minnesota, should be imitated in other states and countries (Poss et al., 1987). Prevention programs should include some form of incentive to growers, dealers, and retailers, to report suspected AI cases, which would help to contain infected birds to small areas.
Epidemiological surveillance is the second line of defense against AI. Surveillance efforts during the 1983-1984 outbreaks in Pennsylvania were able to track the origin of the virus to live bird markets. Likewise, the constant monitoring of influenza activity in the live bird markets of Hong Kong helped to quickly characterize the 1997 H5N1 virus that transmitted to humans from chickens (Sims et al., 2003). During the recent H7N2 outbreak in Virginia in 2002, which led to the culling of more than four million turkeys and chickens, it was possible to track the origin of the virus to live bird markets of New York and New Jersey, where the virus is endemic (Bulaga et al., 2003; Spackman et al., 2003; Suarez et al., 2003). Although the 2002 H7N2 virus was of low pathogenicity (no polybasic amino acid region in the HA cleavage site), eradication efforts were implemented to prevent further spread and the possibility that the virus would become highly pathogenic. Adequate emergency response plans must be in place in every state where poultry represents an economically important commodity. State and federal authorities are responsible for developing surveillance, quarantine, stamping out, and indemnification programs and policies. Additional information and recommendations about biosecurity, surveillance, and reporting AI activity can be found at the Animal and Plant Health Inspection Service, United States Department of Agriculture (APHIS-USDA).
Economic Importance
The economic losses associated with HPAI have been of varying importance depending on the magnitude of the outbreak and the measures taken to eradicate the virus. Epizootics of HPAI that have expanded into commercial farms or into large live bird market systems have resulted in the greatest economic losses. For instance, during the 1983-1984 H5N2 outbreak in Pennsylvania, it cost the U.S. government more than $60 million to eradicate the disease, including $40 million in indemnities (Swayne and Halvorson, 2003). Producers had to absorb an additional $15 million in non-indemnified losses. Consumers, on the other hand, had to overcome approximately $350 million in increased food costs. More recently, the eradication of the H7N1 virus responsible for the HPAI outbreak in Italy in 1999-2000 carried approximately $100 million in compensations and total indirect losses have been calculated in excess of $500 million (Swayne and Halvorson, 2003). Without any doubts, the massive HPAI epidemic of H5N1 Asia dwarfs previous figures: More than 100 million birds have either died or been killed to contain the outbreak. For Southeast Asian countries, the economic implications of the 2004 H5N1 virus are tremendous. Besides the time it can take to eradicate the virus, it will take several years until some of these countries can restore export of their poultry products. China and Thailand, which are the two most important poultry exporters in Asia, have been particularly hit with the outbreak.
Public Health Significance
In recent years HPAI infections have taken a new dimension after the realization that some of these viruses have acquired the capacity to transmit directly from birds to humans, with the consequent potential for causing a pandemic. During the 20th century, humans experienced three influenza pandemics: the 1918-1919 influenza pandemic known as ?Spanish flu?, which killed approximately 40-50 million people, and the pandemics of 1957 ?Asian flu? and the 1968 ?Hong Kong flu?. In each instances, pandemics have been characterized by the emergence of new HA subtypes, a process known as antigenic shift. There are basically two mechanisms that can result in antigenic shift: transfer of an avian virus in toto or through reassortment with a human influenza virus. Transfer in toto is thought to have occurred during the pandemic of 1918, the virus carrying the H1N1 subtypes. Antigenic shift through reassortment occurred during the 1957 and 1968 pandemics. The 1957 H2N2 pandemic virus was a reassortant that inherited three genes (HA, NA, and PB1) from the avian influenza pool and the other genes from the H1N1 human influenza virus circulating at that time. The 1968 H3N2 virus inherited two genes (HA and PB1) from an avian influenza virus and the rest from the H2N2 virus circulating at that time. Pandemics occur because the human population has no immunity against a new virus subtype that can be transmitted efficiently from human to human. Once in the human population, the virus perpetuates itself by evading the immune system through small antigenic changes, a process called ?antigenic drift.? Currently, two influenza subtypes circulate in the human population: H3N2 and H1N1, the latter re-introduced in humans in 1977 and named ?Russian flu? (although the virus may have originated in China or Mongolia). Interestingly, the 1977 H1N1 virus was almost identical to the H1N1 virus that circulated until 1957. An accidental laboratory release or the perpetuation of the virus in a different animal host have been proposed as potential causes for the re-emergence of the 1977 H1N1 virus. The occurrence of influenza pandemics is unpredictable; however co-mingling of avian and mammalian species is considered a potential factor in the emergence of pandemic viruses. Pigs have been proposed as potential intermediate hosts or ?mixing vessels? where avian and human influenza can reassort, although humans could act as mixing vessels themselves. In addition, other animal species, particularly terrestrial birds, have been proposed as potential ?amplifiers? of avian/human reassortant influenza viruses. Historical evidence suggests that Southeast Asia is an epicenter of influenza pandemics, although a pandemic virus could emerge virtually from any place where humans come in contact with carriers of AI. Of the 15 avian influenza virus subtypes, H5N1 is of particular concern because of the propensity to infect humans. Its ability to cause severe disease in humans has now been documented on three occasions. In 1997, 18 people became infected with an H5N1 virus during an outbreak of HPAI in Hong Kong. Patients developed symptoms of fever, sore throat, and cough and, in several of the six fatal cases, severe respiratory distress secondary to viral pneumonia. Previously healthy adults and children, and some with chronic medical conditions, were affected. Culling of more than 1 million poultry is thought to have prevented new cases. However, there is evidence that H5N1 viruses may have become endemic in Southeast Asia. H5N1 outbreaks repeated in Hong Kong in 2001, and 2002-2003, even though several preventative measures have been taken: The separation of aquatic and poultry species in different wholesale markets, the prohibition of selling live aquatic birds in retail markets, and the incorporation of monthly rest days, where markets are completely emptied and disinfected before new birds are brought in. In February 2003, two new human cases of H5N1 where documented in Hong Kong, one of them fatal. In mid-December 2003, an outbreak of H5N1 initiated in poultry in South Korea and, by early January 2004, quickly spread to Japan, Taiwan, Viet Nam, Thailand, and China. In Viet Nam, more than 20 people died of respiratory disease and, in at least five of these cases, an H5N1 virus caused it. In Thailand, fewer human cases were reported but they have been mostly lethal (6 out 8). Culling of poultry has been implemented in major live bird markets and big poultry operations in all these countries to prevent new cases. In addition, H5N1 viruses have a propensity to reassort with influenza viruses circulating in other animal species. Therefore if it reassorts with a human virus it may gain the capacity for human-to-human transmission and cause a pandemic. Birds that survive infection excrete virus for at least 10 days, orally and in feces, thus facilitating further spread at live poultry markets and by migratory birds.
Other viruses that are of pandemic concern are H9 and H7 viruses, which in recent years have also crossed to humans. In 1999 in China, 7 cases of bird-to-human transmission of H9N2 viruses were documented. Individuals infected with the H9N2 viruses have mild respiratory symptoms and fully recovered. In 2003, a new episode of H9N2 virus was recorded in one patient with mild respiratory distress. Interestingly, H9 viruses are endemic in poultry in Asia and display typical human-like receptor specificity, binding preferably sa-a2, 6-gal receptors (as opposed to the sa-a2, 3-gal receptor binding of waterfowl influenza viruses, Matrosovich et al., 2001). In early 2003, an HPAI H7N7 outbreak occurred in poultry in the Netherlands. Bird-to-human transmission of the H7N7 virus occurred in at least 82 cases. Conjunctivitis was the most notorious disease symptom in people infected with the H7 virus, with only 7 cases displaying typical flu-like illness. Fortunately, the H7 was not highly pathogenic for humans and only one fatal case was observed. Other viruses with pandemic potential are H2, because of its past history as a pandemic virus, and H6 because of its high incidence in poultry species in Asia and North America.
A major challenge for scientists and public health officials is to avert influenza pandemics. Is it possible? The same measures taken to prevent the spread of AI in the poultry population can reduce opportunities for human exposure to the virus. In addition, vaccination of persons at high risk of exposure to infected poultry, using existing vaccines effective against currently circulating human influenza strains, can reduce the likelihood of co-infection of humans with avian and influenza strains, and thus reduce the risk of reassortment. Workers involved in the culling of poultry flocks should wear protective clothing and also receive antiviral drugs as a prophylactic measure. The World Health Organization, through its Global Influenza Surveillance Program, together with other national and international agencies, assists in reducing the risks of AI for public health. It remains to be seen whether all these activities will effectively avert the emergence of a pandemic strain.
