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Rev. sci. tech. Off. int. Epiz., 2000, 19 (1), 197-225 Recent zoonoses caused by influenza A viruses D.J. Alexander & I.H. Brown Virology Department, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom Summary Influenza is a highly contagious, acute illness which has afflicted humans and animals since ancient times. Influenza viruses are part of the Orthomyxoviridae family and are grouped into types A, B and C according to antigenic characteristics of the core proteins. Influenza A viruses infect a large variety of animal species, including humans, pigs, horses, sea mammals and birds, occasionally producing devastating pandemics in humans, such as in 1918, when over twenty million deaths occurred world-wide. The two surface glycoproteins of the virus, haemagglutinin (HA) and neuraminidase (NA), are the most important antigens for inducing protective immunity in the host and therefore show the greatest variation. For influenza A viruses, fifteen antigenically distinct HA subtypes and nine NA subtypes are recognised at present; a virus possesses one HA and one NA subtype, apparently in any combination. Although viruses of relatively f e w subtype combinations have been isolated from mammalian species, all subtypes, in most combinations, have been isolated from birds. In the 20th Century, the sudden emergence of antigenically different strains in humans, termed antigenic shift, has occurred on four occasions, as follows, in 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each resulting in a pandemic. Frequent epidemics have occurred between the pandemics as a result of gradual antigenic change in the prevalent virus, termed antigenic drift. Currently, epidemics occur throughout the world in the human population due to infection with influenza A viruses of subtypes H1N1 and H3N2 or with influenza B virus. The impact of these epidemics is most effectively measured by monitoring excess mortality due to pneumonia and influenza. Phylogenetic studies suggest that aquatic birds could be the source of all influenza A viruses in other species. Human pandemic strains are thought to have emerged through one of the following three mechanisms: - genetic reassortment (occurring as a result of the segmented genome of the virus) of avian and human influenza A viruses infecting the same host - direct transfer of whole virus from another species - the re-emergence of a virus which may have caused an epidemic many years earlier. Since 1996, the viruses H7N7, H5N1 and H9N2 have been transmitted from birds to humans but have apparently failed to spread in the human population. Such incidents are rare, but transmission between humans and other animals has also been demonstrated. This has led to the suggestion that the proposed reassortment of human and avian viruses occurs in an intermediate animal with subsequent transference to the human population. Pigs have been considered the leading contender for the role of intermediary because these animals may serve as hosts for productive infections of both avian and human viruses and, in addition, the evidence strongly suggests that pigs have been involved in interspecies transmission of influenza viruses, particularly the spread of H1N1 viruses to humans. Global surveillance of influenza is maintained by a network of laboratories sponsored by the World Health Organization. The main control measure for influenza in human populations is immunoprophylaxis, aimed at the epidemics occurring between pandemics. Keywords Birds - Ecology - Influenza - Interspecies transmission - Mammals - Pandemics Prophylaxis - Public health - Reassortment - Zoonoses. Rev. sci. tech. Off. int. Epiz., 19(1) 198 Introduction and history Influenza in h u m a n s is a highly contagious, acute, primarily respiratory illness. T h e s y m p t o m s and epidemiological characteristics are sufficiently distinct that accounts of e p i d e m i c s c a n b e dated b a c k to ancient times. T h e s e accounts s h o w clearly the occurrence of fairly regular e p i d e m i c s in m o s t populations, with occasional severe e p i s o d e s in w h i c h the disease not only a p p e a r e d to b e m o r e serious and to infect a larger proportion of the population, but also a p p e a r e d to spread world-wide, thus reaching p a n d e m i c status. T h e three types of influenza virus, namely: A, B and C, are all capable of causing infections of h u m a n s . Influenza type C viruses are relatively mild, causing only c o m m o n cold-like s y m p t o m s . Influenza type B viruses cause significant flu-like illnesses w h i c h are nevertheless milder than influenza type A infections. Only influenza A viruses h a v e b e e n k n o w n to cause the devastating infections w h i c h spread throughout the world and this review is limited to these viruses. The first influenza p a n d e m i c for w h i c h m o r e than descriptive reports exist, was that of 1889. Retrospective research h a s partially identified the virus responsible b y testing for influenza antibodies in the serum of p e o p l e w h o w e r e alive at that time (174). By far the worst p a n d e m i c of those recognised to date b e g a n in 1918. During the p a n d e m i c , an estimated 2 0 to 4 0 million p e o p l e died. In well-developed countries, s u c h as the United States of America (USA), approximately 0.5% of the population died. However, in s o m e communities in Alaska and the Pacific islands, half the population p e r i s h e d (85). Subsequent influenza p a n d e m i c s occurred in 1957, 1968 and 1977; while these resulted in far less loss of life, in world-wide terms, than the 1918 p a n d e m i c , significant deaths did occur and the impact o n society in terms of morbidity and consequent e c o n o m i c factors was e n o r m o u s . During the interval b e t w e e n these p a n d e m i c s , lesser epidemics occurred in m o s t h u m a n populations, often with significant impacts o n those populations in terms of morbidity, mortality and e c o n o m y . Despite the importance of influenza as a disease of humans, the earliest recognition of influenza virus in animals related to infections of poultry. A disease capable of causing extremely high mortality in infected domestic fowl was first defined in 1878 and b e c a m e k n o w n as 'fowl plague'. As early as 1 9 0 1 , the causative organism of this disease was s h o w n to b e an ultra-filterable agent (i.e. a 'virus'), although not until 1955 was the close relationship b e t w e e n this agent (and other, milder, viruses isolated from birds) and m a m m a l i a n influenza A viruses demonstrated (137). Isolation of influenza virus as the causative organism of a disease termed 'swine influenza', w h i c h was applied to a 'new' disease of pigs with clinical signs similar to those in h u m a n s first described at the time of the 1918 h u m a n p a n d e m i c (39), also p r e c e d e d the isolation of h u m a n influenza virus. In the late 1920s, S h o p e demonstrated the ability to transmit swine influenza b e t w e e n pigs using ultrafiltered material (148). A virus demonstrated to b e related to the pig virus was eventually isolated from a h u m a n patient in 1 9 3 3 , b y inoculating a filtrate of throat washings into the n o s e s of ferrets (160). The demonstration, in 1955, that 'fowl plague' virus was an influenza A virus, was followed in 1956 b y e v i d e n c e that an emerging respiratory disease in h o r s e s was also caused b y an influenza type A virus (69, 137, 162). In the next two years, respiratory disease caused b y this virus in h o r s e s b e c a m e w i d e s p r e a d in Europe. T h e similarities and association of these viruses was n o t e d b y influenza scientists, a n d the W o r l d Health Organization ( W H O ) encouraged and co-ordinated w o r k o n the e p i d e m i o l o g y of animal viruses, particularly in relation to h u m a n influenza (84). However, the vast reservoirs of influenza viruses that exist in animals, particularly birds, w e r e not fully discovered until the 1970s. Importance for public and animal health Public health Although infection with influenza A viruses m a y b e considered a serious illness with potentially fatal c o n s e q u e n c e s for susceptible individuals, the incidence of influenza A infections in h u m a n populations varies significantly from year to year. Incidence ranges from years of devastating p a n d e m i c s to years w h e n the n u m b e r of cases in a given population d o e s not reach e p i d e m i c status. O n average, e a c h individual case of influenza in the USA is associated with an estimated five to six days of restricted activity, three to four days of b e d disability and approximately three days lost from w o r k or s c h o o l (140). T h e overall effect o n public health will therefore b e generally related to the n u m b e r of individuals that b e c o m e infected, although this in turn m a y b e related to the degree of immunity of individuals in the population w h i c h c a n also m o d i f y the severity of the clinical signs. Clinical attack rates during an e p i d e m i c are difficult to estimate d u e to the p r o b l e m s in m a k i n g a clinical diagnosis, especially w h e n cases of influenza A infection occur at a time w h e n other respiratory viruses, including influenza type B, m a y b e circulating. In the United K i n g d o m (UK) during the 1918 p a n d e m i c , an estimated 2 3 % of the population d e v e l o p e d influenza, in 1957 and 1958, 17% (8 million cases) and in 1969 and 1970, 8% (10). During the p a n d e m i c of 1969 and 1970, serological evidence suggested that a larger proportion of the population of the UK was infected, presumably often subclinically. In interpandemic years, clinical strike rates are highest in the elderly, although the highest infection rates are in s c h o o l children. However, in the p a n d e m i c s of 1918-1919 and 1957, the groups showing Rev. sci. tech. Off. int. Epiz., 19 (1) the highest proportions of clinical disease w e r e children u n d e r fourteen years o l d (10). Influenza A infections significantly affect overall mortality in human populations, and until the occurrence of h u m a n immunodeficiency virus (HIV) infections, w e r e the only viruses to h a v e this effect. Excess mortality d u e to p n e u m o n i a and influenza (P&I) is usually a reliable m a r k e r for assessing the beginning and e n d of an influenza e p i d e m i c , although this is probably an underestimation of influenza-associated deaths (158). Although mortality rates during the 1918 p a n d e m i c were extremely h i g h (3,000 per 1,000,000 in 1918 and 1,170 per 1,000,000 in 1919 in England and W a l e s [10]), significant mortality also occurs during the interpandemic epidemics. T h e c o m b i n e d p a n d e m i c s of 1957 and 1968, in the USA, a c c o u n t e d for approximately 9 8 , 0 0 0 excess deaths; however, the e p i d e m i c s from 1957 to 1975, excluding the pandemic years, accounted for over twice that n u m b e r of excess deaths (41). T h e impact of the p a n d e m i c s is amplified by the difference in the distribution of deaths b y age group c o m p a r e d to the interpandemic e p i d e m i c s . Simonsen et al. observed that during the p a n d e m i c years of 1 9 6 8 - 1 9 7 0 , approximately 5 0 % of the excess P&I deaths in the USA w e r e recorded in p e o p l e less than 65 years old. In contrast, over the entire d e c a d e , only 0%-7% of the excess P&I deaths occurring each year w e r e in the u n d e r 65 age group (158). Similar figures w e r e obtained for the p a n d e m i c of 1957. Thus, while the 1918 p a n d e m i c was particularly n o t e d for greater mortality in young adults than in the m o r e elderly, this m a y be a feature of p a n d e m i c s in general. Human influenza e p i d e m i c s m a y also affect public health as a result of the s o c i o - e c o n o m i c impact and the m o n o p o l i s a t i o n of healthcare resources during an e p i d e m i c . In the past, pandemics and significant e p i d e m i c s have h a d e n o r m o u s effects o n hospital resources. During the e p i d e m i c caused b y the 1957 p a n d e m i c virus, hospital admissions in the UK rose by as m u c h as 2 5 0 % in s o m e areas, while at the p e a k of the epidemic, u p to o n e third of nurses w e r e absent from w o r k (10). Influenza infections m a y leave a legacy o n c e the e p i d e m i c or pandemic is over. Ravenholt and F o e g e h a v e a d v a n c e d a compelling argument that the global p a n d e m i c of a Parkinson-like disease t e r m e d encephalitis lethargica, affecting more than 1,000,000 p e o p l e b e t w e e n 1919 and 1928, was a direct c o n s e q u e n c e of the influenza p a n d e m i c of 1919 (128). Animal health Pigs The disease caused b y influenza viruses in pigs is essentially similar to that r e c o r d e d in h u m a n s , although generally somewhat milder, consisting of an acute febrile, respiratory disease characterised b y fever, apathy, anorexia, coughing, laboured breathing, sneezing, nasal discharge, l o w mortality rate and rapid recovery, usually five to s e v e n days after the onset of clinical signs (148, 115, 4 3 ) . T h e extremely h i g h 199 morbidity h a s serious e c o n o m i c c o n s e q u e n c e s d u e to the increased time n e e d e d to attain slaughter weight as a result of the influenza virus infection. T h e cost h a s b e e n estimated at u p to £7 per pig, accounting for a financial loss to the pig industry in the U K of approximately £ 6 5 million e a c h year (89). Horses Equine influenza is a clinical disease of horses, d o n k e y s and m u l e s caused b y influenza A viruses of subtypes H 7 N 7 and H3N8. T h e disease in fully susceptible animals is similar to that s e e n in pigs and h u m a n s , presenting as a severe respiratory infection with a harsh c o u g h , nasal discharge and pyrexia. Infections with subtype H 3 N 8 are generally considered to b e m o r e severe than H7N7 infections (65). W h e n e