EVALUATION OF DIFFERENT STRATAGIES FOR THE DIFFERENTIATION OF INFECTED AND VACCINATED ANIMALS (DIVA) IN CHICKENS VACCINATED

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1 EVALUATION OF DIFFERENT STRATAGIES FOR THE DIFFERENTIATION OF INFECTED AND VACCINATED ANIMALS (DIVA) IN CHICKENS VACCINATED WITH AVIAN INFLUENZA OIL EMULSION VACCINES by GLORIA E AVELLANEDA (Under the Direction of David L Suarez) ABSTRACT The use of avian influenza vaccination in poultry would have greater world-wide acceptance if a reliable test that clearly discriminates naturally infected from vaccinated only animals (DIVA) was available. In order to evaluate the non-structural protein 1 (NS1) and neuraminidase (NA) proteins of avian influenza virus as potential markers for infection, an indirect ELISA using recombinant baculovirus purified NS1 protein as antigen and the neuraminidase inhibition (NI) test using MUN (2 -(4- methylumbelliferyl)-_-d-nacetylneuraminic acid sodium salt hydrate) as NA substrate, were used as differential diagnostic serological tests. Because the NS1 protein is only produced when the virus is replicating in cells and it is not packed in the infectious virion, theoretically it is not present in the killed vaccines. Therefore, vaccinated birds will be negative for NS1 antibody as opposed to infected birds that will develop antibodies against NS1 during virus replication. Antibodies against NS1 protein were first

2 detected three weeks after infection of naïve birds, but decreased rapidly by 5 weeks after infection. In inactivated oil emulsion immunized birds, antibodies against NS1 were detected only in a small percentage of birds after homologous LPAI virus challenge. Vaccinated birds challenged with highly pathogenic AI showed a better NS1 antibody response in some but not all challenged birds. The use of killed adjuvanted vaccines containing virus with homologous hemagglutinin and heterologous NA subtype to the challenge virus can be used effectively to protect birds against the disease and allow the possibility of using the antibody response to the heterologous viral NA protein as marker of infection. In two groups of birds vaccinated prior to challenge, the NI test discriminated chickens receiving different vaccine antigens (e.g., N8 or N9), and two weeks post-challenge with a highly pathogenic avian influenza virus subtype H5N2, 100% of the vaccinated birds had significant levels of N2 NI activity with high specificity. Comparing the two DIVA strategies, the heterologous NA method gave a more consistent response with earlier detection of infection under these experimental conditions. However, further research is needed to evaluate how this approach works under different field conditions before it can be adopted on a commercial scale. INDEX WORDS: Avian influenza virus, chickens, DIVA strategy, avian influenza vaccine, heterologus neuraminidase, NS1 protein, ELISA

3 EVALUATION OF DIFFERENT STRATAGIES FOR THE DIFFERENTIATION OF INFECTED AND VACCINATED ANIMALS (DIVA) IN CHICKENS VACCINATED WITH AVIAN INFLUENZA OIL EMULSION VACCINES by GLORIA E AVELLANEDA DVM, University of Caldas, Colombia, 1980 M.S., University of Georgia, 1993 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2008

4 2008 Gloria E Avellaneda All Rights Reserved

5 EVALUATION OF DIFFERENT STRATAGIES FOR THE DIFFERENTIATION OF INFECTED AND VACCINATED ANIMALS (DIVA) IN CHICKENS VACCINATED WITH AVIAN INFLUENZA OIL EMULSION VACCINES by GLORIA E AVELLANEDA Major Professor: Committee: David L Suarez David E Swayne Maricarmen Garcia Charles L Hofacre Amelia Woolums Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2008

6 DEDICATION To the loving memory of my mother Gloria To my children Juan Camilo and Juliana To my father Luis iv

7 ACKNOWLEDGEMENTS I want to express my gratitude to the following people that contributed with their knowledge, support, or friendship to the achievement of this goal My committee Dr. David Suarez Dr. David Swayne Dr. Charles Hofacre Dr. Maricarmen Garcia Dr. Amelia Wooloms My coworkers: Suzanne DeBlois Joan Beck James and Kira Aniko Zsak Scott Lee Tim Oliver Dr. Carlos Estevez Dr. Samadhan Jadhao Dr. Claudio Alfonso Joyce Bennett Melissa Scott Cam Greene Tracy Smith-Faulkner Roger Brock Ronald Gram Johnny Doster Special thanks and appreciation to: Dr Egbert Mundt Dr. Matt Sylte v

8 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS... v CHAPTER 1 INTRODUCTION LITERATURE REVIEW DIFFERENTIATION OF INFECTED AND VACCINATED ANIMALS (DIVA) USING THE NS1 PROTEIN OF AVIAN INFLUENZA VIRUS A HETEROLOGOUS NEURAMINIDASE SUBTYPE STRATEGY FOR THE DIFFERENTIATION OF VACCINATED AND INFECTED ANIMALS (DIVA) STRATEGY FOR AVIAN INFLUENZA VIRUS USING A MORE FLEXIBLE NEURAMINIDASE INHIBITION TEST DISCUSSION REFERENCES vi

9 CHAPTER I INTRODUCTION The outbreak of human influenza with H5N1 avian influenza viruses by direct transmission from poultry in Hong-Kong in 1997 (1) provided compelling evidence that avian influenza viruses could be transmitted directly to humans (2). The number of cases involving bird-to-human transmission and the resulting production of clinically severe and fatal human infections has slowly increased (3), and the cumulative number of confirmed human cases of avian influenza (H5N1) reported to WHO as of March 8, 2008 was 372 with 235 deaths (3). The number of reported outbreaks caused by H5N1 highly pathogenic avian influenza (HPAI) in poultry (4-6) has increased dramatically, but outbreaks of clinical disease has also occurred in waterfowl species including geese, ducks and swans (7), species that normally do not show clinical signs. All these events have changed the field of avian influenza and its epidemiology during the last decade, and have led to considerable pressure to use vaccination as part of control policies, either as an emergency measure or prophylactically for both highly pathogenic avian influenza (HPAI) and low pathogenicity avian influenza (LPAI) of H5 or H7 subtypes (8-11). Vaccination plays an important role in the control of diseases in poultry. However, for avian influenza several factors have limited its use as routine part of control programs. The main concern is that vaccination with traditional killed oil emulsion vaccines will interfere with the traditional serologic tests that are used for surveillance of

10 influenza infection such as the AGP, HI and nucleoprotein- specific ELISA (12). These tests cannot differentiate whether the antibodies are from infection or vaccination, when conventional killed vaccines are applied (13). Several different strategies to differentiate vaccinated from infected animals (DIVA) have been proposed (12), and require the use of appropriate vaccines and specific serological discriminatory tests. DIVA strategies for avian influenza are important not just for detecting any outbreak of the virus in vaccinated birds (14), but also for trade purposes as it enables restrictions on vaccinated poultry meat to be lifted once the flocks can be clearly shown to be free of infection (12, 15). The DIVA strategy has been accepted by some countries as a way to monitor and provide assurance on the infection free status of vaccinated poultry (10). Ideally a DIVA companion test would not strictly be limited to a particular AI vaccine but it should be able to be used with different vaccines depending on the needs. At the present there is no universal DIVA test readily available that can be used in emergency situations with any subtype of available commercial oil emulsion vaccines. One potential target for a DIVA strategy that will fit this situation is the nonstructural protein 1 (NS1). The NS1 protein is encoded by the smallest gene segment of influenza virus, segment 8. The NS1 protein is a conserved protein amongst type A influenza viruses, but it exists as two major subtypes, A and B, that differ by about 35% in nucleotide sequence (16). The NS1 protein is a true non-structural protein. It is produced in infected cells during the virus replication cycle and accumulates in the infected cells but it is not packaged into the infectious viral particle (17). Traditionally, killed influenza vaccines for poultry are made by inoculating the virus in embryonating chicken eggs and harvesting the allantoic fluid, which contains high titers of whole viral 2