HPAI as a bioweapon
The accidental or intentional introduction of HPAI into a flock can have devastating consequences as discussed earlier. Fortunately, the same characteristics that are hallmarks of HPAI can be used for the early detection of the virus and develop an efficient and rapid response to contain it. More problematic is the introduction of the low pathogenic forms of H5 and H7 viruses since they have the potential to become HPAI; however can be spread sometimes without significant or noticeable signs of disease. Outbreaks of HPAI have been caused by naturally occurring highly pathogenic forms of the disease. Attempts in the laboratory to reproduce the generation of HPAI from non-virulent forms have proven difficult at best. In recent years, a new technology known as reverse genetics has allowed the generation of influenza viruses entirely from plasmid DNA. Such technology allows for the complete manipulation of the influenza virus genome. The construction of tailor-made influenza viruses can be used to pinpoint the viral markers for pathogenesis and transmission. The plasmid-based system shows also promise for the rapid generation of vaccines for pandemic preparedness, although at least four months would be needed to produce a new vaccine, in significant quantities, capable of conferring protection against a new virus subtype. Certainly, this same DNA technology could be used to generate a virus with undesirable features; however genetic engineering of potentially harmful influenza viruses is in itself a challenge to which nature has always anticipated.
Literature Cited
Acland, H. M., Silverman Bachin, L. A., and Eckroade, R. J. (1984). Lesions in broiler and layer chickens in an outbreak of highly pathogenic avian influenza virus infection. Vet Pathol 21, 564-569.
Alexander, D. J. (1982). Ecological aspects of influenza A viruses in animals and their relationship to human influenza: a review. J R Soc Med 75, 799-811.
Alexander, D. J. (2000). A review of avian influenza in different bird species. Vet Microbiol 74, 3-13.
Banks, J., and Plowright, L. (2003). Additional glycosylation at the receptor binding site of the hemagglutinin (HA) for H5 and H7 viruses may be an adaptation to poultry hosts, but does it influence pathogenicity? Avian Dis 47, 942-950.
Beaton, A. R., and Krug, R. M. (1986). Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end. Proc Natl Acad Sci U S A 83, 6282-6286.
Bright, R. A., Ross, T. M., Subbarao, K., Robinson, H. L., and Katz, J. M. (2003). Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine. Virology 308, 270-278.
Brown, D. W., Kawaoka, Y., Webster, R. G., and Robinson, H. L. (1992). Assessment of retrovirus-expressed nucleoprotein as a vaccine against lethal influenza virus infections of chickens. Avian Dis 36, 515-520.
Bulaga, L. L., Garber, L., Senne, D. A., Myers, T. J., Good, R., Wainwright, S., Trock, S., and Suarez, D. L. (2003). Epidemiologic and surveillance studies on avian influenza in live-bird markets in New York and New Jersey, 2001. Avian Dis 47, 996-1001.
Capua, I. (2003). Development of a DIVA (Differentiating Infected from Vaccinated Animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza. Avian Pathol 32, 47-55.
Ehlers, M., Moller, M., Marangon, S., and Ferre, N. (2003). The use of Geographic Information System (GIS) in the frame of the contingency plan implemented during the 1999-2001 avian influenza (AI) epidemic in Italy. Avian Dis 47, 1010-1014.
Fynan, E. F., Robinson, H. L., and Webster, R. G. (1993). Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine. DNA Cell Biol 12, 785-789.
Gambaryan, A. S., Marinina, V. P., Tuzikov, A. B., Bovin, N. V., Rudneva, I. A., Sinitsyn, B. V., Shilov, A. A., and Matrosovich, M. N. (1998). Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs. Virology 247, 170-177.
Gambaryan, A. S., Piskarev, V. E., Yamskov, I. A., Sakharov, A. M., Tuzikov, A. B., Bovin, N. V., Nifant'ev, N. E., and Matrosovich, M. N. (1995). Human influenza virus recognition of sialyloligosaccharides. FEBS Lett 366, 57-60.
Gambaryan, A. S., Tuzikov, A. B., Piskarev, V. E., Yamnikova, S. S., Lvov, D. K., Robertson, J. S., Bovin, N. V., and Matrosovich, M. N. (1997). Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine). Virology 232, 345-350.
Halvorson, D. A. (2002a). The control of H5 or H7 mildly pathogenic avian influenza: a role for inactivated vaccine. Avian Pathol 31, 5-12.
Halvorson, D. A. (2002b). Is avian influenza research meeting the needs of the poultry veterinarian? Vet J 164, 173-175.
Hay, A. J., Lomniczi, B., Bellamy, A. R., and Skehel, J. J. (1977). Transcription of the influenza virus genome. Virology 83, 337-355.
Helenius, A. (1992.). Unpacking the incoming influenza virus. Cell 69, 577-578.
Herlocher, M. L., Bucher, D., and Webster, R. G. (1992). Host range determination and functional mapping of the nucleoprotein and matrix genes of influenza viruses using monoclonal antibodies. Virus Res 22, 281-293.
Hinshaw, V. S., Webster, R. G., and Rodriguez, R. J. (1979). Influenza A viruses: combinations of hemagglutinin and neuraminidase subtypes isolated from animals and other sources. Brief review. Arch Virol 62, 281-290.
Hirst, G. K. (1941). The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus. Science 94, 22-23.
Horimoto, T., and Kawaoka, Y. (1994). Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. J Virol 68, 3120-3128.
Horimoto, T., and Kawaoka, Y. (1997). Biologic effects of introducing additional basic amino acid residues into the hemagglutinin cleavage site of a virulent avian influenza virus. Virus Res 50, 35-40.
Kawaoka, Y., Naeve, C. W., and Webster, R. G. (1984). Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology 139, 303-316.
Kawaoka, Y., Nestorowicz, A., Alexander, D. J., and Webster, R. G. (1987). Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain. Virology 158, 218-227.
Kawaoka, Y., and Webster, R. G. (1988). Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells. Proc Natl Acad Sci U S A 85, 324-328.
Kodihalli, S., Haynes, J. R., Robinson, H. L., and Webster, R. G. (1997). Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin. J Virol 71, 3391-3396.
Kodihalli, S., Sivanandan, V., Nagaraja, K. V., Shaw, D., and Halvorson, D. A. (1994). A type-specific avian influenza virus subunit vaccine for turkeys: induction of protective immunity to challenge infection. Vaccine 12, 1467-1472.
Krug, R. M., Alonso-Caplen, F. V., Julkunen, I., and Katze, M. G. (1989). Expression and Replication of the Influenza Virus Genome. In The Influenza Viruses, K. R.M., ed. (New York, Plenum), pp. 89-152.
Lamb, R. (1989). Genes and Proteins of the Influenza Viruses. In The Influenza Viruses, R. M. Krug, ed. (New York, Plenum Press), pp. 1-67.
Lamb, R. A., and Lai, C. J. (1980). Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus. Cell 21, 475-485.
Lamb, R. A., and Lai, C. J. (1984). Expression of unspliced NS1 mRNA, spliced NS2 mRNA, and a spliced chimera mRNA from cloned influenza virus NS DNA in an SV40 vector. Virology 135, 139-147.
Lamb, R. A., Lai, C. J., and Choppin, P. W. (1981). Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc Natl Acad Sci U S A 78, 4170-4174.
Liu, M., He, S., Walker, D., Zhou, N., Perez, D. R., Mo, B., Li, F., Huang, X., Webster, R. G., and Webby, R. J. (2003a). The influenza virus gene pool in a poultry market in South central china. Virology 305, 267-275.
Liu, M., Wood, J. M., Ellis, T. M., Krauss, S., Seiler, J. P., Johnson, C., Hoffmann, E., Humberd, J., Hulse, D., Zhang, Y., et al. (2003b). Preparation of a standardized, efficacious Agricultural H5N1 vaccine by reverse genetics. In Virology (in press).
Lu, H. (2003). A longitudinal study of a novel dot-enzyme-linked immunosorbent assay for the detection of avian influenza virus. Avian Dis 47, 361-369.
Matrosovich, M., Zhou, N., Kawaoka, Y., and Webster, R. (1999). The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 73, 1146-1155.
Matrosovich, M. N., Gambaryan, A. S., Tuzikov, A. B., Byramova, N. E., Mochalova, L. V., Golbraikh, A. A., Shenderovich, M. D., Finne, J., and Bovin, N. V. (1993). Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides. Virology 196, 111-121.
Matrosovich, M. N., Krauss, S., and Webster, R. G. (2001). H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity. Virology 281, 156-162.
McGeoch, D., Fellner, P., and Newton, C. (1976). Influenza virus genome consists of eight distinct RNA species. Proc Natl Acad Sci U S A 73, 3045-3049.
Mullaney, R. (2003). Live-bird market closure activities in the northeastern United States. Avian Dis 47, 1096-1098.