q u i n e influenza has b e e n introduced into susceptible populations, characteristic rapid spread and h i g h morbidity h a v e occurred. In highly d e v e l o p e d countries, equine species are largely k e p t for sport or as c o m p a n i o n animals. Thus, while influenza infections can b e a nuisance or, for the h o r s e racing industry, an e c o n o m i c p r o b l e m , the disease is m a n a g e d largely b y vaccination and b y resting clinically infected animals (111). However, in m a n y p o o r e r parts of the world, horses, d o n k e y s and m u l e s remain the principal w o r k i n g animals. As a c o n s e q u e n c e , vaccination and rest m a y not b e an option, and this in turn, results in m o r e severe disease, often with secondary bacterial infections. In relatively recent years, s u c h severe infections have occurred three t i m e s , in India, in 1987 (176), and in the People's Republic of China from 1989 to 1990 (60) and from 1993 to 1 9 9 4 (154). In e a c h case, the disease spread rapidly and widely. In the 1 9 9 3 - 1 9 9 4 o u t b r e a k in the People's Republic of China, an estimated 2,245,000 horses w e r e affected clinically and 2 4 , 6 0 0 (1%) died (154). In the e p i d e m i c of 1 9 8 9 - 1 9 9 0 in the north-east of the People's Republic of China, morbidity was estimated at 8 1 % , with mortality varying in different h e r d s , but reaching 2 0 % or m o r e in s o m e (60). Birds In birds, the clinical signs and disease o b s e r v e d following infection with influenza A viruses vary according to the host species, age, the presence of o t h e r micro-organisms and environmental factors. In susceptible avian species, u n c o m p l i c a t e d infections can b e divided too t w o forms b a s e d o n the severity of the clinical disease p r o d u c e d . T h e very virulent viruses cause a disease formerly k n o w n as fowl plague and n o w t e r m e d highly pathogenic avian influenza (HPAI), in w h i c h mortality m a y b e as h i g h as 1 0 0 % . T h e s e virases have b e e n restricted to subtypes H5 and H7, although not all viruses of these subtypes cause HPÄ1. Seventeen primary isolates of such viruses h a v e b e e n reported in domestic poultry since 1959 (5). Apart from s u d d e n onset of mortality, clinical signs associated with HPAI vary enormously and n o n e are p a t h o g n o m o n i c . Generally, all or s o m e of the following m a y b e o b s e r v e d : cessation of egg laying, respiratory signs, 200 excessive lachrymation, sinusitis and, m o r e characteristically, o e d e m a of the h e a d face and n e c k and cyanosis of the unfeathered skin (46). Infections of poultry with HPAI are often self-limiting or rapidly controlled b y slaughter. However, o n three occasions in recent years, in Pennsylvania, USA, from 1983 to 1984 (187), in Mexico from 1994 to 1995 (31, 179) and in Pakistan from 1994 to 1995 (113), the viruses have b e c o m e w i d e s p r e a d in c h i c k e n s and have h a d severe implications for animal health and the e c o n o m y of the country or region in w h i c h they occurred. T h e impact of these outbreaks is best m e a s u r e d in losses of birds and financial terms. For example, in the e p i d e m i c in Pennsylvania, m o r e than 17,000,000 birds w e r e slaughtered or died. Lasley estimated that this cost the Government of the USA US$60,000,000, with a further US$15,000,000 b o r n e b y the farmers, and that subsequent rises in f o o d prices cost the consumer US$349,000,000 (99). All other viruses cause a m u c h milder disease consisting primarily of respiratory disease, depression and egg production p r o b l e m s in laying birds. In addition, these n o n pathogenic avian influenza viruses m a y replicate in the epithelial cells of the intestine of birds without inducing signs of disease (83), although virus m a y b e s h e d in high concentrations in the faeces (159, 8 3 , 90). Infections such as Pasteurella multocida, avian pneumovirus, avian paramyxovirus type 3, infectious bronchitis virus and e v e n live bacterial or virus vaccines or environmental conditions m a y result in significant severe disease, s o m e t i m e s with mortalities sufficiently h i g h to b e mistaken for HPAI. Typical of these extremes was the infected turkey b r e e d e r flock reported b y Alexander and S p a c k m a n as infected with virus of H7 subtype, in w h i c h the only sign was 2 % white, m i s s h a p e n eggs (7); c o m p a r e d to the outbreaks caused b y a virus of H4N8 subtype in flocks of c h i c k e n s in Alabama, USA, in 1975, in w h i c h u p to 6 9 % mortality was reported, including one flock with 3 1 % mortality in a single day (80). Viruses of low pathogenicity m a y b e c o m e w i d e s p r e a d in a given area, incurring substantial e c o n o m i c losses. During 1995, 178 turkey farms in Minnesota, USA, were affected b y influenza A virus of H 9 N 2 , resulting in the worst e c o n o m i c loss due to influenza infections r e c o r d e d in a single year in Minnesota (approximately US$6,000,000) (63). Aetiology Taxonomy The influenza viruses are m e m b e r s of the Orthomyxoviridae family w h i c h includes three genera, o n e consisting of types A and B influenza viruses, a s e c o n d containing type C influenza viruses, and a third containing 'Thogoto-like viruses' (78). Influenza viras types A, B and C infect h u m a n s but, e x c e p t for occasional reports, infections of other animals are restricted to type A influenza viruses. Only influenza A viruses have b e e n isolated from birds. Rev. sci. tech. Off. int. Epiz., 19(1) Influenza A virus particles appear roughly spherical or filamentous, with diameter of 80 n m to 120 n m . The nucleocapsid s h o w s helical symmetry and is e n c l o s e d within a protein matrix. External to the matrix is a lipid m e m b r a n e , the surface of w h i c h is c o v e r e d b y two types of glycoprotein projections, or spikes, with w h i c h haemagglutinin (HA) and neuraminidase (NA) activities are associated. T h e s e two surface glycoproteins, particularly the HA, appear to b e the most important antigens in terms of stimulation of protective immunity in the host. Consequently, considerable antigenic variation is s e e n in these p o l y p e p t i d e s while other polypeptides are antigenically m o r e stable. T h e classification and nomenclature of influenza viruses take account of the antigenic variation that exists (193). Influenza viruses are g r o u p e d into types A, B and C o n the basis of the antigenic nature of the internal nucleocapsid or the matrix protein. B o t h these antigens are c o m m o n to all viruses of the s a m e type (138). Viruses of influenza A type are further divided into subtypes o n the basis of the HA and NA antigens. At present, fifteen HA (H1-H15) and nine NA (N1-N9) subtypes are k n o w n ; a virus possesses o n e HA and o n e NA subtype, apparently in any c o m b i n a t i o n (72). T h e system of nomenclature for influenza viruses consists of several c o m p o n e n t parts. T h e information is presented as follows: type, host of origin (for n o n - h u m a n species), geographical origin, strain n u m b e r and year of isolation. This is followed by the antigenic subtype in parentheses, e.g. A/duck/Alberta/ 3 5 / 7 6 (H1N1). Molecular biology The g e n o m e s of influenza A and B viruses consist of eight unique segments of single-stranded ribonucleic acid (RNA) w h i c h are of negative polarity. Influenza C viruses possess seven segments of RNA. T h e viral RNA is transcribed to complementary messenger RNA b y a virus-associated polymerase c o m p l e x (designated PB1, PB2 and PA). T o b e infectious, a single virus particle must contain e a c h of the eight unique RNA segments. It is likely that the incorporation of RNA into the virion is at least partly r a n d o m . T h e r a n d o m incorporation of RNA segments allows the generation of progeny viruses containing novel combinations of genes w h e n cells are concurrently infected with two different parent viruses. This p h e n o m e n o n is referred to as genetic reassortment ( 7 4 , 1 8 2 ) . T h e HA protein is an integral m e m b r a n e protein and the major surface antigen of the influenza virus virion. This protein is responsible for binding of virions to host cell receptors and for fusion b e t w e e n the virion e n v e l o p e and the host cell. Newly synthesised HA is cleaved to r e m o v e the amino-terminal h y d r o p h o b i c s e q u e n c e w h i c h is the signal s e q u e n c e for transport to the cell m e m b r a n e . Carbohydrate side chains are a d d e d ; the n u m b e r and position of these chains vary with the virus strain. Fusion activity requires post-translational cleavage of HA b y cellular proteases into the disulphide-linked fragments HAI and HA2. This cleavage of 201 Rev. sci. tech. Off. int. Epiz., 19(1) the HA d o e s not affect the antigenic or receptor-binding properties of the protein (105), but is essential for the virus to be infectious (93) and is an important determinant in pathogenicity (134). Molecules of H A form h o m o t r i m e r s during maturation. T h e three-dimensional structure of the complete trimer h a s b e e n determined, consisting of a globular head (HA1) o n a stalk (HA1/2). T h e h e a d contains the receptor-binding cavity in addition to m o s t of the antigenic sites of the m o l e c u l e . T h e carboxy-terminus of HA2 anchors the glycoprotein in the cell or virion m e m b r a n e . T h e HA is subject to a h i g h rate of mutation d u e to error p r o n e viral RNA polymerase activity. Selection for a m i n o acid substitutions in the variants p r o d u c e d is driven at least in part b y i m m u n e pressure, as the HA is the major target of the host i m m u n e response. T h e amino acids m a k i n g u p the receptor binding site are highly conserved but the remainder of the HA molecule is highly mutable. T h e fifteen subtypes of HA recognised currently differ b y at least 3 0 % in the a m i n o acid sequence of HA1 and are not cross-reactive serologically. Subtypes m a y include several variant strains w h i c h are only partially cross-reactive in serological assay. The NA is the s e c o n d major surface antigen of the virus which, like HA, is an integral m e m b r a n e glycoprotein. T h e protein functions to free virus particles from host cell receptors, to enable progeny virions to e s c a p e from the cell in which they arose, and s o facilitate virus spread. This activity destroys the HA receptor o n the host cell (27), preventing progeny virions reabsorbing to the host cell (123). Like HA, the NA is highly mutable with variant selection driven b y host immune pressure. T h e nine subtypes identified in nature to date are not cross-reactive serologically, although variants within subtypes are partially cross-reactive serologically. Ecology Influenza A viruses infect a large variety of animal species, including h u m a n s , pigs, h o r s e s , sea m a m m a l s and birds (Tables I and II) (2, 189). Recent phylogenetic studies of influenza A viruses h a v e revealed species-specific lineages of viral genes and h a v e demonstrated that the prevalence of interspecies transmission is d e p e n d e n t o n the animal species involved (189). In the early 1970s, the W H O initiated long-term global studies o n the influenza viruses of l o w e r animals and birds to determine as far as possible w h e t h e r a finite n u m b e r of influenza A viruses existed in nature and w h e t h e r it w a s possible to isolate a future p a n d e m i c strain of virus from a m o n g these viruses, in advance of the appearance of a n e w strain in h u m a n s . A vast n u m b e r of viruses h a v e b e e n isolated from a w i d e variety of birds and a range of terrestrial and sea m a m m a l s . Although an e n o r m o u s diversity of animal species h a v e b e e n s h o w n to b e susceptible to influenza A virus infections, three groups of animals appear to b e far m o r e important in terms of n u m b e r s a n d the e p i d e m i c / e n d e m i c nature of the disease than other animals, namely: birds, pigs and horses. Influenza viruses in humans Despite the recognition of fifteen distinct haemagglutinin subtypes (H1-H15) and nine neuraminidase subtypes (N1-N9), the n u m b e r of subtype combinations found to b e Table I Haemagglutinin (H) subtypes of influenza A viruses isolated from humans, pigs, horses and birds Subtype Examples(a) of viruses of the subtype isolated from the specified host group Humans Pigs Horses Birds m PR/8/34 (H1N1) Swìne/lowa/15/30 (H1N1) _(b) Duck/Alberta/35/76 (H1N1) H2 Singapore/1/57 (H2N2) - - Duck/Gennany/1215/73 (H2N3) H3 Hong Kong/1/68 (H3N2) Swine/Taiwan/70 (H3N2) Equine/Miami/1/63 (H3N8) H4 -Hong Kong/156/97 (H5N1) -Swine/HK/9/98 (H9N2) - Duck/Utaine/1/63fH3H8) Duck/CzeehoslovakR/56 (H4N6) H5 H6 H7 H8 -England/268/96 (H7N7) H9 -Hong Kong/1073/99 (H9N2) H10 - H11 - H12 H13 H14 H15 - - - - a) The reference strains of influenza viruses, or the first isolates of the subtype from the host group b) Not found in this host group FPV: fowl plague virus - Tem/S.Africa/61 (H5N3) Turkey/Massacfousetts/3?40'/S5 (H6N2) Equine/Prague/1/56(H7N7) FPV/Dutch/27 (H7N7) - Turkey/0ntario/8118/6B (H8N4) - Duck/England/56 (H11N6) - Duck/Albcrta/60/76(H12N5) - Gull/Maryland/7Q4/77 (H13N6) - Duck/Gurjev/263/82 (H14N5) Turkey/Wiscoosin/1/66 (H9N2) Chicken/Germany/N/49 (H10N7) Duck/AtJStralia/341/83 (H15N8) Rev. sci. tech Off. int. Epiz.. 19 (1) 202 Table II Neuraminidase (N) subtypes of influenza A viruses isolated from humans, pigs, horses and birds Examples Subtype (a) of viruses of the subtype isolated from the specified host group Humans Pigs Horses N1 PR/8/34 (H1N1) Swine/lowa/15/30(H1N1) -(b) Chicken/Scotland/59 (H5N1) N2 Singapore/1/57 (H2N2) Swine/Taiwan/70 (H3N2) N3 N4 _ _ _ N6 _ -- Turkey/0ntarlo/6118/68 (H8N4) N5 - -- Turkey/Massachusetts/3740/65(H6N2) N7 England/268/96 (H7N7) Swine/England/92(H1N7) Equine/Prague/1/56 (H7N7) FPV/Dutch/27 (H7N7) N8 - Duck/Ukraine/1/63 (H3N8) N9 - - Equine/Mlaml/1/63 (H3N8] - Duck/Memphis/546/74 (H11N9) - Birds Tern/S.Africa/61 (H5N3) Shearwater/Australla/1/72 (H6N5) Duck/Czechoslovakia/56 (H4N6) a) The reference strains of influenza viruses, or the first isolates of the subtype from the host group b) Not found in this host group FPV: fowl plague virus prevalent in h u m a n s and other m a m m a l s at any o n e time appears to b e severely limited. Pandemics of influenza occur w h e n a virus is introduced w h i c h is fully capable of replication and spread, and for w h i c h all or a large proportion of the population have h a d n o immunological experience of at least the functionally important haemagglutinin. This is termed antigenic shift and has occurred four times during the 20th Century, e a c h time resulting in a true p a n d e m i c . T h e first of these shifts was, almost certainly, in 1918, w h e n virus of H1N1 entered the h u m a n population, resulting in the most devastating p a n d e m i c k n o w n to m a n . T h e s e c o n d was in 1957, w h e n H 2 N 2 virus appeared. T h e third, in 1968, with a H 3 N 2 virus and the fourth in 1977, with the re-emergence of the H1N1 virus. In 1957 and 1968, the introduction of a n e w influenza virus coincided with the disappearance of the earlier subtype, but this did not occur in 1977, and currently b o t h H 3 N 2 and H1N1 viruses are circulating. The emergence of n e w subtypes of influenza virus and the ensuing p a n d e m i c s are unmistakable, but the e p i d e m i c s occurring b e t w e e n the p a n d e m i c s m a y nevertheless b e severe. These are a result of the gradual change of the antigenicity of the circulating virus b y the accumulation of point mutations until the virus is antigenically sufficiently different from earlier strains that a large proportion of the population is susceptible and cases reach e p i d e m i c levels. This accumulative variation is termed antigenic drift, and the size, severity and spread of the virus will d e p e n d o n the degree to w h i c h the virus is antigenically different from viruses already experienced b y the population. Examination of serum samples taken from elderly p e o p l e prior to the p a n d e m i c s of 1957 or 1968 has b e e n u s e d to assess virus subtypes present in h u m a n s before H 1 N 1 . 'Sero-archaeology' studies h a v e indicated that the two prevalent viruses preceding 1918 w e r e H 2 N 2 in 1889 and H 3 N 8 in 1900 (189), further confirming the limited range of subtypes k n o w n to cause influenza e p i d e m i c s in humans. Influenza viruses in pigs Swine influenza (SI) was first o b s e r v e d at the time of the p a n d e m i c in h u m a n s in 1918 and the viruses responsible are k n o w n to b e closely related, possibly having derived from a c o m m o n ancestor (130). Although the disease was described in pigs during the years w h i c h followed, not until 1930 was the virus isolated and identified. This H 1 N 1 virus was the prototype strain of a group of viruses n o w k n o w n as classical viruses, w h i c h h a v e b e e n reported in pig populations world-wide. Influenza A viruses of subtypes H 1 N 1 and H 3 N 2 h a v e b e e n reported widely in pigs and h a v e b e e n frequently associated with clinical disease. T h e s e include classical swine H1N1, avian-like H1N1 and h u m a n - and avian-like H 3 N 2 viruses. Influenza is widespread and e n d e m i c in pig populations world-wide and is responsible for o n e of the most prevalent respiratory diseases in pigs. Occasionally, influenza infections of pigs m a y result in e p i d e m i c s through the introduction of virus to an immunologically naïve population or due to significant antigenic drift in the HA or NA of e n d e m i c viruses. Swine influenza is related to the m o v e m e n t of animals from infected to susceptible herds, clinical disease generally appears with the introduction of n e w pigs into a herd. Once a h e r d is infected, the virus is likely to persist through the production of young susceptible pigs and the introduction of n e w stock. Outbreaks of disease occur throughout the year but usually p e a k in the colder m o n t h s (45). Infection is frequently subclinical, and often typical signs are s e e n in only 2 5 % to 3 0 % of a herd. Swine husbandry practices influence directly the evolution of SI viruses through r e d u c e d immune pressure and constant availability of susceptible hosts, generally leading to r e d u c e d genetic drift in the genes encoding HA and NA, c o m p a r e d to those of similar viruses in the h u m a n population. However, mixing of pigs from multiple-sources and the h i g h frequency of contact with other 203 Rev. sci. tech. Off. int. Epiz., 19 (1) species, particularly h u m a n s , provides an opportunity for co-circulation of viruses and genetic reassortment. Serological studies of pigs w o r l d - w i d e have s h o w n that classical swine H1N1 influenza virus is prevalent throughout the major pig populations, with approximately 2 5 % of animals demonstrating e v i d e n c e of infection (24, 70). Classical swine H 1 N 1 influenza virus isolates in the USA h a v e remained conserved b o t h genetically (104) and antigenically (146), but viruses w h i c h are antigenically distinguishable, although closely related, h a v e b e e n reported b y Olsen e£ al. (120) and W e n t w o r t h et al. (190). In Canada, an H1N1 virus was associated with a n e w and distinctive pathology in pigs which a p p e a r e d in 1990. However, the virus was m o s t closely related to classical swine influenza viruses (131). In Europe, classical viruses r e a p p e a r e d in 1976 (116), but h a v e b e e n largely r e p l a c e d since 1979 b y 'avian-like' swine H 1 N 1 viruses which are antigenically and genetically distinguishable from the classical swine H 1 N 1 influenza viruses of North America (125, 142). Influenza A viruses of H 3 N 2 subtype, related closely to early human strains, h a v e b e e n widely reported from pigs, particularly in E u r o p e and Asia w h e r e they continue to circulate long after disappearing from the h u m a n population. These viruses, following years of adaptation to pigs, are associated with clinical signs in pigs w h i c h are typical of SI (61). Seroprevalence is usually in the range 3 0 % to 5 0 % (98, 132), but can b e as h i g h as 7 5 % (175). This apparently h i g h level of H 3 N 2 infections in Europe is in sharp contrast to the low prevalence in pigs in North America. S o m e of the H 3 N 2 viruses isolated from pigs in Asia are entirely avian-like (91), but repeated introductions from h u m a n s appear to occur regularly. The prevailing h u m a n H1N1 strains are transmitted frequently to pigs and are occasionally associated with respiratory epizootics in pigs (24, 8 7 ) . However, most strains are not readily transmitted a m o n g pigs in the field and are not therefore maintained entirely independently of the h u m a n population (70). Influenza A H 1 N 2 viruses, derived from classical swine H 1 N 1 and 'human-like' swine H 3 N 2 viruses h a v e b e e n isolated in Japan (169) a n d France ( 5 5 ) . In J a p a n , these viruses a p p e a r to have spread widely within pig populations and are associated frequently with respiratory epizootics (121). Subsequently, an H1N2 influenza virus related antigenically to h u m a n and 'human-like' swine viruses h a s e m e r g e d and b e c o m e e n d e m i c in pigs in the UK, often in association w i t h respiratory disease (23). Influenza viruses in horses Influenza is a c o m m o n disease of h o r s e s throughout the world. Apart from rare reports of isolations or serological evidence of infection with other subtypes, only two subtypes of influenza A virus, H 7 N 7 and H3N8, have b e e n identified as infecting a n d causing disease in horses. Subtype H 7 N 7 viruses m a y h a v e d i s a p p e a r e d largely from the h o r s e population, as n o substantiated reports of virus isolated from horses h a v e b e e n r e c o r d e d since 1980 (180). However, recent w o r l d - w i d e serological surveillance has suggested that H 7 N 7 m a y b e circulating in Eastern Europe ( 1 1 1 , 106) and Central Asia (189). Phylogenetic analyses of H 3 N 8 (equine-2) viruses h a s revealed the existence of two distinct evolutionary lineages, E u r o p e a n and American, the circulation of w h i c h was originally centred largely o n the geographical origin (112). However, in Europe, b o t h lineages n o w appear to b e co-circulating, with the American type viruses predominant in s o m e areas (122). T h e s e findings h a v e importance for control, since vaccines including o n e H 3 N 8 virus type m a y not provide protection against infection with viruses from the heterologous group. Recently, equine influenza o u t b r e a k s d u e to infection with H 3 N 8 virus w e r e o b s e r v e d in South Africa, India, the People's Republic of China, Hong K o n g a n d Nigeria, w h e r e e q u i n e influenza viruses w e r e not k n o w n to b e circulating. Recent outbreaks in the People's Republic of China h a v e b e e n d u e to b o t h conventional strains of H 3 N 8 virus, and viruses w h i c h contained the s a m e surface antigens as the other e q u i n e viruses of this subtype, but w h i c h h a d genetic features that w e r e avian-like, indicating that the virus h a d b e e n introduced from birds (60, 188). Sporadic outbreaks typically occur and may, in part, b e d u e to antigenic drift, w h i c h m a y c o m p r o m i s e the efficacy of the vaccines available ( 1 8 , 1 1 2 ) . Influenza viruses in birds W i l d birds T h e first isolation of influenza virus from feral birds was in 1961, from c o m m o n terns (Sterna hirundo) in South Africa (14). However, the e n o r m o u s p o o l s of influenza viruses w h i c h were present in the wild bird population w e r e not revealed until systematic investigation of influenza in feral birds was u n d e r t a k e n in the 1970s, following the 1968 p a n d e m i c in h u m a n s . Isolations of virus from other wild birds h a v e b e e n completely o v e r s h a d o w e d b y t h e n u m b e r , variety and w i d e s p r e a d distribution of influenza viruses i n waterfowl, order Anseriformes. In the surveys listed b y Stallknecht a n d Shane, a total of 2 1 , 3 1 8 s a m p l e s from all s p e c i e s resulted i n the isolation of 2,317 (10.9%) viruses ( 1 6 3 ) . Of these s a m p l e s , 14,303 w e r e from b i r d s of the o r d e r Anseriformes a n d y i e l d e d 2,173 (15.2%) isolates. T h e next highest isolation rates w e r e 2.9% and 2 . 2 % from the Passeriformes a n d Charadriiformes. T h e overall isolation rate from all b i r d s o t h e r than d u c k s and geese was 2 . 1 % . In a three-year study of waterfowl congregating o n lakes in Alberta, Canada, prior to migration, Hinshaw et al. found an overall isolation rate of 2 6 % , with 60% of juveniles excreting virus in the first year of the life of the bird (71). T h e large n u m b e r s and concentration of waterfowl o n s u c h lakes, c o u p l e d with the facts that d u c k s h a v e b e e n reported to excrete u p to 1 0 m e a n egg infectious d o s e s of virus p e r g r a m of faeces (184), and that this virus 8 . 7 Rev. sci. tech. Off. int. Epiz., 19 (1) 204 remains viable for considerable periods in l a k e water (164), must represent a unique level of viral contamination of a large environment. C a g e d ' p e t ' birds Since 1975, w h e n the first isolates from caged birds w e r e r e c o r d e d , isolates from all sources have b e e n mainly of H 4 or H 3 subtypes. T h e majority of influenza viruses from caged birds c o m e from passerine species and only rarely are psittacines infected. Although the presence of influenza viruses in birds h e l d in quarantine is monitored continually in several countries around the world, periods w h e n n o isolations have b e e n m a d e h a v e b e e n r e c o r d e d , often lasting several years. Africa in 1995, and the Republic of K o r e a in 1996 (9). Since 1997, serious p r o b l e m s associated with H 9 N 2 virus have b e e n reported in Iran, Saudi Arabia, Pakistan, the People's Republic of China and other countries of Asia. In Minnesota, USA, during 1 9 9 5 , 1 7 8 turkey farms w e r e infected with virus of subtype H 9 N 2 (63). The influenza status of c o m m e r c i a l d u c k s in m o s t countries is poorly understood or h a s not b e e n investigated fully. However, c o m m e r c i a l d u c k s , especially fattening d u c k s , are generally reared o n p o n d s or fields outdoors and w h e n surveillance of c o m m e r c i a l d u c k s h a s b e e n undertaken, e n o r m o u s p o o l s of virus and m a n y subtype combinations h a v e b e e n detected (3). D o m e s t i c poultry At the e n d of the 19th Century and in the early 20th Century, 'fowl plague', the highly pathogenic form of avian influenza, was often reported in c h i c k e n s and was probably enzootic in several countries. However, in the s e c o n d half of the 20th Century, reports of influenza infections of c h i c k e n s have b e e n rare c o m p a r e d to infections of other domestic poultry, despite the m u c h higher populations of chickens. For e x a m p l e in the USA, despite frequent influenza epizootics in turkeys in s o m e States, b e t w e e n 1964 and 1982, only three outbreaks w e r e r e c o r d e d in c h i c k e n s (127). Ratites T h e increase in trade in ostriches and other ratites during the 1990s led to the m o v e m e n t of large n u m b e r s of s u c h birds around the world. T h e testing for viruses, including influenza, has resulted in the regular isolation of influenza viruses from these birds. Since these birds are mainly k e p t in o p e n fields, influenza infections p r o b a b l y represent spread from feral birds. Despite the l o w incidence of influenza infections of c h i c k e n s throughout the world, twelve outbreaks of HPAI h a v e occurred since 1959, with significant spread of infection in Pennsylvania and neighbouring States of the USA during 1983 and 1984, and in