11 particles, for the vaccine preparation with little purification. Vaccinated animals theoretically should not have an antibody response to the NS1 protein, but naturally infected animals should develop antibodies to the NS1 protein (18). This is the rationale behind the Differentiation of Infected and Vaccinated Animal (DIVA) approach first proposed for equine influenza viruses (19), where purified NS1 protein was used to detect antibodies to NS1 in serum samples from ponies and horses experimentally infected with influenza virus (19, 20). Later on, the approach was tested on chickens and turkeys and the antibody levels against NS1 appear to be higher in infected than in vaccinated birds allowing differentiation between these two populations (21, 22). This experimental data produced using the recombinant NS1 protein on indirect ELISA (21, 22) appeared very promising and laid the background for this research. There were three specific aims of this study: Specific aim I: To understand the kinetics of the antibody response against NS1 protein after infection with LPAI virus. Groups of eight 38-day-old birds were infected by the intranasal route with LPAI virus A/CK/California/K Ct/03 (H6N2) or A/TK/Wisconsin/68 (H5N9) at 10 5 EID 50 and 10 6 EID 50 EID/0.2ml, respectively. Serum samples were taken for serological analysis before the infection and every week after the infection for five consecutive weeks. To measure the response of antibodies against NS1 protein, recombinant NS1 protein was produced in baculovirus infected Sf9 (Spodoptera frugiperda) insect cell line and used to develop an indirect NS1-ELISA. NS1 antibodies were detected in only 2/8 (25%) birds infected with the CK/CA LPAI virus in spite of the high HI antibody titer detected in 100% of the birds. A plot of the serological results 3

12 obtained after the infection with CK/CA virus shows three important difference between the HI and the NS1 response: 1) The onset of the antibody response to NS1 protein appears to be later than the onset of the antibody response to HA; 2) The antibody response to NS1 reaches its peak almost immediately after its onset while the level of HA antibodies reach its peak slower over a period of 3 weeks, and 3) NS1 antibody response consistently declines after reaching its peak level at 3 weeks after infection, as opposed to the antibody response to the HA protein that after reaching its peak level forms a plateau suggesting longer half-live than antibodies against NS1. Specific aim II: To determine the usefulness of the NS1- indirect-elisa as accompanying test for the Differentiation of infected and Vaccinated Animals (DIVA) strategy. The approach used for this specific aim was the vaccination of different groups of birds with experimental recombinant vaccine (rh5n8-sepns1) or commercial inactivated vaccine (subtype H5N9). Two weeks after vaccination, the birds were challenged with homologous low pathogenic avian influenza (LPAI) virus (A/CK/Pennsylvania/13609/93 [H5N2]) or high pathogenic avian influenza (HPAI) virus (A/CK/Queretaro/ /95 [H5N2]). Vaccinated birds challenged with homologous LPAI virus showed a low percentage of birds with a detectable immune response against NS1. In the group vaccinated with the recombinant vaccine, samples from only one bird taken at 2 and 3 weeks after the challenge were positive for NS1 antibodies in the indirect ELISA. Vaccinated and then challenged birds with homologous HPAI virus had a higher detection rate than the LPAI virus challenged groups, with 20% (2/10) and 30% (3/10) 4

13 positive birds with antibodies against NS1 protein at week 4 after challenge in the two vaccinated groups. The response started to be detected at 2 weeks, but did not last longer than 1 or 2 weeks, and the levels of NS1 antibodies were low. The use of the NS1 DIVA strategy to differentiate infected from vaccinated and then infected birds had a better response with HPAI virus as compared to the LPAI virus challenge. The level of antibody response to the challenge NS1 seen in this experiment was better to HPAI virus challenge. However, the duration was short and may not allow the test to be performed on a practical basis. Specific aim III: Analysis of the antibody response to the neuraminidase (NA) protein as marker of HPAI virus infection in chickens immunized with inactivated oil emulsion vaccines containing virus with heterologous NA. To achieve this objective, a quantitative neuraminidase inhibition (NI) test using MUN (2 -(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid sodium salt hydrate) as NA substrate was used to discriminate the NI activity to the challenge AIV strain from the NI activity to the NA of the vaccines. Groups of chickens vaccinated with inactivated rh5n8-sepns1 or commercial vaccine H5N9 were challenged with 10 5 EID 50 /bird of the HPAI virus A/CK/Queretaro (H5N2). The serum samples were taken at 2 weeks after vaccination shortly before challenge (W-0), and every week for 4 consecutive weeks after challenge (W-1, W-2, W-3 W-4), and evaluated for NI activity. The NI activity of the anti-neuraminidase antibodies was assayed against three inactivated antigens A/CK/Queretaro (H5N2), rn8h5-sep-ns1 (H5N8) and A/TK/Wisconsin/68 (H5N9). At week 0 (2 weeks after vaccination and shortly before challenge), both vaccinated groups showed positive NI activity only to the homologous vaccine virus with 5

14 NI log 2 titers 4.6 (N8) for the group immunized with the recombinant vaccine and 5.7 (N9) for the group immunized with the commercial vaccine. No N2 antibodies were detected in both vaccinated groups as well as in the sham vaccinated group control. During the weeks following challenge (W-1, W-2, W-3 W-4) the NI activity as measured with each vaccine homologous NA antigen increased significantly (p<0.001) until 3 and 2 weeks post-challenge in the recombinant and commercial vaccinated group respectively. From this point until the end of the experiment, there were not significant changes in NI titers to the vaccines in either vaccinated group. The vaccine efficacy was evaluated with the HI test using BPL inactivated HI antigen homologous to each vaccine strain. The HI data showed a strong antibody response to the vaccine in both immunized groups at day of challenge (2 weeks after vaccination) with an HI log 2 titer of 5.4 and 8.0 for the group immunized with the recombinant and commercial vaccine, respectively. The HI titers in both groups increased significantly (P<0.001) after the challenge and remained high during the testing period. The birds in the sham vaccinated group did not survive the challenge and 100% mortality was observed before day three after challenge. 6

15 CHAPTER 2 LITERATURE REVIEW I Avian Influenza virus classification The family Orthomyxoviridae contains viruses that have negative sense, single stranded, segmented RNA genomes. The definition of negative-sense RNA was given by David Baltimore who showed that the packaged viral RNA was complementary to the mrna which is defined as positive sense (23). The Orthomyxoviridae family consists of five genera: Influenza virus A, Influenza virus B, Influenza virus C, Thogotovirus, and Isavirus. The Thogotovirus are tick borne arboviruses that have been isolated form both humans and livestock (24), and the Isavirus group includes the important fish pathogen infectious anemia virus (25). Only viruses of the Influenza virus A genus are known to infect birds. Viruses of this genus also infect mammals whereas type B and C are essentially restricted to human beings (26). The viruses in the Influenza virus A genera are further classified based on the antigenicity of two proteins found on the surface of the virus. One is the hemagglutinin (H), of which there are 16 different subtypes (H1-H16); the other is the neuraminidase (N), of which there are 9 different subtypes (N1-N9) (23). Each virus has one HA and one NA antigen subtype, for a total of 144 possible combinations. The majority of these possible combinations of subtypes have been isolated from avian species (26). The present nomenclature system includes the type of the virus, the host of origin (except for humans), geographic site of isolation, the strain number or laboratory identification,