Murphy, B. R., Buckler-White, A. J., London, W. T., and Snyder, M. H. (1989). Characterization of the M protein and nucleoprotein genes of an avian influenza A virus which are involved in host range restriction in monkeys. Vaccine 7, 557-561.
OIE (2003). Highly Pathogenic Avian Influenza. In Terrestrial Animal Health Code (Office International des Epizzoties. World Organization for Animal Health).
Palese, P., Ritchey, M. B., and Schulman, J. L. (1977). Mapping of the influenza virus genome. II. Identification of the P1, P2, and P3 genes. Virology 76, 114-121.
Parkin, N. T., Chiu, P., and Coelingh, K. (1997). Genetically engineered live attenuated influenza A virus vaccine candidates. J Virol 71, 2772-2778.
Penn, C. (1989). The role of RNA segment 1 in an in vitro host restriction occurring in an avian influenza virus mutant. Virus Res 12, 349-359.
Perkins, L. E., and Swayne, D. E. (2002a). Pathogenicity of a Hong Kong-origin H5N1 highly pathogenic avian influenza virus for emus, geese, ducks, and pigeons. Avian Dis 46, 53-63.
Perkins, L. E., and Swayne, D. E. (2002b). Susceptibility of laughing gulls (Larus atricilla) to H5N1 and H5N3 highly pathogenic avian influenza viruses. Avian Dis 46, 877-885.
Perkins, L. E., and Swayne, D. E. (2003). Varied pathogenicity of a Hong Kong-origin H5N1 avian influenza virus in four passerine species and budgerigars. Vet Pathol 40, 14-24.
Perroncito, E. (1878). Epizoozia tifoide neigalliancei. Annali della Academia d'agricoltora di Torino 21, 87-126.
Pinto, L. H., Holsinger, L. J., and Lamb, R. A. (1992). Influenza virus M2 protein has ion channel activity. Cell 69, 517-528.
Plotch, S. J., Bouloy, M., Ulmanen, I., and Krug, R. M. (1981). A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, 847-858.
Poss, P. E., Friendshuh, K. A., and Ausherman, L. T. (1987). The control of avian influenza. In Proceedings of the Second International Symposium on Avian Influenza (Richmond, VA, U.S. Animal Health Association), pp. 318-326.
Report of Committee on Recombinant DNA Molecules: ?Potential biohazards of recombinant DNA molecules,? Proc. Nat. Acad. Sci. USA 71:2593-2594.
Robertson, J. S., Schubert, M., and Lazzarini, R. A. (1981). Polyadenylation sites for influenza virus mRNA. J Virol 38, 157-163.
Rogers, G. N., Paulson, J. C., Daniels, R. S., Skehel, J. J., Wilson, I. A., and Wiley, D. C. (1983). Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304, 76-78.
Rott, R., Klenk, H. D., Nagai, Y., and Tashiro, M. (1995). Influenza viruses, cell enzymes, and pathogenicity. Am J Respir Crit Care Med 152, S16-19.
Schafer, W. (1955). Vergleichende sero-immunologische untersuchungen uber die viren der influenza unf klassichen geflugelpest. Z Naturforsch 10B, 81-91.
Scholtissek, C. (1994). Source for influenza pandemics. Eur J Epidemiol 10, 455-458.
Scholtissek, C. (1995). Molecular evolution of influenza viruses. Virus Genes 11, 209-215.
Scholtissek, C. (1997). Molecular epidemiology of influenza. Arch Virol Suppl 13, 99-103.
Scholtissek, C., Koennecke, I., and Rott, R. (1978). Host range recombinants of fowl plague (influenza A) virus. Virology 91, 79-85.
Scholtissek, C., and Murphy, B. R. (1978). Host range mutants of an influenza A virus. Arch Virol 58, 323-333.
Sims, L. D., Guan, Y., Ellis, T. M., Liu, K. K., Dyrting, K., Wong, H., Kung, N. Y., Shortridge, K. F., and Peiris, M. (2003). An update on avian influenza in Hong Kong 2002. Avian Dis 47, 1083-1086.
Skehel, J. J., and Wiley, D. C. (2000). Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531-569.
Slemons, R. D., Condobery, P. K., and Swayne, D. E. (1991). Assessing pathogenicity potential of waterfowl-origin type A influenza viruses in chickens. Avian Dis 35, 210-215.
Slemons, R. D., and Easterday, B. C. (1977). Type-A influenza viruses in the feces of migratory waterfowl. J Am Vet Med Assoc 171, 947-948.
Slemons, R. D., and Easterday, B. C. (1978). Virus replication in the digestive tract of ducks exposed by aerosol to type-A influenza. Avian Dis 22, 367-377.
Smith, W., Andrewes, C. H., and Laidlaw, P. P. (1933). A virus obtained from influenza patients. Lancet II, 66-68.
Spackman, E., Senne, D. A., Davison, S., and Suarez, D. L. (2003). Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States. J Virol 77, 13399-13402.
Spackman, E., Senne, D. A., Myers, T. J., Bulaga, L. L., Garber, L. P., Perdue, M. L., Lohman, K., Daum, L. T., and Suarez, D. L. (2002). Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 40, 3256-3260.
Steinhauer, D. A. (1999). Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258, 1-20.
Suarez, D. L., Spackman, E., and Senne, D. A. (2003). Update on molecular epidemiology of H1, H5, and H7 influenza virus infections in poultry in North America. Avian Dis 47, 888-897.
Subbarao, E. K., London, W., and Murphy, B. R. (1993). A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol 67, 1761-1764.
Subbarao, E. K., Park, E. J., Lawson, C. M., Chen, A. Y., and Murphy, B. R. (1995). Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine. J Virol 69, 5969-5977.
Subbarao, K., Chen, H., Swayne, D., Mingay, L., Fodor, E., Brownlee, G., Xu, X., Lu, X., Katz, J., Cox, N., and Matsuoka, Y. (2003). Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305, 192-200.
Swayne, D. E., Beck, J. R., and Mickle, T. R. (1997). Efficacy of recombinant fowl poxvirus vaccine in protecting chickens against a highly pathogenic Mexican-origin H5N2 avian influenza virus. Avian Dis 41, 910-922.
Swayne, D. E., Beck, J. R., Perdue, M. L., and Beard, C. W. (2001). Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza. Avian Dis 45, 355-365.
Swayne, D. E., Garcia, M., Beck, J. R., Kinney, N., and Suarez, D. L. (2000). Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 18, 1088-1095.
Swayne, D. E., and Halvorson, D. A. (2003). Influenza. In Diseases of Poultry, Y. M. Saif, ed. (Ames, IA, Iowa State Press. Blackwell Publishing Co), pp. 135-160.
Swayne, D. E., and Suarez, D. L. (2000). Highly pathogenic avian influenza. Rev Sci Tech 19, 463-482.
Takada, A., Kuboki, N., Okazaki, K., Ninomiya, A., Tanaka, H., Ozaki, H., Itamura, S., Nishimura, H., Enami, M., Tashiro, M., et al. (1999). Avirulent Avian influenza virus as a vaccine strain against a potential human pandemic. J Virol 73, 8303-8307.
Ulmanen, I., Broni, B., and Krug, R. M. (1983). Influenza virus temperature-sensitive cap (m7GpppNm)-dependent endonuclease. J Virol 45, 27-35.
Vines, A., Wells, K., Matrosovich, M., Castrucci, M. R., Ito, T., and Kawaoka, Y. (1998). The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction. J Virol 72, 7626-7631.
Walker, J. A., and Kawaoka, Y. (1993). Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus. J Gen Virol 74 ( Pt 2), 311-314.
Wang, X., Castro, A. E., Castro, M. D., Lu, H., Weinstock, D., Soyster, N., Scheuchenzuber, W., and Perdue, M. (2000). Production and evaluation criteria of specific monoclonal antibodies to the hemagglutinin of the H7N2 subtype of avian influenza virus. J Vet Diagn Invest 12, 503-509.
Webster, R. G. (1997). Influenza virus: transmission between species and relevance to emergence of the next human pandemic. Arch Virol Suppl 13, 105-113.
Weltzin, R., J. Liu, K. V. Pugachev, G. A. Myers, B. Coughlin, P. S. Blum, R. Nichols, C. Johnson, J. Cruz, J. S. Kennedy, F. A. Ennis, and T. P. Monath. 2003. Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med 9:1125-30.
Wharton, S. A., Weis, W., Skehel, J. J., and Wiley, D. C. (1989). Structure, Function, and Antigenicity of the Hemagglutinin of Influenza Virus. In The Influenza Viruses, R. M. Krug, ed. (New York, Plenum Press), pp. 153-174.
Wood, J. M., Kawaoka, Y., Newberry, L. A., Bordwell, E., and Webster, R. G. (1985). Standardization of inactivated H5N2 influenza vaccine and efficacy against lethal A/Chicken/Pennsylvania/1370/83 infection. Avian Dis 29, 867-872.
