Swine Influenza Virus: Zoonotic Potential and Vaccination Strategies for the Control of Avian and Swine Influenzas

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1 SUPPLEMENT ARTICLE Swine Influenza Virus: Zoonotic Potential and Vaccination Strategies for the Control of Avian and Swine Influenzas Eileen Thacker 1 and Bruce Janke 2 Departments of 1 Veterinary Microbiology and Preventive Medicine and 2 Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames Influenza viruses are able to infect humans, swine, and avian species, and swine have long been considered a potential source of new influenza viruses that can infect humans. Swine have receptors to which both avian and mammalian influenza viruses bind, which increases the potential for viruses to exchange genetic sequences and produce new reassortant viruses in swine. A number of genetically diverse viruses are circulating in swine herds throughout the world and are a major cause of concern to the swine industry. Control of swine influenza is primarily through the vaccination of sows, to protect young pigs through maternally derived antibodies. However, influenza viruses continue to circulate in pigs after the decay of maternal antibodies, providing a continuing source of virus on a herd basis. Measures to control avian influenza in commercial poultry operations are dictated by the virulence of the virus. Detection of a highly pathogenic avian influenza (HPAI) virus results in immediate elimination of the flock. Low-pathogenic avian influenza viruses are controlled through vaccination, which is done primarily in turkey flocks. Maintenance of the current HPAI virus free status of poultry in the United States is through constant surveillance of poultry flocks. Although current influenza vaccines for poultry and swine are inactivated and adjuvanted, ongoing research into the development of newer vaccines, such as DNA, live-virus, or vectored vaccines, is being done. Control of influenza virus infection in poultry and swine is critical to the reduction of potential cross-species adaptation and spread of influenza viruses, which will minimize the risk of animals being the source of the next pandemic. INFLUENZA VIRUSES AND PIGS The influenza viruses that infect pigs are influenza A viruses in the Orthomyxoviridae family. The presence of influenza in pigs was first recognized clinically during the summer of 1918 in the United States, at about the time of the Spanish influenza pandemic [1]. Influenza virus was first isolated from pigs in North America in Potential conflicts of interest: none reported. Presented in part: Harvard University Asian Flus and Avian Influenza Workshop, Cambridge, Massachusetts, 8 10 December Financial support: supplement sponsorship is detailed in the Acknowledgments. Reprints or correspondence: Dr. Eileen Thacker, Dept. of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, 2118 Vet Med Bldg., Iowa State University, Ames, IA (ethacker@iastate.edu). The Journal of Infectious Diseases 2008; 197:S by the Infectious Diseases Society of America. All rights reserved /2008/19704S1-0005$15.00 DOI: / [2]. Isolation of a swine influenza virus from humans in 1974 confirmed that swine influenza viruses are zoonotic in nature [3]. The genetic makeup of swine influenza viruses is identical to that of other influenza A viruses and consists of a negative-sense, segmented RNA genome. This segmented makeup allows for reassortment between viruses, resulting in the generation of new, antigenically novel viruses. The 2 major surface glycoproteins, hemagglutinin (H) and neuraminidase (N), are important for the determination of host range, antigenicity, and potential pathogenicity and serve as targets for diagnostic assays. It is important to recognize that the genetic makeup of swine influenza viruses in swine populations throughout the world can differ significantly. The swine influenza viruses in Europe differ significantly, in their antigenic and genetic makeup, from the viruses currently circulating in North America, even Swine and Avian Influenzas JID 2008:197 (Suppl 1) S19

2 though they consist of the same H and N subtypes [4]. Less is known about the genetic makeup of viruses currently circulating among pigs throughout Asia. Historically, the genetic makeup of swine influenza viruses was relatively stable in North America for nearly 80 years and consisted of 1 predominant subtype, known as classic swine influenza A(H1N1) virus (ch1n1 virus) [1]. ch1n1 virus had a genetic makeup composed primarily of swine influenza virus genes and was genetically and antigenically similar to the first swine influenza virus isolated in In 1998, H3N2 virus emerged in the US swine population and was composed of avian, human, and swine influenza virus genes [5 7]. This H3N2 virus consisted of H, N, and polymerase B1 genes that originated from human influenza virus; matrix and nonstructural protein and nucleoprotein (NP) genes from ch1n1 virus; and polymerase A and polymerase B2 genes that originated from avian influenza virus. Genetically, the human influenza virus genes in this H3N2 virus were similar to those in an influenza virus contained in a human vaccine used in the mid- 1990s. The entrance of this H3N2 virus into the US swine population has resulted in the emergence of multiple reassortant influenza viruses [8]. Since then, influenza viruses that have circulated in US swine-production systems have consisted of H1 viruses with H and N from the ch1n1 virus and internal genes from the