16 and year of isolation followed by the antigenic description of the HA and NA in parenthesis (23). For example, the influenza virus 220 th sample accession from chickens in Hong-Kong in 1997 is designated: influenza A/chicken/Hong-Kong/220/97 (H5N1) virus (23). Avian influenza viruses (AIV) are further classified based on the severity of the disease they cause in chickens and are classified as either low or highly pathogenic avian influenza viruses (27). Low pathogenic avian influenza (LPAI) viruses can be of many different hemagglutinin and neuraminidase subtypes. They cause mild or asymptomatic infections in chickens in experimental infections, although in some field situations serious diseases with some mortality have been reported. In contrast, highly pathogenic avian influenza (HPAI) viruses have been restricted to the H5 and H7 subtypes, but most H5 and H7 influenza viruses are of low pathogenicity. The HPAI viruses cause a severe and extremely contagious illness and death among infected chickens. Mortality rates for chickens infected with HPAI can be as high as 90 to 100 percent (27). II. Structure of Influenza Virus All influenza viruses have different gene segments that encode at least 10 different proteins. The surface proteins in the mature virion include the hemagglutinin (HA), the neuraminidase (NA), and the M2 proteins. These proteins radiate outwards from the surrounding lipid envelop derived from the plasma membrane of the host cell that protects the integrity of the virion during the transmission process (23). Influenza virus protein M2 is a small (97-residue) integral membrane protein, inserted into the hostderived lipid envelop. The M2 protein acts as an ion channel during the virus-uncoating process in endosomes, permitting a flow of protons into the interior of virus particles to 8

17 disrupt protein-protein interactions. The M2 ion channel is specifically inhibited by the antiviral drug amantadine (28, 29), The internal proteins include the nucleoprotein (NP), the matrix protein M1, which is the most abundant protein of the virion and underlines the lipid bilayer and interacts with the ribonucleoprotein (RNP) (23). The RNP contains the 8 different segments of the viral RNA and each one is associated with the polymerase complex which is comprised of the polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase acidic protein (PA) (23). Two additional proteins produced by influenza viruses are the non-structural proteins, non-structural protein 1 (NS1) and the non-structural protein 2 (NS2), also known as the nuclear export protein (NEP)(23). NS2 is also associated with the M1 protein and plays a role in the export of the ribonucleoprotein complex (RPN) from the nucleus to the cytoplasm (23). A novel 87- residue protein, PB1-F2, is expressed by some viruses in infected cells and localized in mitochondria, and appears to be involved in apoptosis of host cells (30). However, its exact role in pathogenicity is still under investigation (30). III. Virus Life Cycle The HA surface protein is a trimer and the major protective antigenic determinant. It binds to sialic acid, which is commonly expressed on host cells and is the receptor that allows virus entry into the host cell. Sialic acid is a generic term for the N- or O- substituted derivatives of neuraminic acid. These linkages take the name according to how they are linked to the sugar by the alpha 2 carbon. These different sialic acid linkages result in different conformation of the host receptor that affects virus binding. Some viruses bind preferentially to terminal sialic acids containing α-(2,6) linkages, 9

18 whereas others bind to α-(2,3)-linked sialic acid (31). The receptor binding specificity correlates with a specific amino acid at position 226 of HA(32). HAs that have leucine at position 226 selectively bind to α-(2,6) sialic acid and occur preferentially in human strains (33). HAs that have glutamine at position 226 are specific for α-(2,3) linkages, and occur mostly in avian and equine strains of the virus (34). Both α-(2,3)- and α-(2,6)- linked sialic acid occur in the trachea of swine accounting for the ability of pigs to be infected with both avian and human strains (35). After binding, the virus is internalized by endocytosis into the endosome (cytoplasmic vesicles) (36). The low ph of the endosome triggers a conformational change of the viral HA allowing the fusion domain to become active. The channel activity of the M2 integral membrane protein allows H+ ions to go from the endosome to the interior of the virus cavity acidifying the ph helping to dissociate the RNP complex and completing the uncoating process (37). Acidification causes a conformational change of HA exposing the fusion peptide that brings together the endosome and the viral membrane. These membranes fuse together and by an unknown mechanism separate to allow the exit of the viral nucleic acid to the cytoplasm. The uncoated vrnps then enter the nucleus of the host cell for virus replication. Influenza viruses are one of the few RNA viruses to undergo replication and transcription in the nucleus of their host cells (38, 39). The polymerase complex must synthesize three different viral RNA species: viral mrna; the crna replication intermediate; and negative sense vrna. The polymerase complex is formed by the three polymerase proteins: PB1, PB2, and PA. In the initial steps PB2 binds to the CAP (i.e. contain a methylated 5' guanosine residue) of host mrna. The CAP is cleaved from the host mrna by the nuclease activity of PB1. 10

19 PB2 remains attached to the CAP structure which serves as a primer for viral mrna synthesis. Therefore, the 5' end of the viral mrna is composed of a cap structure and nucleotides derived from the host cell mrna, which are used as primers for initiation (40). The PB2 protein has been implicated in pathogenicity in mammals. The 1918 virus and subsequent human isolates have a Lys residue at position 627. This residue has been implicated in host adaptation (41), and has previously been shown to be crucial for high pathogenicity in mice infected with the 1997 H5N1 virus (42). Reverse genetics studies classified the 1997 H5N1 human isolates in two groups based on their pathogenicity in mice: viruses of low pathogenicity that contains glutamic acid at position 627 (found in most influenza viruses isolated from avian species) and highly pathogenic viruses that contain lysine at this position (42). In general, this classification also corresponds to the severity of the diseases in humans (43). The positive sense viral mrna then migrates from the nucleus to the cytoplasm to begin viral protein translation using cellular machinery. The nuclear export of viral mrna utilizes the machinery of the host cell, but is selective; export is controlled by the viral non-structural protein NS1 (44). The NS1 protein is efficiently targeted into the nucleus, and two nuclear localization signals (NLSs) have been identified in the H3N2 subtype influenza A/Alaska/6/77 virus NS1 (45). The molecular mechanisms of the nuclear import of NS1 proteins has been shown to take place via the classical importin α/β nuclear import pathway, where basic amino acids 35, 38, and 41 are critical for importin α binding and nuclear translocation (45). 11

20 The positive sense RNA also serves as a template to produce the negative sense viral RNA that will be packed into the virion. Most of the protein remains in the cytoplasm or becomes associated with the cell membrane. However, NP, M1, NS2 and the polymerases go back to the nucleus where they become associated with the newly synthesized vrna to form RNPs. Following virus replication, the vrnps leave the nucleus and move to the plasma membrane, where they associate with viral glycoproteins before final budding and release. Both trafficking events, nuclear import and export of vrnps for virus assembly occurs through nuclear membrane pores (46). This trafficking appears to rely on the host machinery since the nuclear export is blocked by the antibiotic leptomycin B (47). Leptomycin B binds to the chromosome region maintenance 1 (CRM1) export receptor, the major receptor in protein-based nuclear export pathways in the cell. The trafficking of the new vrnps rely on the export signals on the NS2 protein (48, 49) while the signals for nucleus import are found in the NPs. The level of NP is believed to control whether mrna or vrna is made (48, 50). The viral assembly process occurs at the plasma membrane. M1 is central to this interaction by the binding of the M1 molecules to vrnps and the plasma membrane to form a shell beneath the virus envelope. The envelope proteins, HA and NA, and small amounts of the M2 protein, enter the endoplasmic reticulum, where they are folded and glycosylated before eventually moving to the apical plasma membrane (51). The formation of viruses at these sites appears to rely on the presence of the cytoplasmic tails of both HA and NA. These glycoproteins, along with M1, M2 and host-cell factors 12