</TD></TR></TBODY></TABLE>
In the dawn of a new century one pathogen remains still as the paradigm of emerging diseases: The Influenza A virus.
http://www.agnr.umd.edu/avianflu/
http://www.agnr.umd.edu/avianflu/
Our mission at the Avian Influenza Virus Program at the Virginia-Maryland Regional College of Veterinary Medicine is to determine the molecular basis for interspecies transmission and pathogenesis of avian influenza viruses.
Our Program is located in the Department of Veterinary Medicine at the Avrum Gudelsky Bldg.,College of Agriculture and Natural Resorces, University of Maryland, College Park Campus.
Research Interests
The molecular basis of interspecies transmission, pathogenesis, and cross-protection of influenza A viruses.
Aquatic birds are the natural reservoir of influenza A viruses. Occasionally, some of these viruses cross to other animal species, in which they can cause severe disease outbreaks that include pandemics. In the 20th century, humans experienced three major pandemics. The most devastating was the 1918 "Spanish flu" that killed between 20 and 40 million people. The emergence of another influenza pandemic is considered imminent. Unfortunately, little is known about the molecular features that allow an influenza A virus to cause pandemic disease, although multiple genes, encoding both the surface and internal proteins of the virus, are thought to be involved. We have recently developed a plasmid-based reverse genetics system for influenza A viruses that allows the complete manipulation of the viral genome. We are currently using this system to map specific amino acid residues involved in interspecies transmission of H9, H2 and H7 influenza viruses, which are believed, along with H5 influenza viruses, to have the greatest potential to become pandemic viruses. Our studies are elucidating the most important factors in the spread and pathogenesis of influenza viruses from the aquatic bird reservoir to land-based birds and mammals.
Want to know more? Check the background information below and our publications. Click on the icons on the left for more information about resources and our program.
The information below has been extracted from the book "Biodefense: Principles and Pathogens" edited by M.S. Bronze and R.A. Greenfield, chapter 21 "Miscellaneous threats: Highly pathogenic avian influenza, and novel bio-engineered organisms." by D.R. Perez, S.H. Nazarian, G. McFadden, and M.S. Gilmore. Copyright ? 2005, Horizon Bioscience, Norfolk, England.
- Introduction
- Highly Pathogenic Avian Influenza
- Etiology
- Brief Overview of the Influenza Life Cycle
- Natural Reservoir of Avian Influenza
- Genesis of HPAI
- Manifestations of HPAI
- Transmission and Control of HPAI
- Economic Importance
- Public Health Significance
- HPAI as a bioweapon
- Literature Cited
Influenza infections have been recognized in humans since ancient times. The name influenza refers to the ancient belief that it was caused by a malign and supernatural influence (Latin influentia). Year-to-year epidemics in humans, recurrent epidemics in swine and horses, sporadic cases in minks, seals and whales, and devastating outbreaks in poultry constantly remind us that influenza is a non-eradicable disease.
Highly Pathogenic Avian Influenza
Avian influenza was defined as ?fowl plague? in 1878 as a disease causing high mortality in chickens in Italy (Perroncito, 1878). It was not until 1955 that it was demonstrated that fowl plague was an avian influenza virus whose genomic composition was virtually identical to the one found in the human influenza virus (Schafer, 1955). By then it was already shown that human influenza viruses, identified as a virus in 1933, (Smith et al., 1933) shared with their avian counterparts many biological properties including the ability to grow in chick embryos and agglutinate red blood cells (Hirst, 1941). The terminology ?highly pathogenic avian influenza? was officially adopted in 1981 at the First International Symposium on Avian Influenza to designate the highly virulent forms of avian influenza. The Office International des Epizooties (OIE) that codifies sanitary and health standards, and is affiliated to the World Trade Organization (WTO), has included HPAI as a List A reportable disease (OIE, 2003). Countries with representation in the WTO are obliged to report outbreaks of HPAI. Any AI virus that fits into either one of the following criteria is considered an HPAI virus
1) Lethal for six, seven, or eight 4 to 6 week-old chickens within 10 days following intravenous inoculation with 0.2 ml of a 1:10 dilution of virus in a bacteria-free, allantoic fluid.
2) It has a polybasic amino acid region at the hemagglutinin cleavage site and is of the H5 or H7 subtype (see below).
3) It is not an H5 or H7 virus but kills one to five chickens and grows in cell culture in the absence of trypsin.
Etiology
The influenza A viruses belong to the family Orthomyxoviridae (Lamb, 1989). Other members of the family include influenza viruses type B and C, the insect viruses Togotho and Dhori, and Salmon Anemia virus. Influenza A virions are 80-120 nm in diameter. The influenza viruses are relatively unstable in the environment. Heat, extreme changes of pH, or nonisotonic conditions and dryness can readily inactivate the influenza viruses. The virus has an envelope with a host-derived lipid bilayer and covered with about 500 projecting glycoprotein spikes with hemagglutinating and neuraminidase activities. These activities correspond to the two major surface viral glycoproteins: the hemagglutinin (HA) and neuraminidase (NA), present as homotrimers and homotetramers, respectively. Within the envelope, a matrix protein (M1) and a nucleocapsid (NP) protein protect the viral RNA (Lamb, 1989). The type designation (A, B, or C) is based upon the antigenic features of the M1 and NP proteins. Eight segments of single stranded RNA of negative polarity, totaling approximately 14 kilobases, encode for at least 10 viral proteins (McGeoch et al., 1976). Approximately half of the total genome encodes for the three viral polymerase proteins (segments 1, 2 and 3; (Palese et al., 1977). Segment 5 encodes the NP protein. The three-polymerase subunits, the NP and the vRNA are associated in virions and infected cells in the form of viral ribonucleoprotein particles (vRNPs). Segments 4 and 6 encode for the HA and NA genes, respectively. The two smallest segments (7 and 8) encode two genes each with overlapping reading frames, which are generated by splicing of the co-linear mRNA molecules (Lamb and Lai, 1980; Lamb and Lai, 1984; Lamb et al., 1981). In addition to M1, segment 7 encodes for the M2 protein, which has ion channel activity and is embedded in the viral envelope. Segment 8 encodes for NS1, a nonstructural protein that blocks the host?s antiviral response, and NS2 or NEP that participates in the assembly of virus particles.
Brief Overview of the Influenza Life Cycle
Influenza virus attachment to the susceptible cell is mediated by the interaction between the viral hemagglutinin and sialic acid receptors present on glycolipids and glycoproteins on the cell surface (Lamb, 1989). At this stage, the sialidase activity of the neuraminidase prevents binding of the HA to sialic acids present in mucopolysaccharadies, which would otherwise interfere with the virus binding to the adequate cellular receptors. The virus is internalized by endocytosis and, upon acidification of the endosome, conformational changes on the hemagglutinin lead to the fusion between the viral and the endosomal membranes (Lamb, 1989). Acidification of the endosomal lumen also activates the ion channel activity of the viral membrane protein M2 (Pinto et al., 1992). Activation of M2 generates an inward current of protons into the virion?s interior that triggers the disassembly of M1 from the vRNPs, which are transported to the nucleus, the site of influenza virus transcription and replication (Holsinger et al., 1994; Martin and Helenius, 1991a). A minimal set of four viral proteins is essential for influenza virus transcription and replication: PB1, PB2, PA - referred to as P-proteins-, and the NP protein (Huang, Palese, and Krystal, 1990). Two different populations of positive sense RNAs are synthesized from vRNA templates: messenger RNAs (mRNAs) and complementary RNAs (cRNAs). Viral mRNAs are primed by 5? capped (m7GpppNm-containing) fragments derived from newly synthesized host-cell RNA polymerase II transcripts (Beaton and Krug, 1986; Krug et al., 1989; Plotch et al., 1981; Ulmanen et al., 1983)Viral mRNAs are polyadenylated by a stuttering mechanism involving the viral polymerase and a stretch of uridines, which are located 17-22 nucleotides before the 5? end of the vRNAs (Hay et al., 1977; Robertson et al., 1981); Synthesis of cRNA is the first step in influenza virus replication. Transcription of cRNAs occurs in the absence of primer or polyadenylation and they represent full-length copies of vRNAs (McGeoch et al., 1976). The second step in viral replication is the synthesis of progeny vRNA molecules from cRNAs templates (McGeoch et al., 1976). Towards the end of the infection cycle and once enough molecules of M1 and NEP have been produced, the newly synthesized vRNPs are exported out of the nucleus and assembled into full virus particles. The final assembly steps occur at the plasma membrane exposing the newly synthesized hemagglutinin, neuraminidase proteins, and M2 (Helenius, 1992.) Once the final assembly events are completed, new virus particles bud from the plasma membrane. The activity of the neuraminidase becomes again important by disrupting viral aggregates and thus releasing viral particles that can start a new cycle of infection.