H3N2 virus (i.e., rh1n1 virus) or with H from the ch1n1 virus and N and internal genes from the H3N2 virus (i.e., H1N2 virus) [6, 9]. An increased rate of genetic change has occurred among both H1 and H3 subtypes, with multiple genetically and antigenically diverse viruses of both major subtypes (H1 and H3) circulating in swine herds. Recently, there have been reports of H3N1 viruses circulating in Asia and the United States [10 12]. Even more recently, an H1N1 virus composed of only human influenza virus genes has entered the US swine population [13]. In addition to the influenza viruses described above, which are isolated fairly commonly from US swine herds, H3 and H1N1 avian influenza virus subtypes have been isolated from pigs in Canada [14]. As a result of the constantly changing genetic makeup of influenza viruses in pigs, the US swine industry is continually scrambling to respond to the influenza viruses circulating within individual production systems. The relationship between swine and human influenza disease has long been recognized as important. Pigs often are thought to be mixing vessels or to act as transmission agents in the adaptation of avian influenza viruses to mammals, including humans. Infection by influenza viruses is initiated by a binding event between viral H and sialic acid containing moieties on target cells. Different host species display differing virus-binding receptors with preferences for either Neu5Aca2-3Gal or Neu5Aca2-6Gal epitopes [15, 16]. H1, H2, and H3 subtypes of human influenza virus bind to the NeuAca2-6Gal linked sialic acid found in the human respiratory tract. In contrast, avian influenza viruses bind to the NeuAca2-3Gal receptors found primarily in the enteric tract of waterfowl [17]. The receptor specificity of avian influenza viruses is due to conserved amino acids at positions 190, 194, 225, 226, and 223 [16]. Swine influenza viruses have been reported to bind to both Neu5Aca2-3Gal and NeuAca2-6Gal receptors in the swine respiratory tract [16, 18, 19]. In addition, the N-glycolyl analog (Neu5Gc), a receptor that binds to swine influenza viruses with increased affinity, has been shown to be expressed abundantly in pigs [20]. More recently, both swine and avian influenza viruses have been shown to bind, with high affinity, to sulfated forms of sialyloligosaccharide receptors [21, 22]. Much of the research regarding influenza viruses has used viruses propagated in embryonated hen eggs, which has been shown to alter virus receptor specificity, increasing the affinity of human viruses for a2-3 linked sialic acid [17, 23, 24]. Recent research has demonstrated that the binding abilities of influenza viruses propagated by use of Madin-Darby canine kidney cells differ according to their H subtype [18]. Thus, either H3 subtypes of human influenza virus or ch1n1 preferentially bind to Neu5Aca2-6Gal, a pattern typical of human influenza viruses. In contrast, avian-like H1 and H3 subtypes of swine influenza virus bind to Neu5Aca2-6Gal and Neu5Aca2-3Gal. Transmission of influenza viruses between host species is of obvious concern, and transmission of human and swine influenza viruses between the 2 species has been well documented. The fear that such transmission can generate was demonstrated by the concern for a swine influenza pandemic in 1974 that was raised when an influenza outbreak caused by ch1n1 virus was documented in Fort Dix, New Jersey. During this outbreak, 1 soldier died, and 12 additional soldiers were hospitalized owing to respiratory disease [25 28]. Interestingly, no known exposure to pigs was ever documented. A subsequent investigation found that up to 230 soldiers had been infected with or exposed to the virus [25, 26]. Since then, a number of studies have found that people in contact with swine have higher antibody levels due to exposure to swine influenza viruses, although cases of clinical disease associated with swine influenza viruses are sporadic [29, 30]. In addition to transmission between humans and pigs, swine influenza viruses also have been isolated from turkeys on a fairly regular basis, indicating transmission between pigs and avian species [31 34]. It has been documented that pigs can be infected with the current highly pathogenic avian influenza (HPAI) H5N1 virus that has been responsible for disease in poultry in Asia [35]. These findings suggest that pigs would be the ideal mixing vessel for the creation of new avian/mammalian influenza viruses capable of causing novel diseases with the potential for producing pandemics in the human population. Whether this will happen S20 JID 2008:197 (Suppl 1) Thacker and Janke

3 easily, however, is less clear, although it is apparent that, in the US swine industry, transmission of influenza viruses between swine and humans is fairly common and is bidirectional. The pandemics of the past appear to have involved the introduction of whole or partial avian influenza viruses into a human population directly, rather than through swine. VACCINATION STRATEGIES FOR THE CONTROL OF SWINE INFLUENZA VIRUS INFECTION Two primary obstacles confound programs for successful influenza vaccination of pigs namely, viral antigenic shift and drift and the effect of maternally derived antibodies on vaccine efficacy. The vaccination of sows is a common practice used by the swine industry to increase and prolong maternally derived antibody levels in young pigs, to protect them against clinical disease. However, the presence of maternal antibodies reduces