Mexico and Pakistan in 1994 and 1995. Outbreaks since 1959 have s h o w n n o or extremely limited spread. Eight HPAI outbreaks in b a c k y a r d poultry flocks infected with H 5 N 2 virus w e r e reported in Italy in 1997 and 1998. Outbreaks of H 5 N 1 HPAI occurred o n three farms in H o n g Kong from March to May 1997, with 7 0 % - 1 0 0 % mortality and subsequent spread to live bird markets (35). Differences in pathogenicity a m o n g influenza viruses result in the production of a w i d e spectrum of clinical diseases that range in severity from fatal systemic to mild, sometimes inapparent, respiratory disease (43, 108). Severity of the disease is also determined b y the host species infected (6, 8, 77) and in part b y factors s u c h as host age and sex, virus dose, environment and concurrent infections with other pathogens (46). Turkeys tend to b e reared in less substantial a c c o m m o d a t i o n than c h i c k e n s or o n o p e n range and m o s t of the major turkey-producing countries h a v e h a d disease p r o b l e m s associated with influenza infections. In the USA, in California and Minnesota, w h e r e turkey farms are not only heavily concentrated but are also situated o n migratory waterfowl flyways and are reared o n range, influenza virus infections have b e e n s e e n regularly and m a y represent a significant e c o n o m i c loss (63). In other countries w h e r e these birds are generally reared indoors, infections of turkeys are not s e e n regularly and in the years w h e n infection h a s occurred, these have usually b e e n restricted to o n e or two isolated incidents. During the period b e t w e e n 1994 and 1999, infections of poultry with influenza viruses of H 9 N 2 subtype appear to have b e e n c o m m o n world-wide. Outbreaks occurred in Italy in 1994, Germany from 1995 to 1996, Ireland in 1997, South Pathogenesis T h e pathogenesis of infection with influenza virus is difficult to define precisely, since it is c o m p l i c a t e d frequently b y the effects of infection with secondary bacteria. Severe damage to the epithelium of the respiratory tract is the main feature in infections of m a m m a l s with influenza virus. T h e resulting d a m a g e to the epithelium facilitates secondary infection by bacterial respiratory pathogens. Humans In h u m a n s , the disease incubation p e r i o d will vary b e t w e e n one and three days, d e p e n d i n g o n the virus strain and dose. Typically, the onset of clinical signs is rapid, characterised by malaise, fever, nasal s y m p t o m s , an unproductive cough, myalgia and h e a d a c h e . Initially, typical s y m p t o m s are rhinitis followed b y tracheobronchitis and, infrequently, an interstitial pneumonitis (40). T h e disease p r o c e s s usually damages the respiratory tract from the n o s e to the small b r o n c h i , but rarely damages alveolar cells (100). T h e illness is rapidly prostrating and usually lasts from three to five days, b e i n g m o s t severe in children and the elderly. Complications include primary viral or secondary bacterial p n e u m o n i a (68, 101) and these often 205 Rev. sci. tech. Off. int. Epiz.. 19 (1) confuse the respiratory pathology following infection with influenza virus. Significant pathological changes in other organs have not b e e n o b s e r v e d consistently. Influenza A virus infection of h u m a n s and other m a m m a l s can result in gross lung lesions w h i c h are patchy and randomly distributed throughout the l o b e s . T h e altered lung areas are d e p r e s s e d and consolidated, d a r k red or purple red in colour, contrasting sharply with n o r m a l lung tissue. Infection of h u m a n s with an avian influenza A virus of H 5 N 1 subtype in H o n g K o n g in 1997, a p p e a r e d to result in a higher rate of complications in patients hospitalised w h e n c o m p a r e d to infections with the prevailing h u m a n subtypes H 1 N 1 and H3N2. F e w of the severely affected patients h a d active underlying disease, but additional manifestations s u c h as gastrointestinal and renal failure w e r e prominent, an unusual feature of influenza infections in h u m a n s (197). Other mammalian species Disease signs in pigs and h o r s e s a p p e a r suddenly in all or a large n u m b e r of animals of all ages k e p t within a single unit, following an incubation p e r i o d of o n e to three days. In pigs, morbidity is high, resulting in serious e c o n o m i c implications for the farmer. Secondary bacterial infections in b o t h pigs and horses can often increase the severity of the illness and m a y result in complications s u c h as p n e u m o n i a , in addition to strangles in horses (81). In pigs, the b r o n c h i and b r o n c h i o l i are dilated a n d filled with exudate. Bronchial and mediastinal l y m p h n o d e s are usually hyperaemic and enlarged (148, 177). Histologically, widespread degeneration and necrosis of the epithelium occurs in the b r o n c h i and bronchioli. T h e l u m e n of b r o n c h i , bronchioli and alveoli are filled with exudate containing desquamated cells and neutrophils progressing to mainly monocytes. Furthermore, dilatation of the capillaries and infiltration of the alveolar septae w i t h l y m p h o c y t e s , histiocytes and plasma cells occurs. W i d e s p r e a d interstitial pneumonia and e m p h y s e m a a c c o m p a n y these lesions, although the severity of the former is d e p e n d e n t o n the infecting strain (21). In North America, a proliferative necrotising p n e u m o n i a of pigs is characterised b y w i d e s p r e a d hyperplasia of type II epithelial cells (51). Influenza infection of other animals, s u c h as ruminants, sea mammals, d o g s and mustelids usually results in subclinical disease. However, H7N7 and H 4 N 5 influenza A viruses w e r e associated with a h i g h mortality in Harbour seals at C a p e C o d , USA, in 1979 (97) and 1982 (167), respectively, and w e r e isolated from the lungs and brains of d e a d animals. Avian species In birds, the disease signs can vary considerably as d e s c r i b e d earlier. Infection of birds with highly virulent avian viruses is characterised b y haemorrhagic, necrotic, congestive and transudative changes. Haemorrhagic changes are frequently severe in the oviducts a n d intestines. S o m e t i m e s , despite a h i g h titre of virus, n o lesions occur in the lung (135). Encephalitis m a y d e v e l o p in the c e r e b r u m and c e r e b e l l u m , especially in broilers (1). Alterations to myocardial tissues h a v e b e e n o b s e r v e d following infection with highly pathogenic strains (126). T h e haemagglutinin glycoprotein for influenza viruses is produced as a precursor, HAO, which requires post-translational cleavage b y host proteases b e f o r e it is functional and virus particles are infectious (134). T h e HAO precursor proteins of avian influenza viruses of l o w virulence for poultry have a single arginine at position - 1 from the cleavage site and another at position - 4 . T h e s e viruses are limited to cleavage b y h o s t proteases s u c h as trypsin-like e n z y m e s and are thus restricted to replication at sites in the host w h e r e s u c h e n z y m e s are found, i.e. the respiratory and intestinal tracts. T h e HPAI viruses possess multiple basic a m i n o acids (arginine and lysine) at the HAO cleavage sites, either as a result of apparent insertion or apparent substitution (145, 178, 191), and appear to b e cleavable b y a ubiquitous protease(s), p r o b a b l y o n e or m o r e proprotein-processing subtilisin-related e n d o p r o t e a s e s of w h i c h furin is the leading candidate (165). T h e s e viruses are able to replicate throughout the bird, damaging vital organs and tissues w h i c h results in disease and d e a t h ( 1 3 4 ) . Typical cleavage site amino acid s e q u e n c e s for H5 viruses of h i g h and l o w virulence are s h o w n in T a b l e III. Table III Amino acid sequences at the HAO cleavage site of H5 influenza A viruses in relation to their virulence for chickens (4) Virus - Amino acids at HAO cleavage site (marked by * ) H5 viruses low pathogenicity Various sources > 100 isolates -PQRETR*GLF- H5 viruses high pathogenicity 1994/1995 Mexican isolates (H5N2) -PORKRKTR*GLF- Chicken/Scotland/59(H5Nt! -PORKKR*GLF- Tern/S.Africa/61 (H5N3) -PORETRRROKR*GLF- Chicken/Pennsylvania/1370/83 (H5N2) -PQKKKR*GLF- Turkey/England/50-92/911H5N1) -PQRKRKTR*GLF- HK/156/97 (H5N1) (human) -PQRERRRKKR*GLF- Ck/HK/97 (H5N1) —PQRERRRKKR*GLF— Poultry/ltaly/97 (H5N2) -PQRRRKKR*GLF- HAO: uncleaved haemagglutinin gene Data taken from: Genbank or viruses sequenced at the Veterinary Laboratory Agency-Weybridge Arginine (RI and lysine (K) ara basic amino acids 206 Rev. sci. tech. Off. int. Epiz., 19(1) a) Direct transmission Phylogenetic evidence suggests that an influenza A virus possessing eight gene segments from avian influenza reservoirs may have been transmitted directly humans and pigs before 1918. This virus is believed to have caused the severe Spanish influenza pandemic of 1918. Direct transmissions of H5N1 and H9N2 viruses from chickens to humans, whilst not resulting in a pandemic to date, demonstrate that avian species may be a direct source of virus for humans. b) Genetic reassortment In 1957, the Asian pandemic virus H2N2, appears to have acquired three genes (PB1, HA and NA) from the avian influenza gene pool in wild ducks by genetic reassortment with the circulating human strain from which it retained five other genes. Following the appearance of this virus, the H1N1 strains disappeared from humans. In 1968, the Hong Kong pandemic virus H3N2 probably originated by a similar mechanism. It has been suggested that the reassortment event leading to the production of Asian and Hong Kong pandemic viruses may have occurred in an intermediate host such as the pig, since the latter is receptive to, and allows productive replication of, both human and avian influenza viruses. Fig. 1a & Fig. 1b Theoretical origin of influenza A viruses circulating in humans since 1918 Rev. sci. tech. Off. int. Epiz., 19(1) 207 Epidemiology Theories of pandemic human influenza Three major pandemics occurred in the 20th Century, caused by viruses antigenically 'new' to the host population (antigenic shift), interspersed with both minor and major epidemics. Pandemic strains generally appear through genetic reassortment. Because viral RNA is segmented, genetic reassortment can readily occur in mixed infections with different strains of influenza A viruses (182,183). This means that when two viruses infect the same cell, progeny viruses may inherit sets of RNA segments made up of combinations of segments identical to those of either of the parent viruses. This gives a theoretically possible number of 2 (256) different combinations that can form a complete set of RNA segments from a concurrent infection, although in practice, only a few progeny virions possess the correct gene constellation required for viability (74,189). The new subtypes of influenza viruses which appeared in humans in 1957 (Asian influenza), 1968 (Hong Kong influenza) and 1977 (Russian influenza), 8 had several features in common. The appearance of these subtypes was sudden, the viruses were antigenically distinct from the influenza viruses then circulating in humans, they were confined to HI, H2 and H3 subtypes and the first outbreaks occurred in South-East Asia. Phylogenetic evidence suggests that these pandemic strains were derived from avian influenza viruses either after reassortment (Fig. la) or by direct transfer (Fig. lb) (189). The appearance of the H2N2 and H3N2 subtypes was paralleled by the disappearance from the human population of the subtypes circulating previously, H1N1 and H2N2, respectively. This phenomenon probably occurred in 1918 when emerging H1N1 viruses replaced H3-like viruses. The reasons for the sudden disappearance of human strains circulating previously are unknown, but the earlier strain is possibly disadvantaged compared with the new strain because it has already elicited widespread immunity in the human population. This may explain the failure of the H1N1 virus to replace H3N2 on re-emergence in 1977 (Fig. le), as a large proportion of the population would have been infected with H1N1 prior to 1957, and would have retained some immunity. c) Re-emergence In 1977, the Russian influenza virus H1N1 that had circulated in humans prior to 1950 reappeared and has continued to co-circulate with H3N2 viruses in the human population. The origin of the virus is a mystery and a number of sources have been proposed, including the possibility that the virus re-emerged following escape from a laboratory. Fig. 1c Theoretical origin of influenza A viruses circulating in humans since 1918 208 Rev. sci. tech. Off. int. Epiz., 19 (1) T h e e m e r g e n c e of p a n d e m i c strains f o l l o w i n g g e n e t i c reassortment The detection of vast p o o l s of influenza virases of m a n y different subtypes a m o n g animals, particularly aquatic birds, gave considerable impetus to research a i m e d at determining w h e r e n e w subtypes, particularly those that cause p a n d e m i c s , emerge. A n u m b e r of theories has b e e n suggested, of w h i c h the m o s t widely a c c e p t e d is that b y adaptation, s o m e t i m e s involving genetic reassortment, transference of virus from animals to h u m a n s occurs, resulting in an antigenically novel virus with the ability to infect and spread in h u m a n s (incidents are summarised in Table IV). Following genetic reassortment, viruses m a y arise that possess the genes necessary to enable infection of h u m a n s but m a y have surface antigens n e w to the host i m m u n e system. Table IV Summary of influenza A hosts and the potential for recombination and/or spread to humans Influenza host Contact with humans Infections w i t h human influenza viruses Spread from host to humans Pigs Yes Yes: H1N1, H3N2 Yes: natural Horses Yes None known None known None known None known Birds Waterfowl lndirectly Caged birds