</TD></TR><TR height=875><TD vAlign=top width=759 height=875>
Natural Reservoir of Avian Influenza The antigenic diversity of influenza A viruses occurs primarily at the surface glycoproteins. Differences of up to 80% have been found in these proteins (Wharton et al., 1989). The viral glycoproteins are classified into different subtypes on the basis of their reaction with hyperimmune sera using the letters H and N with the appropriate numbers in the virus designation e.g., H5N1. Among the influenza A viruses, 15 hemagglutinin and 9 neuraminidase subtypes have been described. (Wharton et al., 1989). Wild aquatic birds (Orders Anseriformes and Charadriiformes) are the major natural reservoir of influenza A and from which viruses corresponding to all15 HA and 9 NA subtypes have been isolated, reviewed in (Alexander, 2000). Given the segmented nature of their genome, influenza A viruses easily exchange genomic information, a process known as genetic reassortment. Thus, when a single cell is infected by two strains of influenza A with different antigenic subtypes reassortment can occur, further increasing the diversity of these viruses. Influenza A viruses in many different combinations of HA and NA subtypes have been isolated from wild and domestic birds supporting the notion that reassortment occurs freely in nature (Hinshaw et al., 1979). The intestine is the primary site of replication of influenza A virus in waterfowl, where the infection is usually asymptomatic and large quantities of viral particles are excreted with the feces into the water, perpetuating a cycle of oral-fecal transmission (Slemons and Easterday, 1977; Slemons and Easterday, 1978).
Genesis of HPAI
It is commonly accepted that migratory waterfowl, including ducks, sea birds, or shorebirds are responsible for introducing AI into domestic poultry (Alexander, 2000). Some of these viruses can establish stable lineages in terrestrial birds (Order Galliformes) and a limited number of mammalian species (Scholtissek, 1994; Scholtissek, 1995; Scholtissek, 1997; Webster, 1997). All highly pathogenic avian influenza viruses (HPAI) identified to date differ from their low pathogenic (LPAI) counterparts in the susceptibility of the HA to host proteases. HPAI are characterized by HAs that are highly susceptible to cleavage by numerous cellular proteases, which are ubiquitous in many cell compartments and organ systems. In contrast, the LPAI HA requires specific active extra-cellular proteases ?such as trypsin- for cleavage and activation of infectivity. Trypsin-like proteases are restricted to the lumen of the respiratory and intestinal sites.
Theoretically, viruses of all HA subtypes have the potential to cause disease. HPAI viruses have been historically restricted to only two HA subtypes, H5 and H7; although not all viruses that belong to these two subtypes are highly pathogenic. Viruses with any other subtype can cause a mild, primarily respiratory disease in poultry, which may be exacerbated by other infections or environmental conditions. An H5 or H7 virus can start out by presenting itself as a low pathogenic virus but can mutate without warning to become highly pathogenic. Unlike other HA subtypes, the highly virulent H5 and H7 viruses possess multiple basic amino acids at the cleavage site of the HA, that increase the spectrum of proteases that recognize the site (Swayne and Suarez, 2000). The cleavage of HA can also be modulated by the presence of additional glycosylation sites spatially located nearby. For example, a loss of a glycosylation site at amino acid 13 and the presence of the polybasic amino acid region in the HA are pathotypic markers of the H5N2 virus that caused the 1983-1984 outbreak in Pennsylvania (Banks and Plowright, 2003; Kawaoka et al., 1984). The exact contributing factors that lead to the accumulation of the polybasic amino acid region at the HA cleavage site remain largely unknown, although it is postulated that viral adaptation to terrestrial birds plays a major role.
Manifestations of HPAI
The most virulent forms of HPAI are characterized by a highly fatal systemic infection that spreads to most organ systems including the cardiovascular and nervous systems (Acland et al., 1984; Gross et al., 1986; Perkins and Swayne, 2002a; Perkins and Swayne, 2002b; Perkins and Swayne, 2003; Slemons et al., 1991). Morbidity and mortality can be as high as 100%, particularly in gallinaceous species. The incubation period is usually between 3 to 7 days depending on the virus isolate, age of the bird and bird species. Death may occur within 24 to 48 hr after the onset of symptoms but it may be delayed for as long as one or two weeks. In chickens, the initial stages of HPAI infections are usually accompanied by marked depression with ruffled feathers, loss of appetite, excessive thirst, watery bright green diarrhea, and markedly lower egg production. Adult chickens frequently have swollen combs, wattles, and edema surrounding the eyes. Cyanosis is often observed at the tips of the combs and it is not uncommon to observe plasma or blood vesicles on the surface with dark areas of hemorrhage and necrotic foci. Laid eggs are frequently without shells. Edema of the head and the neck are usually observed. The conjunctivae are congested and swollen with occasional hemorrhage. Areas of diffuse hemorrhage may be observed on the legs, between the hocks and feet. Respiratory syndrome and mucus accumulation can be significant characteristics of the disease. Some severely affected hens may occasionally recover, although neurological sequelae may be observed, such as torticolis and ataxia. The disease in turkeys, ducks, geese, and quail is similar to that seen in chickens, but it may last 2 to 3 days longer and is occasionally accompanied by swollen sinuses. Mortality rates may be lower in ducks and geese than those observed for chickens or turkeys, although younger birds may exhibit neurologic sequelae (Swayne and Halvorson, 2003).
Histologic lesions may not be apparent in birds that died soon after the onset of the infection other than severe congestion of the musculature and dehydration. However, in birds that survive the acute form of the disease, significant gross lesions are observed, although they may resemble those observed with velogenic viscerotropic Newcastle disease (NDV), and thus, they cannot be used for differential diagnosis of HPAI. During post-mortem examination, fluid and mucous exudates from the nares, oral cavity, the trachea, and subcutaneous edema of the head and neck may be observed. The conjunctivae are severely congested, often presenting petechia. Pinpoint petechiae are frequently observed on the inside of the keel, the abdominal fat, serosal surfaces, and peritoneum. Kidney function is usually severely compromised, with tubules that are plugged with white deposits of urate. In layers, the ovary may be hemorrhagic or degenerated with darkened areas of necrosis. In layers that survive longer than a week, the ova are ruptured and the yolk fills the peritoneal cavity causing severe airsacculitis and peritonitis. Hemorrhages may be present on the mucosal surface of the proventriculus and underneath the gizzard. The intestinal mucosa may have hemorrhagic areas, especially in the cecal tonsils and other lymphoid foci (Swayne and Halvorson, 2003).
Transmission and Control of HPAI
Since ancient times, humans have altered the natural ecosystem and created new ones for many animal species including avian species. By domestication and captivity of many bird species, humans have exposed themselves to the potential biohazards carried and/or transmitted by these animals. Such is the case of diseases like Psittacosis (parrot fever), Campylobacteriosis, Salmonellosis, and Avian Tuberculosis, to name a few. Humans have created niches where disease agents have the chance to jump from natural to non-natural hosts. Such is the case of AI in its many virulent forms. As mentioned earlier, live bird markets have played a major role in the emergence of HPAI. The live bird markets in the East Coast of the U.S, in Hong Kong, and Italy, to name a few, have been the originators of very important outbreaks of HPAI that extended beyond their borders and were brought into vast regions of their respective countries involving many small and large poultry production facilities and farms. Once introduced into a flock, it is very easy to spread the virus from flock to flock if not adequate biosecurity measures are taken. Movement of infected birds, excretions from infected birds, if carried in contaminated equipment, clothing, hands, shoes, egg flats, feed trucks, water, and food are all major contributors in the spread of the virus. GIS and retrospective studies have shown how HPAI viruses can be transmitted from farm to farm simply by the movement of trucks and personnel that work in different farms (Ehlers et al., 2003; Valleron and Vidal, 2002). Airborne transmission may occur but it is rather limited to birds that are in close proximity.
Proper sanitation and biosecurity cannot be over emphasized; they are the first line of defense against AI. Thus, all methods for preventing and controlling the spread of AI are related to controlling the contamination of equipment and personnel. Most HPAI viruses start out from non-virulent AI forms, so efforts should be made to control AI before it is too late. Since wild birds are considered the major source of infection for domestic poultry, particularly those in live bird markets and those raised on open range, it is essential to reduce the contact between these two groups. Appropriate education programs, in different languages and/or dialects, are needed to help people of different ethnic groups become aware of the risk factors involved in having flocks infected with AI. At the industrial level the incorporation of education programs about AI and its risks, monitoring, reporting and ?responsible response? initiatives as they were implemented in Minnesota, should be imitated in other states and countries (Poss et al., 1987). Prevention programs should include some form of incentive to growers, dealers, and retailers, to report suspected AI cases, which would help to contain infected birds to small areas.