vaccine efficacy, making it difficult to vaccinate pigs prior to exposure to the virus and resulting in an increased incidence of disease among pigs as their maternal antibodies decay and they become susceptible to virus infection and disease [36]. As a result, influenza viruses potentially can circulate in swine herds on a regular basis, owing to the constant introduction of immunologically naive animals into the herd. Current swine influenza vaccines are adjuvanted, inactivated, whole-virus vaccines prepared typically from virus propagated in embryonated hen eggs. These vaccines stimulate high titers of IgG in serum and lungs, which protect against clinical disease. Antibodies against the H protein appear to be the most protective. Protection against infection is not complete, but virus multiplication and shedding are greatly reduced. An issue currently under discussion is the need to update US Department of Agriculture clearance procedures for new vaccine licensure to allow the addition of newer isolates into vaccines, to keep pace with changes in virus genetics in the field. At present, the requirements for licensure of swine influenza vaccines are much more laborious and expensive than those in place for human influenza vaccines. Approval of animal vaccines requires demonstration of vaccine safety, efficacy, and potency through multiple experimental and field trials. As a result of the extensive trials required, a vaccine often takes up to 5 years to be licensed. Fortunately, cross-protection against antigenic variants of influenza viruses appears to be broader in pigs, compared with that provided by human influenza vaccines [37]. Currently, modified live influenza virus vaccines are not available for swine, although results of recent studies of genedeleted vaccines have been reported [38]. The advantage of modified live-virus vaccines is enhanced stimulation of cellmediated immunity, directed primarily against conserved NP, thus providing more heterosubtypic immunity (i.e., protection across subtypes). One concern about live-virus vaccines would be possible reassortment between field strains and the vaccine virus, producing new reassortant viruses. Vectored vaccines using vaccinia virus, baculovirus, alphavirus, or adenovirus also are being studied [39, 40]. DNA vaccines also have been studied by using chicken, mouse, ferret, and primate models of influenza virus infection. The theoretical advantage of these vaccines is the production of viral protein with normal conformation, without the risks associated with the use of live virus. Such vaccines stimulate long-lasting immunity through both humoral and cell-mediated systems. DNA vaccines appear to be ideal for use with swine, since they would provide heterosubtypic immunity and the internalization of DNA inside host cells would minimize interference by maternal antibodies. Unfortunately, to date, experimental trials of DNA vaccines in pigs have not proved successful. However, DNA vaccines may prove to be excellent primer vaccines when followed by more-conventional inactivated vaccines [41, 42]. AVIAN INFLUENZA VIRUSES AND VACCINE STRATEGIES FOR DOMESTIC POULTRY Birds, especially waterfowl, can be infected with any influenza virus, regardless of H or N subtype. The receptors to which avian influenza viruses bind were described above. In contrast to influenza virus infection in swine or humans, disease in wild waterfowl is rare and observed only when caused by HPAI viruses. No infection-control strategies are currently used on wild birds; because influenza viruses are constantly circulating among wild waterfowl, a primary goal is to minimize the exposure of either domestic poultry or swine to these viruses. Protective immunity against influenza virus infection in birds is similar to that in mammals that is, as in pigs and humans, antibodies against the H protein are important for protection against virus infection, which is subtype specific. However, within-subtype protection appears to be more effective in birds over a wider range of viral antigenic variation, compared with that provided by human influenza vaccines. In addition, antigenic drift among field strains that affects cross-protection has not been a significant problem with avian influenza viruses; however, over the past decade of vaccine use in commercial poultry operations in Mexico, surveillance and genomic/antigenic analysis of influenza viruses have documented that antigenic drift can occur in avian species as well [43]. Inactivated vaccines most commonly are used to vaccinate commercial poultry against influenza viruses and are fairly effective. Subunit vaccines in which selected influenza virus genes have been inserted into a fowlpox vector virus have been developed and tested in experimental trials but have not been used in field situations [44]. In contrast to human and swine influenza viruses, variation in the pathogenicity of avian influenza viruses and, thus, their Swine and Avian Influenzas JID 2008:197 (Suppl 1) S21