21 appear to control virus morphology, and thus determine the spherical or filamentous nature of the resultant particles (52, 53). The final release of viruses from the cell surface relies on the action of the viral neuraminidase. Neuraminidase cleaves terminal sialic acid residues from carbohydrate moieties on the surfaces of infected cells, preventing the HA protein from attaching to other viral proteins. This promotes the release of progeny viruses from infected cells by preventing aggregation of the viruses (23). IV. Clinical Disease Clinical signs vary greatly and depend on many factors including the age and species of poultry affected, particularly chickens or turkeys, husbandry practices, and the inherent pathogenicity of the influenza virus strain (54). LPAI viruses cause mild or asymptomatic infections in chickens in experimental studies, although in some field situations serious disease with some mortality has been reported. In contrast, HPAI viruses cause a severe and extremely contagious illness and death among infected chickens. Mortality rates for chickens infected with HPAI can be as high as 90 to 100 percent (27). Influenza infections can be asymptomatic, but it often causes production losses and a range of clinical disease from mild to severe in affected flocks. The virus can be generally divided into viruses that only cause mucosal infection, either in the respiratory and/or enteric tract, and those viruses that also cause systemic infections. The viruses that cause mucosal infections are usually referred to as low pathogenicity or mildly pathogenic AI (LPAI) viruses, and typically these viruses do not cause high mortality in affected flocks. The viruses that cause systemic infections usually cause high mortality and are referred to as highly pathogenic AI, high pathogenicity AI, or 13

22 historically as fowl plague viruses (54, 55). The LPAI viruses can cause asymptomatic infections, but typically the most common symptoms are mild to severe respiratory disease. A decrease in feed or water consumption is another common indication of flock infection when careful records of consumption are kept. For layer flocks or breeder flocks, drops in egg production can also be observed. The drops in egg production can be severe with the flocks never returning to full production as is commonly seen in turkey breeders infected with swine-like influenza viruses (56, 57). In large flocks, small increases in daily mortality can be observed as the virus spreads through the flock. The LPAI infection at least contributes to this increased mortality because diagnostic testing of the daily mortality is considered to be a sensitive way to identify AI infection in flocks infected with LPAI virus (13, 58). In some situations infection with LPAI virus may result in high mortality, generally in association with concurrent or secondary pathogens and/or poor environmental conditions (59). On rare occasions, LPAI viruses may cause specific lesions in internal organs, through either direct infection or by other indirect causes (60). V. Diagnosis of avian influenza Diagnosis of avian influenza can be made by a variety of methods including, clinical signs, serological methods, and direct detection of the virus (61). Isolation and identification of the virus is still the definitive diagnostic test, particularly for the index case (62). Virus isolation is traditionally performed using specific pathogen-free (SPF) eggs or cell cultures as substrates but commercial eggs from known non-vaccinated source free of AI can be used as well. Tracheal and cloacal swabs, lung and spleen specimens, are samples of choice. For live ducks, cloacal swabs are recommended; if 14

23 dead, lung and brain samples can be important. If the sample is positive, the diagnosis can be given as early as 24 hours; if the sample is negative at least 2 weeks is necessary to complete the testing with at least one blind passage (63). Laboratories where virus isolation is performed must be equipped with Class 2 biosafety cabinets for sample preparation, egg inoculation and allantoic fluid harvesting (64). Agar gel immunodifussion (AGID) and ELISA tests are suitable for serological surveillance of chicken and turkey flocks since they allow the testing of large numbers of samples (62, 65). Both tests are group-specific for antibody. They are useful on a flock basis for LPAI viruses but of limited use for HPAI strains where mortality is high. AGID is not useful to detect virus infection in waterfowl as the precipitating antibody response to group antigen is generally poor. The competitive ELISA can be used for all species. However, in wild birds its use has only limited value because of the likelihood of exposure. For many minor poultry species, like ducks, geese and pheasants, little work has been performed, and therefore the value of the test is unknown. Immunofluorescence test also is used in some countries for screening by also detecting group antigen. It is performed on impression smears taken at the necropsy. For H5N1 infections antigen in comb and spleen is abundant. It is a relatively quick and cheap test to perform though it requires a fluorescence microscope in good condition and it is a more subjective test. The hemagglutination inhibition (HI) test is the subtype-specific test recommended. It is sensitive and specific when an epidemiologically appropriate antigen is used. It can be used for monitoring the response to vaccination. However, some 15

24 influenza viruses do not hemagglutinate RBC s from commonly used species like chickens. Tests for antibodies against NA include the neuraminidase inhibition (NI), indirect fluorescent antibody and direct ELISA tests. The HI and NI tests can be used to subtype AI viruses into 16 hemagglutinin and 9 neuraminidase subtypes. Such information is helpful for epidemiological investigations and in categorization of AI viruses. Several molecular diagnostic tools are available for AIV that amplify the viral RNA (or cdna) to detectable levels, but the most commonly used molecular test is the real-time reverse transcription polymerase reaction test (RRT-PCR) (61). The test is used in the U.S. screens for Type A influenza using matrix primers and probe, and positive samples are confirmed with a subtype specific test (for H5 and H7 viruses). This test is standardized and is used throughout the U.S. in the National Animal Health Laboratory Network (NAHLN). A nucleic acid sequence-based amplification (NASBA) (66-68) test developed in Hong Kong is also very sensitive but the kits are expensive. However, NASBA has advantages in terms of reducing the risk of laboratory contamination with amplified DNA. VI. Changes from LPAI to HPAI The hemagglutinin protein of all influenza A viruses is produced as a precursor (HA0), which requires post-translational cleavage by host proteases before it is functional and virus particles are infectious (69). Normally trypsin or trypsin like proteases (plasmin, blood clotting factor-like proteases, tryptase clara, bacterial proteases) cleave 16

25 the hemagglutinin by recognizing a single arginine at the cleavage site. Therefore, their replication is restricted to those sites in the host where these trypsin like proteases are found such as the respiratory and intestinal tracts. When the cleavage site has multiple basic amino acids (arginine and lysine), apparently acquired either by amino acid insertion or substitution, the cleavage site becomes suitable for the action of other ubiquitous intracellular proteases such as furin (70). The pathogenicity for chickens correlates directly with the ability of the viruses to produce cleaved HA in infected cells in culture and to form plaques on various cell types in the absence of exogenously added trypsin (71). Sequencing of the cleavage site of isolates in different laboratories have confirmed that pathogenic strains invariably contain multiple basic residues at the cleavage site (72-74). For example insertion of additional basic amino acids were observed at the cleavage site of Chicken/Hong Kong/97 H5N1 (QRERRRKKR/G) (75) The Chicken/Scotland/59 H5N1 virus has four basic amino acids at the cleavage site RKKR/G (76) acquired presumably through site substitution that results in a HPAI phenotype. Other examples by which LPAI viruses become highly pathogenic were seen in Chile (77) 2002 and in Canada 2004 (78) where H7N3 HPAI viruses appeared in these cases to have arisen as a result of non-homologous recombination with nucleoprotein gene and matrix gene, respectively, resulting in an insertion at the cleavage site of 10 amino acids for the Chile virus (77) and 7 amino acids for the Canadian virus (78). The mechanism of insertion of amino acids is not clear, but a duplication event appears likely for several of the H5 HPAI viruses (72). Acquisition of additional glycosylation sites near the receptor binding site of the hemagglutinin protein can also play a role in the avian influenza virus acquisition of 17