Epidemiological surveillance is the second line of defense against AI. Surveillance efforts during the 1983-1984 outbreaks in Pennsylvania were able to track the origin of the virus to live bird markets. Likewise, the constant monitoring of influenza activity in the live bird markets of Hong Kong helped to quickly characterize the 1997 H5N1 virus that transmitted to humans from chickens (Sims et al., 2003). During the recent H7N2 outbreak in Virginia in 2002, which led to the culling of more than four million turkeys and chickens, it was possible to track the origin of the virus to live bird markets of New York and New Jersey, where the virus is endemic (Bulaga et al., 2003; Spackman et al., 2003; Suarez et al., 2003). Although the 2002 H7N2 virus was of low pathogenicity (no polybasic amino acid region in the HA cleavage site), eradication efforts were implemented to prevent further spread and the possibility that the virus would become highly pathogenic. Adequate emergency response plans must be in place in every state where poultry represents an economically important commodity. State and federal authorities are responsible for developing surveillance, quarantine, stamping out, and indemnification programs and policies. Additional information and recommendations about biosecurity, surveillance, and reporting AI activity can be found at the Animal and Plant Health Inspection Service, United States Department of Agriculture (APHIS-USDA).
Economic Importance
The economic losses associated with HPAI have been of varying importance depending on the magnitude of the outbreak and the measures taken to eradicate the virus. Epizootics of HPAI that have expanded into commercial farms or into large live bird market systems have resulted in the greatest economic losses. For instance, during the 1983-1984 H5N2 outbreak in Pennsylvania, it cost the U.S. government more than $60 million to eradicate the disease, including $40 million in indemnities (Swayne and Halvorson, 2003). Producers had to absorb an additional $15 million in non-indemnified losses. Consumers, on the other hand, had to overcome approximately $350 million in increased food costs. More recently, the eradication of the H7N1 virus responsible for the HPAI outbreak in Italy in 1999-2000 carried approximately $100 million in compensations and total indirect losses have been calculated in excess of $500 million (Swayne and Halvorson, 2003). Without any doubts, the massive HPAI epidemic of H5N1 Asia dwarfs previous figures: More than 100 million birds have either died or been killed to contain the outbreak. For Southeast Asian countries, the economic implications of the 2004 H5N1 virus are tremendous. Besides the time it can take to eradicate the virus, it will take several years until some of these countries can restore export of their poultry products. China and Thailand, which are the two most important poultry exporters in Asia, have been particularly hit with the outbreak.
Public Health Significance
In recent years HPAI infections have taken a new dimension after the realization that some of these viruses have acquired the capacity to transmit directly from birds to humans, with the consequent potential for causing a pandemic. During the 20th century, humans experienced three influenza pandemics: the 1918-1919 influenza pandemic known as ?Spanish flu?, which killed approximately 40-50 million people, and the pandemics of 1957 ?Asian flu? and the 1968 ?Hong Kong flu?. In each instances, pandemics have been characterized by the emergence of new HA subtypes, a process known as antigenic shift. There are basically two mechanisms that can result in antigenic shift: transfer of an avian virus in toto or through reassortment with a human influenza virus. Transfer in toto is thought to have occurred during the pandemic of 1918, the virus carrying the H1N1 subtypes. Antigenic shift through reassortment occurred during the 1957 and 1968 pandemics. The 1957 H2N2 pandemic virus was a reassortant that inherited three genes (HA, NA, and PB1) from the avian influenza pool and the other genes from the H1N1 human influenza virus circulating at that time. The 1968 H3N2 virus inherited two genes (HA and PB1) from an avian influenza virus and the rest from the H2N2 virus circulating at that time. Pandemics occur because the human population has no immunity against a new virus subtype that can be transmitted efficiently from human to human. Once in the human population, the virus perpetuates itself by evading the immune system through small antigenic changes, a process called ?antigenic drift.? Currently, two influenza subtypes circulate in the human population: H3N2 and H1N1, the latter re-introduced in humans in 1977 and named ?Russian flu? (although the virus may have originated in China or Mongolia). Interestingly, the 1977 H1N1 virus was almost identical to the H1N1 virus that circulated until 1957. An accidental laboratory release or the perpetuation of the virus in a different animal host have been proposed as potential causes for the re-emergence of the 1977 H1N1 virus. The occurrence of influenza pandemics is unpredictable; however co-mingling of avian and mammalian species is considered a potential factor in the emergence of pandemic viruses. Pigs have been proposed as potential intermediate hosts or ?mixing vessels? where avian and human influenza can reassort, although humans could act as mixing vessels themselves. In addition, other animal species, particularly terrestrial birds, have been proposed as potential ?amplifiers? of avian/human reassortant influenza viruses. Historical evidence suggests that Southeast Asia is an epicenter of influenza pandemics, although a pandemic virus could emerge virtually from any place where humans come in contact with carriers of AI. Of the 15 avian influenza virus subtypes, H5N1 is of particular concern because of the propensity to infect humans. Its ability to cause severe disease in humans has now been documented on three occasions. In 1997, 18 people became infected with an H5N1 virus during an outbreak of HPAI in Hong Kong. Patients developed symptoms of fever, sore throat, and cough and, in several of the six fatal cases, severe respiratory distress secondary to viral pneumonia. Previously healthy adults and children, and some with chronic medical conditions, were affected. Culling of more than 1 million poultry is thought to have prevented new cases. However, there is evidence that H5N1 viruses may have become endemic in Southeast Asia. H5N1 outbreaks repeated in Hong Kong in 2001, and 2002-2003, even though several preventative measures have been taken: The separation of aquatic and poultry species in different wholesale markets, the prohibition of selling live aquatic birds in retail markets, and the incorporation of monthly rest days, where markets are completely emptied and disinfected before new birds are brought in. In February 2003, two new human cases of H5N1 where documented in Hong Kong, one of them fatal. In mid-December 2003, an outbreak of H5N1 initiated in poultry in South Korea and, by early January 2004, quickly spread to Japan, Taiwan, Viet Nam, Thailand, and China. In Viet Nam, more than 20 people died of respiratory disease and, in at least five of these cases, an H5N1 virus caused it. In Thailand, fewer human cases were reported but they have been mostly lethal (6 out 8). Culling of poultry has been implemented in major live bird markets and big poultry operations in all these countries to prevent new cases. In addition, H5N1 viruses have a propensity to reassort with influenza viruses circulating in other animal species. Therefore if it reassorts with a human virus it may gain the capacity for human-to-human transmission and cause a pandemic. Birds that survive infection excrete virus for at least 10 days, orally and in feces, thus facilitating further spread at live poultry markets and by migratory birds.
Other viruses that are of pandemic concern are H9 and H7 viruses, which in recent years have also crossed to humans. In 1999 in China, 7 cases of bird-to-human transmission of H9N2 viruses were documented. Individuals infected with the H9N2 viruses have mild respiratory symptoms and fully recovered. In 2003, a new episode of H9N2 virus was recorded in one patient with mild respiratory distress. Interestingly, H9 viruses are endemic in poultry in Asia and display typical human-like receptor specificity, binding preferably sa-a2, 6-gal receptors (as opposed to the sa-a2, 3-gal receptor binding of waterfowl influenza viruses, Matrosovich et al., 2001). In early 2003, an HPAI H7N7 outbreak occurred in poultry in the Netherlands. Bird-to-human transmission of the H7N7 virus occurred in at least 82 cases. Conjunctivitis was the most notorious disease symptom in people infected with the H7 virus, with only 7 cases displaying typical flu-like illness. Fortunately, the H7 was not highly pathogenic for humans and only one fatal case was observed. Other viruses with pandemic potential are H2, because of its past history as a pandemic virus, and H6 because of its high incidence in poultry species in Asia and North America.
A major challenge for scientists and public health officials is to avert influenza pandemics. Is it possible? The same measures taken to prevent the spread of AI in the poultry population can reduce opportunities for human exposure to the virus. In addition, vaccination of persons at high risk of exposure to infected poultry, using existing vaccines effective against currently circulating human influenza strains, can reduce the likelihood of co-infection of humans with avian and influenza strains, and thus reduce the risk of reassortment. Workers involved in the culling of poultry flocks should wear protective clothing and also receive antiviral drugs as a prophylactic measure. The World Health Organization, through its Global Influenza Surveillance Program, together with other national and international agencies, assists in reducing the risks of AI for public health. It remains to be seen whether all these activities will effectively avert the emergence of a pandemic strain.