Yes None known None known Domestic poultry Yes None known Yes: H5N1, H9N2, H7N7 (a) (b) (b) Mustelids Yes Yes: laboratory Whales No None known None known Seals No None known Yes: laboratory Yes: laboratory a) Contact may occur via lake water and other media contaminated by infective faeces b) The H7N7 virus was isolated from the eye of a woman who kept ducks with which feral waterfowl mingled Genetic and b i o c h e m i c a l studies h a v e s h o w n that the p a n d e m i c viruses of 1957 and 1968 arose b y genetic reassortment. T h e 1957 Asian H 2 N 2 strain obtained HA, NA and PB1 genes from an avian virus and the remaining five genes from the preceding h u m a n H 1 N 1 strain (49, 88, 141). The 1968 Hong K o n g H 3 N 2 strain contained HA and PB1 genes from an avian d o n o r ( 4 7 , 8 8 ) and the NA and other five genes from the Asian H 2 N 2 strain. T h e role of t h e pig Pigs serve as major reservoirs of H 1 N 1 and H 3 N 2 influenza viruses and are frequently involved in interspecies transmission of influenza viruses. T h e maintenance of these viruses in pigs and the frequent introduction of viruses from other species m a y b e important in the generation of 'new' strains of influenza, s o m e of w h i c h m a y h a v e the potential to transmit to other species, including h u m a n s . Pigs h a v e b e e n c o n s i d e r e d the logical intermediate host for reassortment of influenza A viruses, for these animals can serve as hosts for viruses from either birds or h u m a n s (144). This susceptibility is d u e to the p r e s e n c e of receptors for b o t h avian (a2-3-galactose sialic acid) and h u m a n (a2-6-galactose sialic acid) influenza viruses w h i c h , following infection of pigs with an avian virus, can result in receptor modification o n the HA to a 2 - 6 , thereby providing a potential link from birds to h u m a n s (Fig. 2) (79). Furthermore, pigs are susceptible to infection with all of the avian subtypes tested to date (H1 to H13) and those avian viruses w h i c h d o not replicate in pigs can contribute genes in the generation of reassortants w h e n co-infecting pigs with a swine influenza virus (92). Evidence for the pig as a mixing vessel of influenza viruses of n o n swine origin has b e e n demonstrated in Europe b y Castrucci et al., w h o detected reassortment of h u m a n and avian viruses in pigs in Italy (32). Novel reassortant viruses of H 1 N 2 subtype derived from h u m a n and avian viruses (26) or H1N7 subtype derived from h u m a n and equine viruses (22) h a v e b e e n isolated from pigs in the UK. T h e H 1 N 2 viruses derived from a multiple reassortant event and spread widely within pigs in the UK. Other studies of influenza viruses isolated from pigs in North America (195) and the south of the People's Republic of China (156) failed to detect any reassortant viruses containing internal gene segments of non swine origin, although genetic heterogeneity of the HA of swine H3 influenza viruses occurs in nature in the People's Republic of China (91). Since 1998, H 3 N 2 viruses isolated from pigs in the USA have contained combinations of human, swine and avian genes. Furthermore, the HA gene of these viruses was derived from a h u m a n virus circulating in the h u m a n population in 1993 (181). Occassional transmission of influenza viruses from pigs to h u m a n s h a s b e e n demonstrated (see the sub-section below, entitled 'Pigs' in the section 'Spread b e t w e e n h u m a n s and m a m m a l i a n species'). T h e internal protein genes of h u m a n influenza viruses share a c o m m o n ancestor with the genes of s o m e swine influenza viruses. A n u m b e r of authors have p r o p o s e d the nucleoprotein (NP) gene as a determinant of host range w h i c h can restrict or attenuate virus replication, thereby controlling the successful transmission of virus to a 'new' host (143, 172, 161). T h e s e observations support the potential role of the pig as a mixing vessel of influenza viruses from avian and h u m a n sources. T h e pig appears to have a b r o a d e r host range in the compatibility of the NP gene in reassortant viruses than b o t h h u m a n s and birds (143). Direct transfer from another species Alternatively, n e w p a n d e m i c viruses c o u l d occur in the h u m a n population if an avian strain or a strain from another m a m m a l b e c a m e infectious for h u m a n s . T h e appearance of the 'Spanish' influenza virus (H1N1) in 1918 almost certainly involved this m e c h a n i s m . Later, retrospective serological investigations confirmed that the disease in h u m a n s and pigs h a d b e e n caused b y closely related influenza A viruses. The 209 Rev. sci. tech. Off. int. Epiz., 19 (1) a2-3 cc2-6 a2-6 Pigs have been considered the logical intermediate host for reassortment of influenza A viruses, for they can serve as hosts for viruses from either birds or humans. This susceptibility is due to the presence of receptors for both avian (a2-3-galactose sialic acid) and human (a2-6-galactose sialic acid) influenza viruses which, following infection of pigs with an avian virus, can result in receptor modification on the HA to that characteristic of human viruses, thereby providing a potential link from birds to humans. The segments in the centre of each virus particle represent the genome. The subtype of some emerging reassortant viruses may differ from their progenitors through exchange of HA (white in reassorted virus) and/or NA genes. These viruses which are distinct antigenically may be able to spread in an immunologically naïve human population if transmission from pigs occurs. HA: haemagglutinin NA: neuraminidase Fig. 2 The potential role of the pig as an intermediate host causative agent was an H1N1 influenza A viras which was possibly derived from the same avian ancestor (52, 83, 130). For this reason, the avian-like H1N1 viruses circulating in pigs in Europe since 1979 (125), and in South-East Asia since 1993, have been implicated as the precursors of the next human pandemic virus (56, 103). Although a pandemic has not resulted to date, the transmissions of H5N1 and H9N2 viruses from chickens to humans in Hong Kong in 1997 and 1999, respectively, further demonstrate that avian species may directly be the source of human pandemic strains (35, 151). Initially, following the transmission of these viruses from aquatic birds to pigs or chickens, the viruses appeared genetically unstable (103, 198), and in the case of transmission of avian H1N1 virus to pigs, apparently became stable after approximately twelve years (103). R e - e m e r g e n c e of p a n d e m i c s t r a i n s The appearance of pandemic virus may be in fact the re-emergence of a virus which caused an epidemic many years earlier. The appearance of Russian influenza (H1N1) provides support for this concept. The virus, which reappeared in the People's Republic of China in 1977, and subsequently spread world-wide, was genetically identical to the virus which caused a human influenza epidemic in 1950 (114). Webster et al. suggested that this virus was most likely reintroduced to humans from a frozen source, possibly from a laboratory (189), whilst Shoham has proposed a biotic mechanism for the preservation of influenza viruses (147). Influenza viruses of the H3N2 subtype persist in pigs many years after their antigenic counterparts have disappeared from humans (61, 210 152), providing a reservoir of virus w h i c h m a y in the future infect a susceptible h u m a n population. However, these viruses have accumulated many mutations and are therefore genetically distinct from the precursor viruses. Pandemic strains m a y also b e antigenically conserved in the avian reservoir, since counterparts of the Asian p a n d e m i c strain of 1957 continue to circulate with increased prevalence in wild d u c k s , d o m e s t i c fowl and live bird markets and are c o m i n g into closer proximity to susceptible h u m a n populations (136). A n 'influenza e p i c e n t r e ' T h e majority of p a n d e m i c strains have apparently originated in the People's Republic of China, raising the possibility that this region is an influenza epicentre ( 1 5 3 , 1 5 0 ) . In the tropical and subtropical regions of the People's Republic of China, h u m a n influenza occurs throughout the year (129). In the People's Republic of China, influenza viruses of all subtypes are prevalent in d u c k s and in water frequented b y d u c k s (59). Agricultural practices provide close contact b e t w e e n wild aquatic birds, d o m e s t i c d u c k s , pigs and humans, thereby presenting the opportunity for interspecies transmission and genetic exchange a m o n g influenza viruses, with the pig acting as an intermediary b e t w e e n domestic d u c k s and h u m a n s . Aquatic birds migrating or over-wintering in the region might provide a source of virus for domestic d u c k s . Yasuda et al. h a v e s h o w n that d o m e s t i c d u c k s harbour H 3 influenza viruses antigenically and genetically similar to those in pigs, suggesting these birds m a y play a role in the transfer of avian influenza viruses from feral d u c k s to pigs (196). Rhythm of o c c u r r e n c e T w o h y p o t h e s e s h a v e b e e n p r o p o s e d for the rhythm of occurrence of h u m a n influenza A viruses, namely: an influenza circle or cycle, or an influenza spiral ( 1 4 9 ) . T h e circle or cycle theory suggests a recycling of H l , H 2 a n d H 3 subtypes. If this is s o , the HA subtype of the next p a n d e m i c virus w o u l d b e e x p e c t e d to b e H 2 . T h e spiral theory p r e s u p p o s e s that h u m a n s are capable of being infected with all k n o w n HA subtypes of influenza A viruses; these presently n u m b e r fifteen. T h e r e are serological grounds for this in that rural dwellers in the influenza epicentre have b e e n found to possess antibodies to the avian subtypes H 4 to H 1 3 e x a m i n e d (149). It is possible that the h y p o t h e s e s are not mutually exclusive, as there is n o reason that recycling should not occur within the spiral. Spread between humans and mammalian species Pigs Early theories suggesting that the p a n d e m i c of 1918 was a result of the transmission of virus from pigs to h u m a n s were at the time speculative and it was not until 1976 that further evidence for such transmissions b e c a m e available. Pigs w e r e implicated as the source of infection w h e n an H1N1 virus was isolated from a soldier w h o h a d d i e d of influenza at Fort Dix, Rev. sci. tech. Off. int. Epiz., 19(1) N e w Jersey, USA. T h e virus was identical to viruses isolated from pigs in the USA. Furthermore, virus isolation demonstrated that five other s e r v i c e m e n w e r e infected, and serological evidence suggested that s o m e 500 personnel at Fort Dix were, or h a d b e e n , infected with the s a m e virus (76, 173). The incident at Fort Dix cannot b e regarded as e v i d e n c e of zoonosis, since although pigs w e r e the likely source of the virus, this was never established. However, considerable evidence exists to suggest that transmission from pigs to h u m a n s d o e s occur, following the detection of antibodies to swine H1 viruses in p e o p l e w h o h a v e h a d close contact with pigs (95, 139). Final confirmation of the zoonotic nature of H 1 N 1 influenza viruses from pigs c a m e in 1976 w h e n , following an influenza epizootic in pigs, viruses isolated from the pigs and a h u m a n contact w e r e s h o w n to b e b o t h antigenically and genetically identical swine H 1 N 1 influenza viruses (44, 70). Subsequently, there h a v e b e e n several reports from North America of swine virus b e i n g isolated from h u m a n s with respiratory illness (38, 4 2 ) , occasionally with fatal c o n s e q u e n c e s ( 1 3 3 , 190). All cases e x a m i n e d followed contact with sick pigs and w e r e d u e to viruses related most closely to classical swine H 1 N 1 influenza virus. Perhaps of greater significance for h u m a n s is a report in the Netherlands during 1993 of two distinct cases of infection of children with H 3 N 2 viruses w h o s e genes e n c o d i n g internal proteins w e r e of avian origin (34). Genetically and antigenically related viruses h a d b e e n detected in pigs in Europe, raising the possibility of -potential transmission of avian influenza virus genes to h u m a n s following genetic reassortment in pigs (32). These concerns w e r e substantiated further b y the results of serological studies in Italy w h i c h indicated that these 'swine' H 3 N 2 viruses h a d apparently b e e n transmitted to young, immunologically naïve p e o p l e (30). Influenza viruses of subtype H 3 N 2 are ubiquitous and e n d e m i c in m o s t pig populations w o r l d - w i d e , but no apparent evidence exists of pigs b e i n g infected with this subtype prior to the p a n d e m i c in h u m a n s in 1968. Indeed, the appearance of a H 3 N 2 subtype variant strain in the pig population of a country appears to coincide with the epidemic strain infecting the h u m a n population at that time ( 1 1 , 24, 117). Further evidence of the spread of influenza viruses from h u m a n s to pigs was the appearance in pigs of H1N1 viruses (or antibodies to H 1 N 1 ) , related to those circulating in the h u m a n population since 1977 ( 1 1 , 24, 5 3 , 118). Genetic analysis of two strains of H1N1 virus isolated from pigs in J a p a n revealed that the HA and NA genes w e r e m o s t closely related to those of h u m a n H1N1 viruses circulating in the h u m a n population at that time (87). In addition, reassortant viruses with s o m e characteristics of h u m a n H1 viruses have b e e n isolated from pigs in the UK (23, 2 6 ) . Rev. sci. tech. Off. int. Epiz., 19(1) 211 Horses Horses In historical accounts of p a n d e m i c s in h u m a n s , frequent reference is m a d e to similar disease in horses, w h i c h occurs either simultaneously or p r e c e d i n g that in h u m a n s . Beveridge noted s u c h references in the accounts of twelve p a n d e m i c s w h i c h o c c u r r e