26 virulence. The best example is the presence or absence of a glycosylation site at position of the HA1 protein. In 1983 a LPAI H5N2 virus, Chicken/Pennsylavania/1/1983, was isolated that had 4 basic amino acids, QRKKR/G, at the cleavage site. Six months later a HPAI virus emerged in Pennsylvania, Chicken/Pennsylvania/1370/83, which had the same HA cleavage site, but this virus, had lost a glycosylation site at position in the HA1 protein. The glycosylation site is structurally extremely close to the HA cleavage site, and it is believed that the loss of the sugars allowed greater access to the cleavage site making it accessible to the ubiquitous proteases that changed the phenotype of the virus (79, 80). Influenza viruses also can change in two different ways: antigenic drift and antigenic shift. Antigenic drift occurs when point mutations accumulate in the hemagglutinin and neuraminidase genes. Influenza viruses, like other RNA viruses, lack a proof reading mechanism in the replication of viral RNA which results in errors in transcription leading to a high mutation rate (81). The impact of antigenic drift on vaccination with human influenza is a well characterized problem that requires the vaccine seed strain to be evaluated every year to try to achieve the best match with the circulating strain as possible (82). One of the primary selective factors on the HA protein is thought to be antibody pressure from the host, either from previous exposure to the virus or by vaccination (83). For humans, the influenza vaccine seed strains are evaluated yearly to determine if the currently circulating field strains are still neutralized effectively by antibody produced to the vaccine strain. For poultry, antigenic drift was not considered to be a problem with avian influenza, but recent experiences with vaccination 18

27 of birds in Mexico, Guatemala, and El Salvador demonstrate antigenic drift to be a serious concern (84). Antigenic shift is the process by which a new influenza A virus hemagglutinin subtype is introduced into a previous infected or vaccinated population with a different subtype (85). Because the host population has little or no protective immunity to the new virus, it can rapidly spread in the new population causing widespread and sometimes severe outbreaks of influenza called a pandemic. During the 20th century three major pandemics occurred in humans. The Spanish influenza (H1N1 virus) pandemic of was the most notable because of the high mortality in people between years old. More than 40 million people around the world died during this pandemic (86). The 1957 Asian influenza (H2N2 virus) pandemic and the 1968 Hong Kong influenza (H3N2 virus) pandemic were milder than the 1918 pandemic, but both still caused significant morbidity and mortality around the world. The 1957 pandemic was caused by a reassortant virus that was derived from the HA (H2), neuraminidase (N2) and PB1 (polymerase basic protein 1) genes from an avian influenza virus and the remaining gene segments from the previously circulating human H1N1 virus (87, 88). The H3N2 virus that caused the 1968 pandemic consisted of avian HA (H3) and PB1 genes in a background of other internal protein genes of the human H2N2 virus that was circulating at the time (87, 88). The 1918 H1N1 virus appeared to be a completely new virus, but the H2N2 and H3N2 viruses appeared to be reassortants viruses that changed multiple genes, including the HA gene (89). For poultry, antigenic shift has not been a major issue because of the short production lives of most commercially produced poultry. For swine in the U.S., H1N1 19

28 was primarily the only strain of influenza that circulated from 1918 to the late 90s. However, starting in 1998 H3N2 viruses started to be isolated in the U.S. This virus was an unusual reassortant virus that had H1N1 swine influenza virus-like genes, human influenza virus-like genes, and AI virus-like genes. The H1N1, H3N2 and even other reassortant viruses (H1N2 and H3N1) currently co-circulate in the U.S. (90, 91). Because of the antigenic shift, vaccines for swine had to be updated to include the new viruses to achieve protection from vaccination. VII. Epidemiology Influenza A viruses have been isolated from many species, including humans, pigs, horses, mink, felids, marine mammals, and a wide range of domestic birds, but the natural reservoir is thought to be wild birds of the orders Anseriformes (ducks, geese, swans) and Charadriiformes (gulls, terns, sandpipers), which are carriers of the full variety of subtypes (92, 93). In the natural host the virus appear to be evolving slowly with most internal genes highly conserved at the amino acid level (79). The surface glycoproteins, HA and NA, are much more variable in amino acid sequence demonstrating the greater diversity of these genes. For species under immune pressure from natural infection and/or vaccination the changes in the HA and NA genes can occur at an even faster rate (94, 95). In ducks influenza virus replicates primarily in the intestinal epithelium. Excretion usually occurs during the two first weeks of infection. Among the bird population, peak excretion titers of up to % egg-infectious dose (EID 50 ) per gram feces have been measured (96). The excretion of the virus by the fecal route results in the contamination of the environment. This is the case of natural and man-made sources of 20

29 drinking water which represent an additional risk of introduction for the infection of poultry on farms. Typically this occurs when surface water sources, such as a lake or river, are used for drinking water or other purposes. The use of unpurified drinking water was suggested to be the source of AI outbreaks in the United States, Australia, and Chile (77, 97, 98). The exposure of turkeys to pigs infected with the swine influenza is an important source of transmission of influenza virus for this species. Infections with both classical H1N1 swine influenza and the more recent reassortant H1N2 and H3N2 swine influenza viruses in turkeys have been reported (56, 99, 100). Once introduced into new hosts, chickens or mammalian species, the virus can adapt, evolve rapidly and establish itself in the new population. Most introductions of avian influenza to other species fails to maintain itself because of poor replication and transmission (79). Current influenza A viruses circulating in humans are mostly H1N1 and H3N2. The H2 subtype circulated in humans since 1957 (H2N2 Asian flu) but was later replaced in 1968 when a subtype H3N2 caused the Hong Kong flu pandemics. Humans have also been infected by H5, H7 and H9 avian viruses (101) as well as by swine influenza viruses (102) but these subtypes never established themselves in the population. Some viruses normally seen in one species sometimes can cross over and cause illness in another species. classical swine H1N1 influenza viruses from North America routinely crosses the species barrier from swine to turkeys causing costly disease outbreaks (103). More recently, two examples of adaptation to new species have been described: H3N8 viruses from horses have crossed over and caused outbreaks in dogs (104) while H5N1 viruses have been transmitted from birds to cats and wild felids (105). 21

30 Wild waterfowl including wild ducks have always been considered as subclinical carriers of low pathogenic avian influenza (LPAI) viruses, and consequently, wild ducks play an important role in the epidemiology of influenza ( ). Transmission events of LPAI viruses from wild to domestic birds with subsequent mutation to highly pathogenic viruses have been increasingly reported ( ). Transmission of HPAI from domestic to wild avifauna was rare but since the end of 2002 evidence has shown that wild birds have become infected with the HP H5N1 strain in Asia (7, 112). This epidemic appeared to originate in Southern China with the first report of H5N1 in commercial geese in Guangdong, China, in 1996 (113). Outbreaks caused by H5N1 HPAI virus in poultry and fatal infections in humans were reported in Hong Kong in The viruses isolated from the outbreaks in Hong Kong were reassortants that retained only the H5 gene from the original goose virus lineage and were eliminated through total depopulation of all poultry markets and chicken farms in December 1997(114). Over the next few years other reassortants emerged, and, in some cases, disappeared. No new cases of disease associated with H5N1 viruses were reported until 2001 when disease reoccurred in poultry markets in Hong Kong Special Administrative Region (SAR)(114). These outbreaks involved numerous reassortants that retained HA and neuraminidase genes of the GS/GD/96 lineage. In late 2002, wild birds, both free and captive, in zoological collections in Hong Kong were infected and died, including species of Anatidae (7). Before this time, ducks had been considered largely resistant to these viruses. The H5N1 virus began to spread dramatically across Southeast Asia starting in late 2003 and 2004 and resulted in a series of reported outbreaks of highly pathogenic avian influenza (HPAI) in several countries of East and Southeast Asia: 22