HPAI as a bioweapon
The accidental or intentional introduction of HPAI into a flock can have devastating consequences as discussed earlier. Fortunately, the same characteristics that are hallmarks of HPAI can be used for the early detection of the virus and develop an efficient and rapid response to contain it. More problematic is the introduction of the low pathogenic forms of H5 and H7 viruses since they have the potential to become HPAI; however can be spread sometimes without significant or noticeable signs of disease. Outbreaks of HPAI have been caused by naturally occurring highly pathogenic forms of the disease. Attempts in the laboratory to reproduce the generation of HPAI from non-virulent forms have proven difficult at best. In recent years, a new technology known as reverse genetics has allowed the generation of influenza viruses entirely from plasmid DNA. Such technology allows for the complete manipulation of the influenza virus genome. The construction of tailor-made influenza viruses can be used to pinpoint the viral markers for pathogenesis and transmission. The plasmid-based system shows also promise for the rapid generation of vaccines for pandemic preparedness, although at least four months would be needed to produce a new vaccine, in significant quantities, capable of conferring protection against a new virus subtype. Certainly, this same DNA technology could be used to generate a virus with undesirable features; however genetic engineering of potentially harmful influenza viruses is in itself a challenge to which nature has always anticipated.
Literature Cited
Acland, H. M., Silverman Bachin, L. A., and Eckroade, R. J. (1984). Lesions in broiler and layer chickens in an outbreak of highly pathogenic avian influenza virus infection. Vet Pathol 21, 564-569.
Alexander, D. J. (1982). Ecological aspects of influenza A viruses in animals and their relationship to human influenza: a review. J R Soc Med 75, 799-811.
Alexander, D. J. (2000). A review of avian influenza in different bird species. Vet Microbiol 74, 3-13.
Banks, J., and Plowright, L. (2003). Additional glycosylation at the receptor binding site of the hemagglutinin (HA) for H5 and H7 viruses may be an adaptation to poultry hosts, but does it influence pathogenicity? Avian Dis 47, 942-950.
Beaton, A. R., and Krug, R. M. (1986). Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end. Proc Natl Acad Sci U S A 83, 6282-6286.
Bright, R. A., Ross, T. M., Subbarao, K., Robinson, H. L., and Katz, J. M. (2003). Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine. Virology 308, 270-278.
Brown, D. W., Kawaoka, Y., Webster, R. G., and Robinson, H. L. (1992). Assessment of retrovirus-expressed nucleoprotein as a vaccine against lethal influenza virus infections of chickens. Avian Dis 36, 515-520.
Bulaga, L. L., Garber, L., Senne, D. A., Myers, T. J., Good, R., Wainwright, S., Trock, S., and Suarez, D. L. (2003). Epidemiologic and surveillance studies on avian influenza in live-bird markets in New York and New Jersey, 2001. Avian Dis 47, 996-1001.
Capua, I. (2003). Development of a DIVA (Differentiating Infected from Vaccinated Animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza. Avian Pathol 32, 47-55.
Ehlers, M., Moller, M., Marangon, S., and Ferre, N. (2003). The use of Geographic Information System (GIS) in the frame of the contingency plan implemented during the 1999-2001 avian influenza (AI) epidemic in Italy. Avian Dis 47, 1010-1014.
Fynan, E. F., Robinson, H. L., and Webster, R. G. (1993). Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine. DNA Cell Biol 12, 785-789.
Gambaryan, A. S., Marinina, V. P., Tuzikov, A. B., Bovin, N. V., Rudneva, I. A., Sinitsyn, B. V., Shilov, A. A., and Matrosovich, M. N. (1998). Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs. Virology 247, 170-177.
Gambaryan, A. S., Piskarev, V. E., Yamskov, I. A., Sakharov, A. M., Tuzikov, A. B., Bovin, N. V., Nifant'ev, N. E., and Matrosovich, M. N. (1995). Human influenza virus recognition of sialyloligosaccharides. FEBS Lett 366, 57-60.
Gambaryan, A. S., Tuzikov, A. B., Piskarev, V. E., Yamnikova, S. S., Lvov, D. K., Robertson, J. S., Bovin, N. V., and Matrosovich, M. N. (1997). Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine). Virology 232, 345-350.
Halvorson, D. A. (2002a). The control of H5 or H7 mildly pathogenic avian influenza: a role for inactivated vaccine. Avian Pathol 31, 5-12.
Halvorson, D. A. (2002b). Is avian influenza research meeting the needs of the poultry veterinarian? Vet J 164, 173-175.
Hay, A. J., Lomniczi, B., Bellamy, A. R., and Skehel, J. J. (1977). Transcription of the influenza virus genome. Virology 83, 337-355.
Helenius, A. (1992.). Unpacking the incoming influenza virus. Cell 69, 577-578.
Herlocher, M. L., Bucher, D., and Webster, R. G. (1992). Host range determination and functional mapping of the nucleoprotein and matrix genes of influenza viruses using monoclonal antibodies. Virus Res 22, 281-293.
Hinshaw, V. S., Webster, R. G., and Rodriguez, R. J. (1979). Influenza A viruses: combinations of hemagglutinin and neuraminidase subtypes isolated from animals and other sources. Brief review. Arch Virol 62, 281-290.
Hirst, G. K. (1941). The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus. Science 94, 22-23.
Horimoto, T., and Kawaoka, Y. (1994). Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. J Virol 68, 3120-3128.
Horimoto, T., and Kawaoka, Y. (1997). Biologic effects of introducing additional basic amino acid residues into the hemagglutinin cleavage site of a virulent avian influenza virus. Virus Res 50, 35-40.
Kawaoka, Y., Naeve, C. W., and Webster, R. G. (1984). Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology 139, 303-316.
Kawaoka, Y., Nestorowicz, A., Alexander, D. J., and Webster, R. G. (1987). Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain. Virology 158, 218-227.
Kawaoka, Y., and Webster, R. G. (1988). Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells. Proc Natl Acad Sci U S A 85, 324-328.
Kodihalli, S., Haynes, J. R., Robinson, H. L., and Webster, R. G. (1997). Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin. J Virol 71, 3391-3396.
Kodihalli, S., Sivanandan, V., Nagaraja, K. V., Shaw, D., and Halvorson, D. A. (1994). A type-specific avian influenza virus subunit vaccine for turkeys: induction of protective immunity to challenge infection. Vaccine 12, 1467-1472.
Krug, R. M., Alonso-Caplen, F. V., Julkunen, I., and Katze, M. G. (1989). Expression and Replication of the Influenza Virus Genome. In The Influenza Viruses, K. R.M., ed. (New York, Plenum), pp. 89-152.
Lamb, R. (1989). Genes and Proteins of the Influenza Viruses. In The Influenza Viruses, R. M. Krug, ed. (New York, Plenum Press), pp. 1-67.
Lamb, R. A., and Lai, C. J. (1980). Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus. Cell 21, 475-485.
Lamb, R. A., and Lai, C. J. (1984). Expression of unspliced NS1 mRNA, spliced NS2 mRNA, and a spliced chimera mRNA from cloned influenza virus NS DNA in an SV40 vector. Virology 135, 139-147.
Lamb, R. A., Lai, C. J., and Choppin, P. W. (1981). Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc Natl Acad Sci U S A 78, 4170-4174.
Liu, M., He, S., Walker, D., Zhou, N., Perez, D. R., Mo, B., Li, F., Huang, X., Webster, R. G., and Webby, R. J. (2003a). The influenza virus gene pool in a poultry market in South central china. Virology 305, 267-275.
Liu, M., Wood, J. M., Ellis, T. M., Krauss, S., Seiler, J. P., Johnson, C., Hoffmann, E., Humberd, J., Hulse, D., Zhang, Y., et al. (2003b). Preparation of a standardized, efficacious Agricultural H5N1 vaccine by reverse genetics. In Virology (in press).
Lu, H. (2003). A longitudinal study of a novel dot-enzyme-linked immunosorbent assay for the detection of avian influenza virus. Avian Dis 47, 361-369.
Matrosovich, M., Zhou, N., Kawaoka, Y., and Webster, R. (1999). The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 73, 1146-1155.
Matrosovich, M. N., Gambaryan, A. S., Tuzikov, A. B., Byramova, N. E., Mochalova, L. V., Golbraikh, A. A., Shenderovich, M. D., Finne, J., and Bovin, N. V. (1993). Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides. Virology 196, 111-121.
Matrosovich, M. N., Krauss, S., and Webster, R. G. (2001). H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity. Virology 281, 156-162.
McGeoch, D., Fellner, P., and Newton, C. (1976). Influenza virus genome consists of eight distinct RNA species. Proc Natl Acad Sci U S A 73, 3045-3049.
Mullaney, R. (2003). Live-bird market closure activities in the northeastern United States. Avian Dis 47, 1096-1098.
Murphy, B. R., Buckler-White, A. J., London, W. T., and Snyder, M. H. (1989). Characterization of the M protein and nucleoprotein genes of an avian influenza A virus which are involved in host range restriction in monkeys. Vaccine 7, 557-561.