d during the 18th and 19th Centuries (17). Apart f r o m occasional isolated reports of e v i d e n c e of infection of h o r s e s with viruses of subtypes H 1 N 1 , H 2 N 2 and H 3 N 2 (174), influenza infections of h o r s e s h a v e b e e n restricted to H7N7 and H 3 N 8 subtypes of influenza A and these viruses form distinct lineages i n phylogenetic studies (see earlier). However, examination of H 3 N 8 viruses isolated from severe e p i d e m i c s in h o r s e s occurring in the Jilin a n d Heilongjiang Provinces in the north-east of the People's Republic of China in 1989 and 1990, s h o w e d these to b e antigenically a n d genetically distinguishable from other e q u i n e H 3 N 8 viruses circulating in the world. Guo et al. c o n c l u d e d that this virus was of recent avian origin a n d h a d p r o b a b l y spread direcdy to horses without reassortment (60). Despite the rapid geographical spread locally in 1989 and 1990, and the h i g h morbidity, this virus d o e s not appear to h a v e b e c o m e established in the h o r s e population; m o r e w i d e s p r e a d e p i d e m i c s w h i c h occurred in the People's Republic of China from 1993 to 1994 w e r e caused b y 'classical' H 3 N 8 virus (154). Experimental infection of h u m a n volunteers with H 3 (equine-2) viruses h a s p r o d u c e d a n influenza-like illness with virus-shedding and seroconversion (36, 86). N o evidence exists of infection of h u m a n s with the other subtype of influenza, H7 (equine-1), w h i c h has caused w i d e s p r e a d epizootics in h o r s e s . Several isolated cases of infection of h o r s e s with subtypes H 1 N 1 , H 2 N 2 and H 3 N 2 h a v e b e e n reported, usually associated with h u m a n infections (174). Experimental infections of h o r s e s w i t h h u m a n - d e r i v e d H 3 N 2 virus h a v e confirmed the susceptibility of h o r s e s to this virus (19). Cattle Marine mammals Influenza infections of cattle with h u m a n viruses of H 3 N 2 a n d H1N1 subtypes h a v e b e e n reported regularly, b a s e d largely on the detection of antibodies (82), occasionally in association with respiratory epizootics in cattle (25). T h e prevailing h u m a n strains appear to transmit to cattle frequently a n d viruses h a v e b e e n isolated from cattle infected naturally (48, 110), although w h e t h e r or not these viruses are able to persist in cattle remains unclear. B r o w n et al. reported the apparent maintenance of h u m a n influenza viruses in cattle s o m e ten years after the disappearance of the viruses from the h u m a n population, raising the possibility that cattle m a y provide a reservoir for virus w h i c h m a y b e a b l e to transmit b a c k to humans w h e n a susceptible p o p u l a t i o n b e c o m e s available (25). During 1 9 7 9 a n d 1980, approximately 5 0 0 deaths o c c u r r e d in harbour seals (Phoca. vitulina) around the C a p e C o d Peninsula in the USA, representing about 2 0 % of the population. Deaths w e r e primarily d u e to acute haemorrhagic p n e u m o n i a , a n d influenza A viruses of H 7 N 7 subtype w e r e isolated repeatedly from the lungs or brains of d e a d seals (97). T h e virus infecting the seals was s h o w n to b e closely related b o t h antigenically a n d genetically to avian influenza viruses (185) a n d w o u l d a p p e a r to represent direct transmission to the seals without reassortment. Avian influenza infections of mammals Pigs The p r o b a b l e introduction of classical swine H 1 N 1 influenza viruses to turkeys from infected pigs has b e e n reported from North America, and in s o m e cases, influenza-like illness in pigs has b e e n followed immediately b y disease signs in turkeys ( 6 2 , 1 0 9 , 1 2 7 ) . Genetic studies of H 1 N 1 viruses from turkeys in the USA h a s revealed a h i g h degree of genetic exchange and reassortment of influenza A viruses from turkeys and pigs in the former species (195). In Europe, avian H1N1 viruses w e r e transmitted to pigs, b e c a m e established, and have subsequently b e e n reintroduced to turkeys from pigs, causing e c o n o m i c losses ( 1 0 2 , 192). An independent introduction of H 1 N 1 virus from birds to pigs occurred in Asia in the early 1990s; these viruses are genetically distinct from the viruses in Europe (56). Recently, H 9 N 2 viruses h a v e b e e n introduced to pigs in South-East Asia, apparently from poultry, although the potential of these viruses to spread and persist in pigs remains u n k n o w n (20). In 1983, further deaths ( 2 % - 4 % ) occurred in harbour seals o n the N e w England coast of the USA and, o n this o c c a s i o n , another influenza A virus of subtype H 4 N 5 was isolated. W h i l e distinguishable from the H 7 N 7 virus, o n c e again, all eight genes of this virus w e r e demonstrably of avian origin (189). Further surveillance of seals o n the Cape C o d peninsula was u n d e r t a k e n and Callan et al. reported isolates of two influenza A viruses of H 4 N 6 subtype m a d e in 1991 and three of H 3 N 3 subtype in 1 9 9 2 from seals found d e a d with apparent viral p n e u m o n i a (28). Following antigenic and genetic characterisation, the authors c o n c l u d e d these too w e r e avian viruses that h a d entered the seal population. Two viruses of H 1 3 N 2 and H 1 3 N 9 subtypes w e r e isolated from a single b e a c h e d pilot whale, and genetic analysis indicated that the viruses h a d b e e n introduced recently from birds (33, 73). Mink In October 1984, outbreaks of respiratory disease-affected approximately 100,000 m i n k o n 3 3 farms situated in close proximity along a coastal region of southern S w e d e n , with 100% morbidity a n d 3 % mortality (94). Influenza A viruses of H 1 0 N 4 subtype were isolated from the m i n k a n d genetic Rev. sci. tech. Off. int. Epiz., 19 (1) 212 analysis indicated that the virases w e r e of avian origin and w e r e very closely related to a virus of the same subtype isolated from c h i c k e n s and a feral d u c k in England in 1985 (16). Earlier experimental infections h a d suggested that m i n k w e r e susceptible to infection with avian influenza viruses (119). Humans Historical background Until 1996, n o reports existed of naturally acquired infections of humans with avian influenza viruses b y direct transmission from birds. Evidence of antibodies to influenza subtypes k n o w n only to infect birds h a d b e e n d e t e c t e d in s e r u m samples t a k e n from families with h o u s e h o l d d u c k s and pigs in the south of the People's Republic of China (149, 157), but this m a y have occurred following passage of the virus through pigs. Beare and W e b s t e r p e r f o r m e d experimental infections o n h u m a n volunteers and reported only transitory infections with little or n o clinical signs and n o antibody production (13). However, reports of ostensibly avian viruses infecting h u m a n s or infections as a result of laboratory accidents have b e e n m a d e o n several occasions. C a m p b e l l et cd. reported the isolation of a virus of H 7 N 1 subtype from a m a n suffering from hepatitis, but with n o antibody response to that virus (29). A female laboratory w o r k e r in Australia d e v e l o p e d kerato-conjunctivitis after accidentally splashing infective allantoic fluids containing A/chicken/Victoria/76 (H7N7) o n h e r face. Virus was isolated b y swabbing the conjunctiva, but significant antibody titres were not obtained (171). A harbour seal infected experimentally with A/seal/Massachusetts/1/80 (H7N7), and k n o w n to b e shedding that virus, s n e e z e d directly into the face and right eye of an investigator w h o subsequently d e v e l o p e d conjunctivitis (186). T h e virus was isolated from the affected eye u p to four days after the incident, but n o antibody response was detected. As discussed a b o v e , the virus was k n o w n to b e of avian origin. Four field w o r k e r s autopsying seals during the o u t b r e a k of 1980 also d e v e l o p e d conjunctivitis, but n o virology was p e r f o r m e d (185). Since 1996, three accounts h a v e b e e n r e c o r d e d of avian influenza viruses apparently affecting h u m a n s as a result of spread directly from birds. H7N7 in England Kurtz et al. reported the isolation of an influenza virus, A/England/268/96 (H7N7), from the eye of a fortythree-year-old w o m a n with conjunctivitis (96). Partial sequencing of the internal protein and the neuraminidase genes of isolate 2 6 8 / 9 6 s h o w e d all s e v e n genes to have closest h o m o l o g y with avian influenza viruses and the entire nucleotide s e q u e n c e of the haemagglutinin gene of 2 6 8 / 9 6 h a d close ( 9 8 . 2 % ) h o m o l o g y with an H 7 N 7 virus isolated from turkeys in Ireland in 1995 (12). Kurtz et al. considered the most likely source of the virus to b e waterfowl, as the w o m a n tended a collection of twenty-six d u c k s of various b r e e d s w h i c h m i x e d freely with feral waterfowl o n a small l a k e (96). T h e infected w o m a n did not have measurable antibodies to H7 five w e e k s after the appearance of conjunctivitis. Cloacal swabs t a k e n from h e r d u c k s o n e m o n t h after the isolation of virus from the affected eye did not yield any virus. H5N1 in Hong Kong Outbreaks of HPAI, c a u s e d b y virus of H 5 N 1 subtype, occurred o n three c h i c k e n farms in H o n g K o n g from March to May 1997, with mortality ranging from 7 0 % - 1 0 0 % , and subsequent spread to live b i r d markets in H o n g K o n g (35, 155). In May 1997, a three-year-old child d i e d of apparent viral p n e u m o n i a with severe complications after admission to hospital in H o n g K o n g (197). A n influenza virus of H5N1 subtype was isolated from respiratory s p e c i m e n s t a k e n from this patient and similar virus infections w e r e confirmed by virus isolation and/or serology in seventeen other cases in h u m a n s w h i c h occurred in H o n g K o n g during N o v e m b e r and D e c e m b e r 1997; a further five patients d i e d (155). Nucleotide sequencing of the virus from the index case s h o w e d all eight genes to b e of avian origin (35), and demonstrated 9 9 % h o m o l o g y of all eight genes with those of the H 5 N 1 virus from c h i c k e n s in H o n g K o n g (168). Analysis of the haemagglutinin and neuraminidase proteins of the sixteen viruses isolated from the eighteen cases indicated that all w e r e essentially similar and a p p e a r e d to have spread directly from poultry with n o cumulative changes that w o u l d indicate adaptation to the h u m a n host (15). All h u m a n isolates h a d the same multiple basic amino acids as the c h i c k e n isolates (15). Investigations of the h u m a n infections with H 5 N 1 in Hong K o n g revealed n o e v i d e n c e of h u m a n to h u m a n transmission and e a c h case was assumed to represent spread from domestic poultry (155). One of the surprising features of these infections was the severe clinical disease caused in humans. Both h u m a n and poultry H 5 N 1 viruses, in c o m m o n with most avian influenza viruses, w e r e s h o w n to bind preferentially to the N-acetylneuraminic acid-a2,3,-galactose linkages o n sialyloligosaccharides ( 1 0 7 ) , whilst conventional h u m a n influenza viruses b i n d to N-acetylneuraminic acid-a2,6Gal linkages; apparently this did not prevent penetrative virus replication in s o m e of the infected humans. There was considerable fear that the virus w o u l d mutate to b e c o m e transmissible either b y mutation in o n e of the infected p e o p l e or b y reassortment with h u m a n influenza in a person infected with h u m a n influenza earlier or subsequently. To prevent this occurring, it was d e c i d e d to r e m o v e the apparent source of the H 5 N 1 virus, and all c h i c k e n s in H o n g Kong, approximately 1,500,000, were slaughtered. H9N2 in Hong Kong and other regions of the People's Republic of China Although n o H5N1 viruses have b e e n isolated from humans since D e c e m b e r 1997, the p r o b l e m s of bird to h u m a n spread in the area w e r e raised again in March 1999, w h e n two young girls aged o n e and four years w e r e admitted to hospital in H o n g Kong with typical influenza-like s y m p t o m s . In b o t h 213 Rev. sci. tech. Off. int. Epiz, 19 (1) cases, influenza A viruses of H 9 N 2 subtype w e r e isolated from respiratory tract samples. Both children m a d e uneventful recoveries. Subsequent to these reports from Hong Kong, it was revealed that five cases of h u m a n H 9 N 2 infection h a d b e e n r e c o r d e d on the mainland of the People's Republic of China in August 1998. Genetic analyses of the two H o n g K o n g H 9 N 2 isolates have s h o w n that these, like the H 5 N 1 viruses, appear to b e avian viruses w h i c h h a v e infected h u m a n s directly (57, 124). As described earlier, influenza A viruses of H 9 N 2 subtype h a v e b e e n w i d e s p r e a d in d o m e s t i c poultry throughout the w o r l d in recent years, but especially in Asia, including the People's Republic of China. Transfer to h u m a n s c o u l d quite possibly have occurred in other areas, but g o n e u n o b s e r v e d , as the level of monitoring in H o n g K o n g and the People's Republic of China h a d b e e n considerably raised as a result of the H 5 N 1 virus isolations from h u m a n s . T h e greatest threat of these infections is p r o b a b l y the risk of a dual infection with a conventional h u m a n virus, resulting in a reassortant virus with H9 subtype haemagglutinin, c o m b i n e d with all or s o m e of the other genes from the h u m a n virus, thereby allowing transmission b e t w e e n h u m a n s . Surveillance Humans Global surveillance of influenza is maintained b y a n e t w o r k of laboratories s p o n s o r e d b y the W H O . T h e two primary goals of the p r o g r a m m e are to gain an understanding of the epidemiology of influenza and to promptly isolate influenza viruses from n e w e p i d e m i c s and distribute t h e m