31 Vietnam, Thailand, Indonesia, Cambodia, Laos, South Korea, Japan, China and later Malaysia. The virus appeared to affect all sectors of the poultry industry, but its presence in free range commercial ducks, village poultry, live bird markets and fighting cocks seemed especially significant in the spread of the virus (26). In the summer of 2005 a H5N1 HPAI virus genetically closely related to an isolate that had killed large numbers of migratory birds in Qinghai, China, was responsible for outbreaks in poultry flocks in Russia (115). This particular strain of virus has now extended its range to Europe and Africa. It caused significant loss of poultry and further human cases of disease in places where there is close association between people and poultry, usually in rural areas. The first report occurred in Kazakhstan in August 2005 where the virus killed 600 geese followed by Romania and Turkey in October and Ukraine in December Isolates from swans and other wild birds from Croatia, Azerbaijan, Iran, Kazakhstan, Georgia and several European countries are indications that these species may have played a key role in the spread of the virus in West Asia and Europe (7, 26, 116). In humans, the transmission of avian influenza was thought to be a rare event. Data indicating that both the 1957 and 1968 human pandemic influenza viruses were a result of reassortment events involving 2 influenza viruses (of avian and human origin) exchanging genetic material, led researchers to believe that the direct transmission of avian influenza viruses to humans was difficult and that a reassortment event needed to occur to result in human infection. The possibility that pigs serve as a mixing vessel for reassortment between avian and human influenza virus was proposed ( ). However, the outbreak of human infections with the H5N1 virus in 1997 in Hong Kong 23

32 showed the human infections were likely the result of exposure to infected poultry (2). Since 1997 LPAI and HPAI of several subtypes have been implicated in human infections. In 2003 a highly pathogenic avian influenza A virus A/chicken/ Netherlands/1/03 (H7N7) (120, 121) was detected in 89 humans with conjunctivitis and influenza-like illness. The H7N7 virus was also thought to be the cause of a fatal case of pneumonia in combination with acute respiratory distress syndrome. In a separate outbreak in Canada in cases of human conjunctivitis caused by an H7N3 poultry virus was reported (122, 123). The subtype H9N2 was involved in outbreaks of human infections in Hong Kong and China ( ) although the clinical disease was not severe. In each of these cases poultry in the region were infected with viruses isolated from humans indicating that the infections occurred as a result of direct transmission from birds to humans. VIII. Immunology During the first stages of viral infection, specialized cellular proteins recognized viral nucleic acids or proteins. This first encounter triggers signaling pathways within the infected cells that culminate in the production of molecules of the innate immune system which are the first line of protection against infection. These molecules are called α/βinterferon, proinflammatory cytokines and chemokines. Dendritic cells (DCs), are the most efficient antigen-presenting cells able to initiate primary immune responses (127). NS1 protein prevents not only the induction of IFN-α/β by human myeloid DCs but also the induction of a transcriptional program associated with DC maturation, resulting in suboptimal stimulation of T cells (128). 24

33 In influenza virus infected cells, the viral molecules of single strand RNA (ssrna) and double-stranded RNA (dsrna) with phosphorylated 5 ends induce the expression of type I interferon (IFN-αβ) (129). These ssrna and dsrna molecules activate several cellular signaling pathways resulting in the activation of several transcription factors such as IFN regulatory factors (IRFs) and stress-induce transcription factor c-jun/ ATF-2 and NFκβ. These activated transcription factors travel from cytoplasm to the nucleus and initiate the expression of IFN-α/β which binds to a common receptor activating the JAK-STAT pathway. The activation of this pathway results in the stimulation of more than 300 genes some of which encode proteins with antiviral activity. The best studied antiviral proteins are protein kinase R (PKR)(130), Mx GTPases(131), 2-5 oligoadenylate synthetase/rnase L system(132). The NS1 protein contains two domains: the N terminal double-stranded RNA (dsrna)-binding domain (133) and a C-terminal effector domain (134) which interacts with the 30-kDa subunit of the cleavage and polyadenylation specificity factor CPSF and with PABII, leading to a block of cellular mrna processing in infected cells ( ). As a result, the processing of cellular pre-mrnas, including beta interferon (IFNβ) premrna and the pre-mrnas of other antiviral proteins, is inhibited, thereby suppressing the amount of mature IFNβ mrna that is produced in infected cells ( ). NS1 binds dsrna and thus prevents activation of the 2-5 oligoadenylate synthetase/rnase L system(132). By sequestering dsrna NS1 prevents activation of the transcription factors IRF3, IRF7, NF-κB, and c-jun/atf2. It directly binds to PKR and blocks PKR activation, leading to stimulation of host protein synthesis in NS1-expressing cells (138). 25

34 NS1 inhibits host cell mrna processing and blocks nuclear export of polyadenylated cellular transcripts. Influenza infection induces both systemic and local antibody (humoral immunity), as well as cytotoxic T-cell responses (cellular immunity), each of which is important in recovery from acute infection and resistance to re-infection. Neutralizing antibodies are produced mostly against the HA glycoprotein, but antibodies are also produced for the NA, M and NP proteins (18). In mice anti-na antibodies do not neutralize virus infectivity, but instead reduces the efficient release of virus from infected cells (139). In chickens recombinant vaccines encoding NA antigen conferred protection when high level of anti-na antibodies were achieved, but failed to affect viral shedding. Antibody against integral membrane protein M2 does not completely protect mice, but does reduce the amount of virus that is shed and provides some protection from disease (18). M2 is well conserved for all influenza Type A viruses which potentially could provide protection for all HA and NA subtypes. However, no work has been published to determine if antibody to the M2 protein would be protective in poultry (18). In influenza infection, CD4 T lymphocytes help B lymphocytes generate anti-ha and anti-na antibodies and may also promote the generation of virus-specific CD8 cytotoxic T lymphocytes (140). During the primary influenza infection, viral clearance depends on CD8 + T lymphocytes. In the mouse model, virus is cleared within 10 days after infection with no indication of persistent antigen or viral antigen (141) Both CD4 and CD8 memory T-cell subsets respond to mediate control of influenza virus reinfection(141). Memory CD8 T cells may play a role in decreasing the severity of disease and increasing resistance to reinfection. However, it appears that the antiviral capacity of 26

35 the virus-specific CD8 cells is strongly dependent on their ability to migrate and to localize to the lung and infected airway epithelium, where they appear 5 7 days after infection (142). The important question is how to achieve the stimulation of these cells through vaccination. Current data suggest that a mucosally delivered vaccine (i.e. delivered via the respiratory tract) might be the best approach (143). In contrast to mammals, chickens do not have encapsulated lymph nodes but instead develop diffuse mucosa-associated lymphoid tissues (MALT) wherever they are antigenically stimulated in the body: (H: head; G: gut; or B: bronchus-associated lymphoid tissue). HALT immunity has been principally evaluated by the detection of specific IgA in the tears, particularly after Newcastle disease virus (NDV) vaccination of chicken (144, 145). In some respiratory diseases, such Newcastle disease and infectious bronchitis, it has been shown that the mucosal local immunity plays an important role against the respiratory infection and replication of these viruses in the upper respiratory tract. IX. Vaccination Vaccination can be a valuable tool for the control of avian influenza outbreaks when done as part of a complete control program that includes: biosecurity, animal movement controls, education, surveillance, and vaccine effectiveness evaluation (13). Subtypes H5 and H7 vaccines have shown efficacy in preventing clinical signs and death in chickens and turkeys following challenge with HPAI viruses ( ). Vaccination reduced both oropharynx and cloacal shedding after challenge with virus containing homologous hemagglutinin protein of LPAI and HPAI viruses(150). Many vaccines are effective after a single injection and provide protection for greater than 20 weeks (150). 27