4 designation as HPAI viruses versus low-pathogenic avian influenza (LPAI) viruses is important when vaccine strategies for domestic poultry are considered. In the United States, HPAI viruses are considered to be foreign animal viruses, with detection resulting in complete elimination of a flock. In contrast, infection by LPAI viruses occurs more commonly in US commercial poultry populations, and the management response to infection depends on the influenza virus subtype involved. Vaccination against LPAI viruses is done primarily in turkey breeder flocks. Infection by LPAI viruses does not result in high mortality rates among turkeys, but egg production can be affected significantly. In the 1980s and early 1990s, most turkeys were raised in the open, and frequent infection of these flocks was attributed to migrating waterfowl, which are natural, typically asymptomatic carriers of avian influenza viruses. Vaccination and controlled marketing were used to eradicate influenza virus from affected turkey flocks. After the mid-1990s, turkey production was moved to confinement facilities, thus greatly reducing potential exposure to wild birds. The open buildings used to house turkeys still expose the birds to prevailing winds, and the H1 and H3 influenza virus subtypes that currently are of concern usually originate from neighboring swine operations. Both broiler and egg-laying chickens are housed in more-controlled environments, and infection with any of the influenza viruses, including LPAI isolates, is rare. In addition, a withdrawal period of at least 43 days between vaccination and slaughter must be preserved with broiler chickens; thus, vaccination of broiler chickens is not done routinely. Infection of poultry flocks with any H5 or H7 influenza virus subtype, even LPAI isolates, elicits a response much different from that for infection by other influenza virus subtypes. All HPAI viruses are H5 or H7 subtype strains, although the opposite is not true that is, not all H5 and H7 subtype strains are HPAI viruses. Past experience has shown that LPAI H5 or H7 viruses are capable of mutating into HPAI viruses; thus, flocks with H5 or H7 influenza virus infections usually are eliminated [45]. Although total eradication of flocks has been used more frequently, vaccination in conjunction with quarantine and surveillance also has been used to successfully eliminate H5 or H7 influenza viruses from domestic poultry flocks. Surveillance programs and trade issues are important considerations in decisions about vaccine use in poultry industries. Serologic surveillance is used to monitor against the introduction of HPAI viruses and to certify flocks free of avian influenza virus, especially for marketing purposes. Loss of the US poultry industry s status as free of HPAI viruses would have a severe impact on exports of birds and poultry products to other countries. In the United States, vaccines against avian influenza typically are oil-adjuvanted, inactivated, whole-virus vaccines that induce antibodies to all viral proteins. The serologic tests used routinely in surveillance programs are agar-gel immunodiffusion and commercial ELISA. Both assays use conserved internal viral proteins (matrix or NP) as the test antigen and, thus, give antibody-positive results whether birds are infected or vaccinated. Thus, with current vaccines and serologic assays, it is impossible to determine whether a vaccinated flock is infected with the virus. Because the vaccine does provide protection against clinical disease, HPAI viruses could circulate undetected within a flock. For this reason, vaccination against H5 and H7 influenza viruses is not done routinely, and the use of these vaccines requires federal approval. Surveillance for influenza viruses in poultry flocks often includes the placement of unvaccinated sentinel birds in the flock, and these birds are monitored serologically for the presence of antibodies or virus by use of polymerase chain reaction assays. This allows detection of infection as well as determination of the influenza virus subtype circulating in the flock. A process for differentiating infected from vaccinated animals (called DIVA ) also is used [46]. One of the first such programs in the control of avian influenza used a heterologous N subtype, H7N3, in the vaccine to immunize against H7N2 influenza virus infection, enabling the differential detection of N3 antibodies in vaccinated birds versus infected birds. Similarly, subunit vaccines are used in which the desired gene, typically the H protein gene, is inserted into a fowlpox vector [44]. With this approach, routine serologic assays that detect matrix protein or NP antibodies can be used for surveillance. SUMMARY Influenza virus infections in swine and poultry are potential sources of viruses for the next pandemic among humans. The bidirectional transmission of influenza viruses between pigs and humans has been documented. Although not common, infection of swine by avian influenza viruses does occur and serves as a potential source of new, genetically diverse influenza viruses capable of infecting humans. As a result, the use of control strategies, especially vaccination, is critical for the control of influenza virus infections among domestic animals, to reduce their potential as sources for outbreaks among humans. In addition, the vaccination of people who work with swine is encouraged, to reduce the chance of human influenza viruses being spread to pigs. Current vaccination strategies for the control of disease in both swine and poultry are fairly effective, but ongoing research needs to be done to ensure the control of both swine and avian influenza virus infections. Acknowledgments The Harvard University Asian Flus and Avian Influenza Workshop was hosted by the Harvard University Department of Anthropology, Harvard School of Public Health, and Harvard Asia Center and was supported by S22 JID 2008:197 (Suppl 1) Thacker and Janke

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