OIE (2003). Highly Pathogenic Avian Influenza. In Terrestrial Animal Health Code (Office International des Epizzoties. World Organization for Animal Health).
Palese, P., Ritchey, M. B., and Schulman, J. L. (1977). Mapping of the influenza virus genome. II. Identification of the P1, P2, and P3 genes. Virology 76, 114-121.
Parkin, N. T., Chiu, P., and Coelingh, K. (1997). Genetically engineered live attenuated influenza A virus vaccine candidates. J Virol 71, 2772-2778.
Penn, C. (1989). The role of RNA segment 1 in an in vitro host restriction occurring in an avian influenza virus mutant. Virus Res 12, 349-359.
Perkins, L. E., and Swayne, D. E. (2002a). Pathogenicity of a Hong Kong-origin H5N1 highly pathogenic avian influenza virus for emus, geese, ducks, and pigeons. Avian Dis 46, 53-63.
Perkins, L. E., and Swayne, D. E. (2002b). Susceptibility of laughing gulls (Larus atricilla) to H5N1 and H5N3 highly pathogenic avian influenza viruses. Avian Dis 46, 877-885.
Perkins, L. E., and Swayne, D. E. (2003). Varied pathogenicity of a Hong Kong-origin H5N1 avian influenza virus in four passerine species and budgerigars. Vet Pathol 40, 14-24.
Perroncito, E. (1878). Epizoozia tifoide neigalliancei. Annali della Academia d'agricoltora di Torino 21, 87-126.
Pinto, L. H., Holsinger, L. J., and Lamb, R. A. (1992). Influenza virus M2 protein has ion channel activity. Cell 69, 517-528.
Plotch, S. J., Bouloy, M., Ulmanen, I., and Krug, R. M. (1981). A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, 847-858.
Poss, P. E., Friendshuh, K. A., and Ausherman, L. T. (1987). The control of avian influenza. In Proceedings of the Second International Symposium on Avian Influenza (Richmond, VA, U.S. Animal Health Association), pp. 318-326.
Report of Committee on Recombinant DNA Molecules: ?Potential biohazards of recombinant DNA molecules,? Proc. Nat. Acad. Sci. USA 71:2593-2594.
Robertson, J. S., Schubert, M., and Lazzarini, R. A. (1981). Polyadenylation sites for influenza virus mRNA. J Virol 38, 157-163.
Rogers, G. N., Paulson, J. C., Daniels, R. S., Skehel, J. J., Wilson, I. A., and Wiley, D. C. (1983). Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304, 76-78.
Rott, R., Klenk, H. D., Nagai, Y., and Tashiro, M. (1995). Influenza viruses, cell enzymes, and pathogenicity. Am J Respir Crit Care Med 152, S16-19.
Schafer, W. (1955). Vergleichende sero-immunologische untersuchungen uber die viren der influenza unf klassichen geflugelpest. Z Naturforsch 10B, 81-91.
Scholtissek, C. (1994). Source for influenza pandemics. Eur J Epidemiol 10, 455-458.
Scholtissek, C. (1995). Molecular evolution of influenza viruses. Virus Genes 11, 209-215.
Scholtissek, C. (1997). Molecular epidemiology of influenza. Arch Virol Suppl 13, 99-103.
Scholtissek, C., Koennecke, I., and Rott, R. (1978). Host range recombinants of fowl plague (influenza A) virus. Virology 91, 79-85.
Scholtissek, C., and Murphy, B. R. (1978). Host range mutants of an influenza A virus. Arch Virol 58, 323-333.
Sims, L. D., Guan, Y., Ellis, T. M., Liu, K. K., Dyrting, K., Wong, H., Kung, N. Y., Shortridge, K. F., and Peiris, M. (2003). An update on avian influenza in Hong Kong 2002. Avian Dis 47, 1083-1086.
Skehel, J. J., and Wiley, D. C. (2000). Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531-569.
Slemons, R. D., Condobery, P. K., and Swayne, D. E. (1991). Assessing pathogenicity potential of waterfowl-origin type A influenza viruses in chickens. Avian Dis 35, 210-215.
Slemons, R. D., and Easterday, B. C. (1977). Type-A influenza viruses in the feces of migratory waterfowl. J Am Vet Med Assoc 171, 947-948.
Slemons, R. D., and Easterday, B. C. (1978). Virus replication in the digestive tract of ducks exposed by aerosol to type-A influenza. Avian Dis 22, 367-377.
Smith, W., Andrewes, C. H., and Laidlaw, P. P. (1933). A virus obtained from influenza patients. Lancet II, 66-68.
Spackman, E., Senne, D. A., Davison, S., and Suarez, D. L. (2003). Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States. J Virol 77, 13399-13402.
Spackman, E., Senne, D. A., Myers, T. J., Bulaga, L. L., Garber, L. P., Perdue, M. L., Lohman, K., Daum, L. T., and Suarez, D. L. (2002). Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 40, 3256-3260.
Steinhauer, D. A. (1999). Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258, 1-20.
Suarez, D. L., Spackman, E., and Senne, D. A. (2003). Update on molecular epidemiology of H1, H5, and H7 influenza virus infections in poultry in North America. Avian Dis 47, 888-897.
Subbarao, E. K., London, W., and Murphy, B. R. (1993). A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol 67, 1761-1764.
Subbarao, E. K., Park, E. J., Lawson, C. M., Chen, A. Y., and Murphy, B. R. (1995). Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine. J Virol 69, 5969-5977.
Subbarao, K., Chen, H., Swayne, D., Mingay, L., Fodor, E., Brownlee, G., Xu, X., Lu, X., Katz, J., Cox, N., and Matsuoka, Y. (2003). Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305, 192-200.
Swayne, D. E., Beck, J. R., and Mickle, T. R. (1997). Efficacy of recombinant fowl poxvirus vaccine in protecting chickens against a highly pathogenic Mexican-origin H5N2 avian influenza virus. Avian Dis 41, 910-922.
Swayne, D. E., Beck, J. R., Perdue, M. L., and Beard, C. W. (2001). Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza. Avian Dis 45, 355-365.
Swayne, D. E., Garcia, M., Beck, J. R., Kinney, N., and Suarez, D. L. (2000). Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 18, 1088-1095.
Swayne, D. E., and Halvorson, D. A. (2003). Influenza. In Diseases of Poultry, Y. M. Saif, ed. (Ames, IA, Iowa State Press. Blackwell Publishing Co), pp. 135-160.
Swayne, D. E., and Suarez, D. L. (2000). Highly pathogenic avian influenza. Rev Sci Tech 19, 463-482.
Takada, A., Kuboki, N., Okazaki, K., Ninomiya, A., Tanaka, H., Ozaki, H., Itamura, S., Nishimura, H., Enami, M., Tashiro, M., et al. (1999). Avirulent Avian influenza virus as a vaccine strain against a potential human pandemic. J Virol 73, 8303-8307.
Ulmanen, I., Broni, B., and Krug, R. M. (1983). Influenza virus temperature-sensitive cap (m7GpppNm)-dependent endonuclease. J Virol 45, 27-35.
Vines, A., Wells, K., Matrosovich, M., Castrucci, M. R., Ito, T., and Kawaoka, Y. (1998). The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction. J Virol 72, 7626-7631.
Walker, J. A., and Kawaoka, Y. (1993). Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus. J Gen Virol 74 ( Pt 2), 311-314.
Wang, X., Castro, A. E., Castro, M. D., Lu, H., Weinstock, D., Soyster, N., Scheuchenzuber, W., and Perdue, M. (2000). Production and evaluation criteria of specific monoclonal antibodies to the hemagglutinin of the H7N2 subtype of avian influenza virus. J Vet Diagn Invest 12, 503-509.
Webster, R. G. (1997). Influenza virus: transmission between species and relevance to emergence of the next human pandemic. Arch Virol Suppl 13, 105-113.
Weltzin, R., J. Liu, K. V. Pugachev, G. A. Myers, B. Coughlin, P. S. Blum, R. Nichols, C. Johnson, J. Cruz, J. S. Kennedy, F. A. Ennis, and T. P. Monath. 2003. Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med 9:1125-30.
Wharton, S. A., Weis, W., Skehel, J. J., and Wiley, D. C. (1989). Structure, Function, and Antigenicity of the Hemagglutinin of Influenza Virus. In The Influenza Viruses, R. M. Krug, ed. (New York, Plenum Press), pp. 153-174.
Wood, J. M., Kawaoka, Y., Newberry, L. A., Bordwell, E., and Webster, R. G. (1985). Standardization of inactivated H5N2 influenza vaccine and efficacy against lethal A/Chicken/Pennsylvania/1370/83 infection. Avian Dis 29, 867-872.
</TD></TR></TBODY></TABLE>