36 Unlike human influenza vaccines, AI vaccine strains do not need to be changed as often to provide adequate protection. Subtype H5 AI vaccines for chickens appear to provide protection against many H5 AI viruses isolated over many years (151). However, vaccines do not completely prevent infection or produce sterilizing immunity (13). Use of vaccination without proper surveillance tools will not provide detection of infections in vaccinated flocks and may hamper eradication efforts (8, 11). Therefore, when vaccination is not used as part of a comprehensive control program the risk of developing an endemic situation (152) exists. A clear example is in Mexico where vaccination with the homologous H5N2 subtype was used during a HPAI and a LPAI outbreak, but vaccination was not used as a part of a larger eradication program, and although the HPAI virus was eradicated, the LPAI virus became endemic in the country (94). Following outbreaks in 1995 (153) homologous immunization with inactivated vaccinate appeared to have eradicated the HPAI virus but the LPAI virus was not eradicated and continues to circulate in Mexico and has spread to several adjoining countries (153). In developing countries the lack of infrastructure and resources can be limiting factors for the establishing control programs that include vaccination (154). In Southeast Asia several countries have instituted vaccination programs for preventing or managing the disease with the intent of eventual eradication. However, only Hong Kong has instituted a vaccination program in conjunction with comprehensive surveillance and biosecurity programs on farms and in live poultry markets, wild birds, recreation park birds and pets (155). Hong Kong introduced vaccination in June 2003 immunizing chicken with a killed H5N2 vaccine using the Mexican H5N2 antigen (114) which successfully protected local farms and live poultry markets from H5N1 outbreaks 28

37 during the regional H5N1 outbreaks in China officially introduced vaccination in 2004, with the use of two main inactivated vaccines: One produced by reverse genetics containing H5 and N1 protein from Gs/GD/96 virus with the multiple basic amino acid cleavage site changed to a low pathogenic cleavage site. The other contained an antigen from H5N2 low pathogenicity strain derived from a turkey in the UK (A/Turkey/England/N-28/73). Vietnam in 2005 and Indonesia started vaccinating poultry against avian influenza. However, the incomplete coverage and poor vaccine quality are thought to be issues in the continuing outbreaks of the diseases specially in China, plus other socio-economic issues in the other countries (114). In the European Union the legislation for avian influenza control had been harmonized in 1992 (156). Due to the absence of major outbreaks, control measures had not been implemented for a number of years. A LPAI strain (H7N1) had circulated for several months in a dense populated poultry area of Northern Italy, mutated to HPAI killing about 16 million birds in 1999 (157). Following the re-emergence of the LPAI (H7N1) in 2000 a vaccination policy was strongly suggested by farmers and the poultry industry (10). European regulations were adopted allowing vaccination providing that the vaccines used were not genetically modified and that they will allow the discrimination of vaccinated from infected birds. In the United States, AI outbreaks in poultry have been controlled mainly through prevention, management, and eradication programs (158). These programs have been accomplished through multidisciplinary approaches including increased surveillance and diagnostics, enhanced biosecurity including quarantine in the infected zone, education of poultry workers, and elimination of infected or suspected infected poultry. Vaccination 29

38 has been used routinely only in turkeys as a strategy to minimize losses and reduce the incidence of the diseases caused by infection with swine influenza virus (H1N1). Prior to 1995, H5 and H7 vaccines were allowed without USDA approval. During 1995, two separate major outbreaks caused by LPAI viruses occurred in turkeys in the USA (Halvorson et al., 1998). In Utah H7N3 subtype affected young turkeys and was associated with about 40% mortality. In most cases, mortality was associated with dual infections with Escherichia coli or Pasteurella multocida. Autogenous inactivated (H7N3) AI vaccine was effective in reducing susceptibility of turkeys to AI virus and, along with biosecurity measures, ending the outbreak. In 1995 in Minnesota H9N2 virus infected 178 turkey farms resulting in the worst economic loss to influenza infections (approximately US$ 6,000,000) recorded in turkey production one year in Minnesota (159). Vaccination with inactivated oil emulsion was included within the strategy to successfully control and eradicate the virus. Since Minnesota is a major turkey producing state and also is a major brooding and gathering place for fall migration of wild waterfowl, the control and eradication programs for AI largely include prevention of exposure by direct or indirect contact with waterfowl and shorebirds and their environments. In 2003, H7N2 LPAI was diagnosed in chickens within a large layer company in Connecticut. A control strategy was developed including vaccination with a single inactivated H7N2 AI-virus vaccination, over the normal production cycle replacement non-infected pullets were twice-vaccinated with inactivated vaccine (H7N3). Evaluation and strict control strategy determined the success of the program (65). X. DIVA strategy for Avian Influenza 30

39 Serological surveillance for avian influenza is typically accomplished by testing for NP/M antibodies with direct and blocking ELISA and/or agar gel immunodiffusion (AGID) test. These tests detect infected birds by detecting antibodies for one of the type specific virus proteins able to stimulate the immune system. Vaccinated poultry with conventional whole virus inactivated vaccines that normally contain nucleoprotein, hemagglutinin, neuraminidase and other viral antigens, will develop an immune response to NP/M proteins. This immune response cannot be distinguished from the immune response induce by natural infection at least with the routine tests used in surveillance programs. The inability to easily differentiate these two immune response makes the vaccination an obstacle not just for the surveillance programs but also for the international commerce of poultry and poultry product, since the trade partners need assurance that the product comes from influenza clean premises (13). The recent joint OIE/FAO summits recommended applying vaccination, using the differentiating infected from vaccinated animals (DIVA) strategy when there is risk of major spread and depopulation is not feasible or desirable. In order for vaccination to be successful in controlling and ultimately in eradicating the infection, vaccination programs must be part of a wider control strategy involving upgraded biosecurity, monitoring vaccine efficacy, identification of field exposure and the appropriate management of infected flocks, regardless of vaccination status, as well as granting financial support for the compensation of farmers (160). The DIVA strategy requires the use of appropriate vaccines and specific discriminatory tests which enables the differentiation to be made between vaccinated and infected birds. Such a strategy is important for detecting any outbreak of the virus in 31

40 vaccinated birds, and also for trade purposes as it enables restrictions on vaccinated poultry meat to be lifted once the flocks could be clearly shown to be free of infection (12, 14). The DIVA strategy has been accepted as a way to monitor and provide assurances on infection free status of vaccinated poultry (5, 6). There are several DIVA strategies that have been proposed with the aim to enable the inclusion of vaccination in the AI control and/or eradication programs (12). The most common is the use of unvaccinated sentinels. A second approach is the use of subunit vaccines targeted to the hemagglutinin protein that allows serologic surveillance to the internal proteins. A third strategy is to vaccinate with a homologous hemagglutinin to the circulating field strain, but a heterologous neuraminidase subtype. Serologic surveillance can then be performed for the homologous NA subtype as evidence of natural infection. The fourth strategy is to measure the serologic response to the nonstructural protein 1 (NS1). All four DIVA strategies have advantages and disadvantages, and further testing is needed to identify the best strategy to make vaccination a more viable option for avian influenza (12). The differentiation between infected/non-infected vaccinated birds and flocks requires the application of a suitable marker vaccine and a companion discriminatory test. Since this condition is not always fulfilled, a monitoring program that includes the use of (unvaccinated) sentinel birds could also be set up (161). Susceptible identifiable birds are placed with the vaccinated flock and routinely tested for infection(12). However, the management of sentinel birds can be time-consuming (identification, bleeding and swabbing). Sentinels birds have been used to detect infection with some other infectious agents in the poultry industry. For instance, The National Poultry 32