for vaccine production (64). T h e p r o g r a m m e is i m p l e m e n t e d through three W H O Collaborating Centres for Influenza Reference and Research located in L o n d o n , Atlanta and Melbourne. T h e functions of these centres are as follows: a) to collect a n d distribute information o n the types of influenza virus w h i c h prevail in various countries world-wide Morbidity and mortality associated with influenza is u s e d as an indicator of influenza activity and impact. Precise quantification of the impact of influenza morbidity and mortality is problematic, since laboratory confirmation is required for exact diagnosis. Many m e t h o d s h a v e b e e n d e v e l o p e d to p r o v i d e estimates of the mortality or morbidity associated with influenza. T h e s e m e t h o d s usually use baseline rates of mortality or morbidity to calculate excess rates of mortality or morbidity attributed to circulating influenza virus. During recent years, the operation of the n e t w o r k h a s b e c o m e increasingly focused o n the annual consultation o n influenza vaccine formulation (see the subsection entitled 'Immunoprophylaxis' b e l o w ) . In addition, surveillance of h u m a n a n d animal influenza in the p r o p o s e d 'influenza epicentre' h a s b e e n intensified a n d i m p r o v e d , thereby contributing to the early identification of two subtypes of influenza virus not r e c o r d e d previously in h u m a n s . Animals Demonstration that the H 2 N 2 and H 3 N 2 p a n d e m i c viruses contained genes from an influenza virus of avian origin l e d to systematic, long-term global surveillance studies o n the influenza viruses of birds and m a m m a l s , to determine the diversity of influenza A viruses in nature and w h e t h e r a future p a n d e m i c strain of virus c o u l d b e isolated from these animals in advance of the appearance of the strain in humans. A vast n u m b e r of viruses have n o w b e e n isolated from a w i d e variety of birds and a range of terrestrial and sea m a m m a l s . T h e s e studies are ongoing but are generally m o r e localised and less formally structured than the equivalent s c h e m e s for the h u m a n population. However, international and national reference laboratories with a similar remit to those in the h u m a n sector h a v e b e e n established for avian and equine influenza viruses. T h e s e facilities have p r o v i d e d invaluable information for dealing with emergencies such as the early identification of a putative vaccine strain from birds following the infection of p e o p l e in Hong K o n g with H5N1 virus, and after vaccine failure in controlling equine influenza. b) to advise o n the strains to b e included in influenza vaccines c) to collect, store and study strains from various outbreaks and distribute these strains to interested laboratories Prophylaxis and treatment d) to educate visiting w o r k e r s in research techniques. The surveillance p r o g r a m m e h a s n o w g r o w n to include 110 designated National Influenza Centres in 80 countries. National centres are involved in the isolation and preliminary characterisation of influenza viruses from suspect cases of infection in h u m a n s and, if appropriate, in collection of viruses from regional laboratories within the country. T h e Centres p r o v i d e the W H O with regular epidemiological reports and forward virus isolates for further analysis to o n e of the three Collaborating Centres. Two control measures are currently available for influenza in h u m a n s , namely: immunoprophylaxis with vaccines and c h e m o p r o p h y l a x i s or therapy with antiviral drugs. Since the late 1940s, the principal preventive measures against influenza have b e e n inactivated virus vaccines. T h e efficacy of vaccines has varied b e t w e e n 6 0 % and 9 0 % and has b e e n d e p e n d e n t o n the closeness of the 'antigenic m a t c h ' b e t w e e n the vaccine virus and the e p i d e m i c virus. However, e v e n for those not completely protected, vaccination r e d u c e s the severity of the disease, thereby reducing costs and mortality. Rev. sci. tech. Off. int. Epiz., 19 (1) 214 Immunoprophylaxis Vaccination of p e o p l e at h i g h risk before e a c h annual influenza season is currently the m o s t effective measure for reducing the impact of h u m a n influenza. W h e n vaccine and e p i d e m i c strains of virus are well m a t c h e d , achieving h i g h vaccination rates a m o n g c l o s e d populations can reduce the risk of outbreaks b y inducing population immunity. This occurs w h e n the overall n u m b e r of susceptible p e o p l e in a population b e c o m e s t o o l o w for virus to spread and infect a significant n u m b e r of the susceptible individuals. To maximise protection of p e o p l e at h i g h risk, they and their close contacts s h o u l d b e targeted for organised vaccination p r o g r a m m e s . Influenza vaccination is strongly r e c o m m e n d e d for any p e r s o n over six m o n t h s of age w h o , b e c a u s e of age or an underlying m e d i c a l condition, is at increased risk for complications of influenza. T h e high-risk group consists of the following: p e o p l e over sixty-five years of age, residents of nursing h o m e s and other chronic-care facilities, p e o p l e with chronic disorders of the pulmonary or cardiovascular systems, and p e o p l e w h o have suffered chronic m e t a b o l i c diseases or i m m u n o s u p p r e s s i o n (including immuno suppression as a result of medication) within the past year. In addition, p e o p l e w h o m a y transmit influenza to those at h i g h risk, i.e. hospital and nursing h o m e personnel and h o u s e h o l d m e m b e r s of p e o p l e in high-risk groups, s h o u l d also b e vaccinated. Other groups m a y b e included o n individual merit, such as pregnant w o m e n , p e o p l e infected with HIV and foreign travellers. Currently, in the UK and the USA, less than 3 0 % of p e o p l e in high-risk groups are vaccinated e a c h year and m o r e effective strategies are required for delivering vaccine to m e m b e r s of high-risk groups. In general, successful vaccination p r o g r a m m e s have c o m b i n e d education for healthcare workers, publicity and education targeted towards potential recipients, and a plan for identifying p e o p l e at h i g h risk. Two basic a p p r o a c h e s for immunisation h a v e b e e n pursued, namely: the use of inactivated virus preparations and the use of live, attenuated viruses. At present, only vaccines p r e p a r e d with inactivated or killed virus particles are licensed for use in the European Union and the USA. N u m e r o u s refinements in the production processes over m a n y years have resulted in vaccines that are m o r e highly purified and m o r e predictable in their reactogenicity and immunogenicity (194). E a c h year, the influenza vaccine is redefined to reflect changes in the antigenicity of circulating virus strains, thereby containing virus strains representative of influenza viruses believed likely to circulate in the forthcoming 'influenza season'. Annual vaccination using the current vaccine is therefore necessary b e c a u s e of potential changes in the circulating viruses, but also d u e to immunity from previous vaccinations b e i n g relatively short-lived. At present, the vaccine consists of two type A viruses, H 1 N 1 and H 3 N 2 , and one type B virus. T h e exact strains of these viruses to b e used are identified b y an international n e t w o r k of laboratories that maintain surveillance for n e w influenza virus variants throughout the world. One d o s e of inactivated vaccine is generally sufficient to induce a protective i m m u n e response, although children occasionally require a b o o s t e r (37). Unless the antigen u s e d is entirely n e w , m o s t adults will m o u n t a b o o s t e r antibody response, e v e n to strains w h o s e antigens are marginally different. Vaccine antibodies react against the HA and NA and the level of s e r u m antibody h a s b e e n s h o w n to correlate inversely with the occurrence of established infection with influenza viruses (75). T h e c o m p o s i t i o n of the vaccine rarely causes systemic or febrile reactions. T h e effectiveness of influenza vaccine in preventing or attenuating illness varies, d e p e n d i n g primarily o n the age and i m m u n o c o m p e t e n c e of the vaccine recipient a n d the degree of antigenic similarity b e t w e e n the virus strains included in the vaccine and those circulating during the influenza season. W h e n the m a t c h b e t w e e n vaccine and circulating viruses is close, influenza vaccine h a s b e e n s h o w n to prevent illness in approximately 7 0 % of healthy children and y o u n g adults, whilst preventing hospitalisation for p n e u m o n i a and influenza a m o n g elderly p e o p l e living in the community. Chemoprophylaxis Antiviral drugs, such as amantadine and rimantadine are effective against all type A influenza viruses (including H5N1 and H 9 N 2 ) . If e m p l o y e d at the beginning of an outbreak, these drugs can prevent disease and spread of the virus. Of those receiving these antivirals within 2 4 to 4 8 h o u r s of the onset of the disease, 7 0 % to 9 0 % h a v e greatly reduced symptoms. However, effectiveness is limited b y the rapid e m e r g e n c e of resistant viral strains a n d b y the adverse effects of the drugs themselves (66, 166). T h e recent addition of neuraminidase inhibitors (GG167 and GS4104) to the portfolio of antiviral drugs is an important development. Zanamivir (GG167) is efficacious against influenza virus (including H 5 N 1 ) infection in h u m a n s and animals without apparent side-effects or the induction of resistant influenza A viruses under clinical trial conditions (58, 67). Antivirals offer the advantage of not interfering with antibody production since infection is not c o m p l e t e l y prevented. Perspectives The next pandemic Prediction of the next p a n d e m i c strain of influenza is important to allow greater preparedness and successful vaccine prophylaxis. However, implicit in predicting the next virus subtype, or at least the next HA subtype, is a k n o w l e d g e of h o w p a n d e m i c viruses emerge. As discussed earlier, several theories exist regarding h o w antigenic shifts occur and p a n d e m i c s arise. T h e p r o b l e m in correctly assessing the actual 215 Rev. sci. tech. Off. int. Epiz., 19 (1) m e c h a n i s m s is that relatively little data is available, as viruses from only three p a n d e m i c s exist, although recently at least s o m e molecular data o n the 1918 p a n d e m i c virus h a s b e c o m e available (130, 170). As this important w o r k progresses, further understanding of p a n d e m i c s in general, and of the singular virulence of the 1 9 1 8 virus in particular, is anticipated. T h e current consensus o p i n i o n appears to b e that p a n d e m i c viruses arise as a result of transfer to h u m a n s of all or part of the g e n o m e of viruses a d a p t e d to another animal species, w h e r e the virus is c a p a b l e or b e c o m e s c a p a b l e of spread in a fully susceptible population, or at least in that proportion of the population that is susceptible. However, this d o e s not explain w h y this transference appears to h a p p e n so rarely and w h y s o f e w subtypes appear to circulate. Equally, if pigs are important as a n intermediate host for the emergence of p a n d e m i c virus as a result of reassortment b e t w e e n avian and h u m a n viruses, an explanation is required to describe w h y the n u m b e r of subtypes of avian influenza virus isolated naturally from pigs is limited to H1 and H 3 . T h e H5 and H9 viruses isolated from h u m a n s in Hong K o n g indicate that direct avian influenza virus infection a n d reassortment with a h u m a n virus c o u l d also result in the emergence of p a n d e m i c virus. This in turn raises the question of w h y this appears to occur so rarely and w h y three natural transmissions w e r e r e c o r d e d b e t w e e n 1996 a n d 1999, but none a p p e a r e d to b e r e c o r d e d before that date. Hosts of influenza A viruses and the potential for r e c o m b i n a t i o n and/or spread to h u m a n s are summarised in Table TV. Data is insufficient to confirm any of the current popular theories o n the e m e r g e n c e of p a n d e m i c influenza viruses or to dismiss any of the less likely theories. In addition, n o n e of the theories are necessarily mutually exclusive, and it c o u l d b e that these viruses arise b y several different m e c h a n i s m s . Virulence As discussed earlier in regard to susceptible birds, a very clear distinction exists b e t w e e n influenza viruses of l o w virulence w h i c h p r o d u c e disease similar to that of m a m m a l s , and the viruses w h i c h cause HPAI w h i c h m a y result in mortality rates approaching 100% in infected flocks. T h e primary reason for this difference is the p r e s e n c e of multiple basic a m i n o acids at the cleavage site of the haemagglutinin precursor, w h i c h in turn allows the virulent viruses to replicate systemically, while those l o w virulence viruses are restricted to replication in the respiratory and intestinal tracts (134). This finding in birds does have s o m e implications for h u m a n infections. W h i l e it is clear that trypsin-like e n z y m e s m a y bring about cleavage of the viruses with a single arginine, furin is the prime candidate for a ubiquitous protease responsible for cleaving HAO with multiple basic a m i n o acids, allowing systemic replication and ultimately death. Mammals, including h u m a n s , also have furin-like proteases c a p a b l e of cleaving at multiple basic amino acid motifs. However, the question as to w h e t h e r or not viruses with multiple basic a m i n o acids at the HAO cleavage site c o u l d cause systemic infections and highly pathogenic disease in h u m a n s remains unanswered, b e c a u s e the viruses k n o w n to h a v e infected h u m a n s ( H l , H 2 and H3 subtypes) all h a v e motifs at the cleavage site indicating they w o u l d only b e cleaved b y trypsin-like e n z y m e s . T h e h i g h mortality associated with the 1 9 1 8 p a n d e m i c virus was thought to h a v e b e e n the result of a multiple basic a m i n o acid HAO cleavage site, but this p r o v e d not to b e the case (130). T h e o b v i o u s inference was that the very h i g h mortality (6/18), amongst the p e o p l e infected with the H 5 N 1 virus in H o n g K o n g occurred b e c a u s e the virus w a s c a p a b l e of systemic infection d u e to the k n o w n p r e s e n c e of multiple basic amino acids at the HAO cleavage site, allowing cleavage to b e m e d i a t e d b y o n e or m o r e furin-like proteases. However, evidence that this w a s the case is lacking. Generally, the eighteen patients presented with severe respiratory s y m p t o m s and p n e u m o n i a a p p e a r e d to b e the principal cause of death, not unlike infections with ' c o m m o n ' influenza viruses. Similarly, infections of other m a m m a l s with avian influenza viruses give few clues to the significance of multiple basic a m i n o acids at the HAO cleavage site. An infection of h a r b o u r seals from 1978 to 1980 off the north-east coast of the USA, with H7N7 avian influenza, resulted in the d e a t h of an estimated 2 0 % of the population. W h i l e this mortality rate is c o m p a r a b l e to that w h i c h occurred in h u m a n s in H o n g Kong, the HAO cleavage site of the H7N7 virus did not h a v e a motif containing multiple basic amino acids (189). Conversely, H7N7 viruses responsible for equine influenza type 1, for w h i c h A/equine/Prague/56 (H7N7) is the type strain, d o have multiple basic amino acids at the HAO cleavage site, and yet in infections of h o r s e s with this strain, virus replication is invariably restricted to the respiratory tract (50). Although the question of the virulence of the 1 9 1 8 p a n d e m i c virus c o u l d not b e answered b y the HAO cleavage site amino acid motif, another m e c h a n i s m for cleaving viral HAO that confers increased virulence of the virus, p r o p o s e d b y G o t o and K a w a o k a , c o u l d have s o m e relevance (54). T h e authors investigated a variant h u m a n H1N1 virus w h i c h is neurotropic for m i c e and discovered that the NA b i n d s and sequesters the ubiquitous protease plasminogen, leading to the production of h i g h levels of plasmin at the surface of the virus. This in turn ensures efficient cleavage of the HA, enabling the virus to invade a w i d e variety of tissues. Plasminogen sequestration has not b e e n found in any other h u m a n influenza virus, but it will b e interesting to see if future genetic analyses reveal that the 1918 virus neuraminidase h a d the genetic markers for this property, namely: a carboxyl-terminal lysine and lack of a glycosylation site at position 146. Surveillance Clearly, careful surveillance c o u l d lead to the early identification of viruses representing candidates for a n e w p a n d e m i c virus. However, s o m e p r o b l e m s arise in achieving the benefits of unrestricted surveillance in all species, and targeted surveillance therefore s e e m s m o r e appropriate. Nevertheless, n o distinct markers for identifying a panzootic 216 Rev. sci. tech. Off. int. Epiz., 19 (1) virus or a potential progenitor p a n d e m i c virus are currently k n o w n . Consequently, identifying a specific, narrow target for surveillance is difficult. T h e n u m b e r s a n d variety of influenza viruses isolated from surveillance of wild birds h a v e not b e e n particularly helpful in the prediction of h u m a n influenza patterns, although this surveillance m a y b e of importance from the point of v i e w of disease in d o m e s t i c poultry. Infections and trends of infection in d o m e s t i c poultry m a y b e of greater significance and importance to h u m a n s . T h e current w i d e s p r e a d infections of c o m m e r c i a l poultry flocks in m a n y countries of Asia with H 9 N 2 (see the subsection entided 'Domestic poultry' in 'Ecology') m a y b e extremely significant in v i e w of the k n o w n infections of h u m a n s with virus of this subtype. Surveillance studies in other domestic animals m a y also b e particularly pertinent, especially in pigs, in v i e w of their k n o w n susceptibility to b o t h avian and h u m a n viruses. Given the apparent origin of three of the four p a n d e m i c s w h i c h occurred in the 20th Century, and possibly s o m e of the earlier p a n d e m i c s , in the south of the People's Republic of China, Shortridge and Stuart-Harris p r o p o s e d the c o n c e p t of an influenza epicentre, i.e., a geographical area w h e r e birds, other animals and h u m a n s live closely together in conditions w h e r e viruses have the greatest opportunity to pass from o n e species to another (153). Surveillance and monitoring of influenza viruses in various species w o u l d s e e m prudent in that geographical region, although not to the exclusion of other countries and geographical areas. Contingency plans Pandemics of influenza exert extreme pressures o n all aspects of society in terms of b o t h public health and e c o n o m y . Even the relatively mild p a n d e m i c of 1968 h a d e n o r m o u s impacts in d e v e l o p e d countries through absenteeism, loss of earnings, pressures o n hospital services and costs of palliative medication. It is essential that governments draw up practicable contingency plans in advance of the next p a n d e m i c to deal with o b v i o u s essential planning s u c h as the production and stockpiling of vaccine and antiviral chemicals, prioritisation of treatment and arrangements for the functioning of hospitals, e m e r g e n c y services and even government in the face of mass incapacitation of the working force. In addition, s h o u l d mortality b e c o m p a r a b l e to the 1918 p a n d e m i c , the potential for hysteria and social upheaval amongst the population must not b e o v e r l o o k e d . It is essential that any contingency plan for p a n d e m i c influenza should b e reviewed frequently and regularly to assimilate n e w scientific k n o w l e d g e in this fast-moving field. Zoonoses récentes dues aux virus influenza A D.J. Alexander & I.H. Brown Résumé La grippe est une maladie aiguë très contagieuse qui frappe les hommes ainsi que les animaux depuis des t e m p s reculés. Les virus influenza, qui f o n t partie de la famille des Orthomyxoviridae, sont répartis en trois types : A , B et C, selon les caractéristiques antigéniques de la nucléoprotéine. Les virus influenza de type A infectent une grande variété d'espèces animales (porcins, équidés, mammifères marins, o i s e a u x ) ; chez l'homme, ils sont à l'origine de pandémies parfois dévastatrices, c o m m e celle qui fit plus de vingt millions de victimes dans le monde en 1918. Les deux antigènes de surface du virus, l'hémagglutinine et la neuraminidase, sont les plus importants par les propriétés immunologiques qu'ils confèrent à l'hôte et sont donc soumis aux plus fortes variations. S'agissant des virus influenza A, quinze sous-types de l'hémagglutinine et neuf sous-types de la neuraminidase, antigéniquement distincts, sont actuellement r é p e r t o r i é s ; un virus possède un sous-type de l'hémagglutinine et un sous-type de la neuraminidase, combinés de manière a p p a r e m m e n t aléatoire. Les virus isolés chez les m a m m i f è r e s présentent relativement p e u de combinaisons de sous-types, tandis que chez les oiseaux, tous les sous-types, dans la plupart des combinaisons, ont été isolés. A u X X siècle, des souches antigéniquement e 217 Rev. sci. tech. Off. int. Epiz., 19 (1) différentes sont apparues soudainement chez l'homme, phénomène désigné comme cassure antigénique (shift : en 1918 (H1N1), en 1957 (H2N2), en 1968 (H3N2) et en 1977 ( H 1 N 1 ) ; ces mutations ont c h a c u n e été à l'origine d'une pandémie. Des épidémies surviennent f r é q u e m m e n t entre deux pandémies, sous l'effet d'une dérive antigénique (drift du virus prévalent. Les épidémies qui sévissent actuellement dans le monde sont dues aux sous-types H1N1 et H3N2 du virus influenza A ou au virus influenza B. Pour mesurer l'impact de ces épidémies la meilleure méthode reste le suivi épidémiologique des épisodes meurtriers de pneumonie et de grippe. Des études phylogénétiques montrent que des oiseaux aquatiques pourraient être la source de tous les virus influenza A affectant les autres espèces. On pense que l'apparition des souches responsables de pandémies chez l'homme est le résultat de l'un des trois mécanismes suivants : - le réassortiment génétique (dû au fait que le génome du virus c o m p o r t e plusieurs segments) de virus influenza A aviaires et humains ayant infecté le même hôte ; - le transfert direct de l'ensemble du virus d'une autre espèce ; - la réémergence d'un virus ayant déjà provoqué une épidémie dans le passé. Depuis 1996, les virus H7N7, H5N1 et H9N2 se sont transmis des oiseaux à l'homme, a p p a r e m m e n t sans se propager dans la population humaine. De rares cas de transmission de l'homme aux animaux ont également été établis. Ces observations ont conduit à l'hypothèse que le réassortiment éventuel des virus humains et aviaires intervient chez une espèce intermédiaire, avec transfert ultérieur à l'homme. Les porcins jouent probablement ce rôle d'intermédiaire : en effet, ces animaux peuvent servir d'hôtes à des virus pathogènes aviaires et h u m a i n s ; de plus, il a été prouvé que les porcins sont impliqués dans la transmission entre espèces des virus influenza, n o t a m m e n t la transmission du virus H1N1 à l'homme. La surveillance mondiale de la grippe est assurée par un réseau de laboratoires placé sous l'égide de l'Organisation mondiale de la santé. La principale mesure prophylactique de la grippe chez l'homme est la v a c c i n a t i o n , qui a pour objectif d'éviter l'apparition d'épidémies entre deux pandémies. Mots-clés Écologie - Grippe - Influenza - Mammifères - Oiseaux - Pandémie - Prophylaxie Réassortiment - Santé publique - Transmission entre espèces - Zoonoses. • Zoonosis recientes causadas por virus de la influenza A D.J. Alexander &I.H. Brown Resumen La influenza (o gripe) es una enfermedad aguda y extremadamente contagiosa que viene afectando a hombres y animales desde tiempos remotos. Los virus causantes de esa enfermedad, pertenecientes a la familia Orthomyxoviridae, se clasifican, según las propiedades antigénicas de sus proteínas de nucleocápside, en tres tipos distintos: A, B y C. Los virus de la influenza de tipo A infectan a un amplio abanico de especies animales, que comprenden el cerdo, el caballo, 218 Rev. sci. tech. Off. int. Epiz., 19 (1) mamíferos marinos y aves, y provocan también ocasionalmente en el hombre pandemias de consecuencias devastadoras, como la de 1918 que causó la muerte de más de veinte millones de personas en todo el mundo. Las dos glucoproteínas de superficie del virus, la hemaglutinina y la neuraminidasa, son los principales antígenos que inducen inmunidad protectora en el huésped, y exhiben por consiguiente el mayor nivel de variación. Para los virus de la influenza A se c o n o c e n actualmente quince subtipos de la hemaglutinina y nueve subtipos de la neuraminidasa antigénicamente distintos. Un virus posee un solo subtipo de cada una de las proteínas, que parecen presentarse en cualquier combinación. Pese a que en especies de mamíferos se han aislado sólo unas pocas combinaciones de subtipos, en las aves se han obtenido todos los subtipos, asociados en casi todas las combinaciones posibles. El siglo XX ha presenciado en cuatro ocasiones la aparición repentina de cepas antigénicamente distintas en el ser humano, un fenómeno llamado "salto a n t i g é n i c o " (shift) que en cada ocasión ha dado lugar a una pandemia: en 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) y 1977 (H1N1). En los intervalos entre esas grandes pandemias también se han declarado frecuentes epidemias, producto de cambios antigénicos graduales en el virus prevalente, proceso conocido como "deriva antigénica" (drift). En la actualidad, las poblaciones humanas de todo el mundo sufren epidemias causadas por los subtipos H1N1 y H3N2 del virus de la influenza A y por el virus de la influenza B. Uno de los procedimientos más eficaces para medir los efectos de esas epidemias consiste en registrar el exceso de mortalidad debida a neumonías o gripes. Ciertos estudios filogenéticos sugieren que tal vez las aves acuáticas sean la fuente de todos los virus de la influenza A que afectan a las demás especies. Se piensa que las cepas causantes de pandemias en el hombre han surgido por uno de los tres mecanismos siguientes: - reordenamiento genético (proceso resultante de la segmentación del genoma vírico) entre virus aviares y humanos de la influenza A presentes en un mismo hospedador; - transferencia directa del virus entero desde otra especie al hombre; - resurgimiento de un virus que pudo haber causado una epidemia muchos años antes. Desde 1996, los virus H7N7, H5N1 y H9N2 se han transmitido de las aves al hombre, aunque en apariencia no han conseguido difundirse entre las poblaciones humanas. Aunque está demostrado que el virus puede transmitirse de animales a humanos, los episodios de este tipo son poco frecuentes. Ello ha llevado a pensar que el hipotético reordenamiento entre virus aviares y humanos tiene lugar en un animal intermediario, con posterior transmisión del virus resultante a la población humana. M u c h o s ven en el cerdo el principal candidato a esa función intermediaria, pues no sólo puede ejercer de hospedador de infecciones productivas de virus tanto humanos como aviares sino que, además, hay sólidas pruebas de su intervención en episodios de transmisión interespecifica de virus de la influenza, y particularmente en la transmisión de virus H1N1 al hombre. 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