41 Improvement Plan (NPIP), has approved the use of unvaccinated sentinel birds (about 350 birds) when a federally licensed Salmonella enteritidis bacterin is used to vaccinate a flock. These birds will be used to conduct the necessary serological tests for Salmonella pullorum and Salmonella gallinarum. The effect of chicken interferon-α administered perorally in drinking water has shown to have an adjuvant effect that causes chickens to rapidly seroconvert after natural infection with low-pathogenicity influenza virus, lowering the threshold of the antigen required to stimulate the adaptive immune response to an LPAI virus. As a consequence the administration of ChIFN-α could create supersentinel chickens that seroconvert in response to levels of antigen that would otherwise go undetected(162). Flocks under traditional vaccination programs with inactivated whole AI vaccines will produce antibodies against nucleoprotein/matrix protein and hemagglutinin. In the absence of sentinel birds in these flocks heterologous neuraminidase testing approaches can be implemented (163). AI vaccines containing a virus of the same H subtype as the challenge strain but a different neuraminidase subtype allow the implementation of serological tests directed to the detection of antibodies to the NA of the field virus. For example, birds vaccinated with H7N3 in the face of a H7N1 epidemic may be differentiated from infected birds (DIVA) by detection of subtype specific NA antibodies of the N1 protein of the field virus. Vaccination with an inactivated heterologous vaccine was in used as one of the measures to control waves of LPAI infection during the Italian H7N1 avian influenza epidemic (10) The "DIVA" strategy was based on the use of an inactivated oil emulsion vaccine containing the same hemagglutinin (H) subtype as the field virus, but a different neuraminidase (N3). To differentiate between 33

42 vaccinated and naturally infected birds, an "ad hoc" indirect immunofluorescence assay based on the detection of specific anti-n1 antibodies was developed. The application of a "DIVA" vaccination, enabled veterinary public health organizations to establish that infection was not circulating any longer, and ultimately resulted in the possibility of marketing meat obtained from vaccinated birds (10). In the USA the heterologous NA DIVA strategy has been used only one time and on a limited basis when a large layer farm in Connecticut (2003) experienced a LPAI outbreak. However, by the time that a suitable DIVA vaccine was used the outbreak was already contained, therefore no evaluation of the program effectiveness could be done (12). Swayne et al, have shown experimental data suggesting that the combination of heterologous neuraminidase inactivated vaccine and a neuraminidase inhibition test as discriminatory test could be useful at the farm level for detecting infection in birds immunized with inactivated vaccines (164). One concern with the heterologous NA DIVA strategy is the availability of the matching vaccine containing the right HA and heterologous NA to the outbreak strains(12) to enable the discriminatory testing. To overcome this problem reverse genetics has been used experimentally to create a virus containing the appropriate surface proteins (12, 165). However, besides regulatory processes there are still proprietary issues that need to be solved before the system can be used. Another concern is the availability and sensitivity of the companion discriminatory serological test to use with all the AI NA subtypes (12). Subunit vaccines have been developed containing the HA protein of influenza virus. Antibodies to the hemagglutinin provide the primary protection against avian 34

43 influenza virus challenge. Therefore, it is possible to protect birds by having only this protein in a vaccine (12). The current serologic surveillance tests, specifically the AGID and commercial ELISA tests that target the matrix (MA) or nucleoprotein (NP) structural proteins can be easily used with these vaccines (12, 166), where the vaccinates will be negative and infected will be positive whether vaccinated or not. Fowlpox-vectored recombinant vaccine for the H5 subtype (rfp-h5) is available commercially (12, 166), and it has shown to induce clinical protection against H5 highly pathogenic viruses (166, 167). Even though this vaccine has a built in DIVA strategy, it has not been used with this purpose in the field. Experimental data has shown that this vaccine enables the AGID or ELISA tests to identify infection among a vaccinated population of chickens, and the HI test with homologous HI antigen to the vaccine strain, to monitor the vaccine effectiveness (166). One potential target for the DIVA strategy for avian influenza is the nonstructural protein (NS1). The NS1 protein is encoded by the smallest gene segment of influenza virus, segment 8. The NS1 protein is a conserved protein amongst type A influenza viruses, but it exists as two major subtypes, A and B, that differ by about 35% in nucleotide sequence (16). The NS1 protein is produced and accumulates in infected cells. Since, influenza vaccines are primarily whole killed viral particles, vaccinated animals theoretically should not have an antibody response to the NS1 protein, but naturally infected animals should develop antibodies to the NS1 protein. This DIVA approach was first proposed for equine influenza viruses, and purified NS1 protein was used to detect antibodies to NS1 in serum samples from ponies and horses experimentally infected with influenza virus (19, 20). NS1 recombinant protein produced 35

44 in Escherichia coli or chemically synthesized NS1 peptides were evaluated on an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis for the ability to detect antibodies from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines (21, 22). Antibodies to the NS1 protein were detected in experimentally infected birds with multiple subtypes of influenza A virus. In contrast, no antibodies against NS1 were detected in animals inoculated with inactivated gradient-purified vaccines. In birds vaccinated with commercial vaccines there were low, but detectable, levels of NS1 antibodies. Dilution of serum samples 1:200 limited the NS1 positive samples only to infected birds, but all the samples were positive for both AGP. These results demonstrate the potential benefit of a simple, specific ELISA test for anti-ns1 antibodies that may have diagnostic value for the poultry industries. 36

45 CHAPTER 3 DIFFERENTIATION OF INFECTED AND VACCINATED ANIMALS (DIVA) USING THE NS1 PROTEIN OF AVIAN INFLUENZA VIRUS. Gloria Avellaneda, Egbert Mundt, Chang-Won Lee, and David L Suarez Prepared for submission to Avian Diseases

46 Key words: influenza A virus, DIVA strategy, NS1 protein, ELISA test, diagnostic, chickens, turkeys Abbreviations: AI=avian influenza; ANOVA=analysis of variance; BPL=βpropiolactone; A/CK/Queretaro = A/CK/Queretaro/ /95 (H5N2); HPAI = high pathogenic avian influenza; LPAI= Low pathogenic avian influenza; DIVA= differentiating infected from vaccinated animals; A/CK/California = A/CK/California/K Ct/03 (H6N2); A/TK/Wisconsin/68 = A/TK/Wisconsin/68 (H5N9) 38

47 Abstract Vaccination against avian influenza (AI) virus, a powerful tool for the control of the disease, may result in issues related to surveillance programs and international trade of poultry and poultry products. The use of AI vaccination in poultry would have greater world-wide acceptance if a reliable test that clearly discriminates naturally infected from vaccinated only animals (DIVA) was available. Because the non-structural protein (NS1) is expressed in infected cells, and is not packaged in the infectious virion, it has been considered as a target candidate for a DIVA differential diagnostic test. The aim of this work was to determine the onset of the antibody response to the NS1 protein in chickens infected with low pathogenic avian influenza (LPAI) virus, and to evaluate the diagnostic potential of a baculovirus expressed purified NS1 protein in an indirect ELISA-based DIVA strategy. An antibody response against NS1 was first detected three weeks after infection, but antibody levels were already decreasing rapidly by 5 weeks after infection. This response was also only detected in one group and in 25% of the birds in spite of high hemagglutination inhibition (HI) antibody titers in this group. In birds vaccinated with inactivated oil emulsion vaccines, antibodies against NS1 were not detected or were detected in only a small percentage of birds after homologous LPAI virus challenge. Vaccinated birds challenged with highly pathogenic AI showed a higher NS1 antibody response. Most birds seroconverted by three weeks after challenge, however, not all challenged birds had a detectable response. The serological response against NS1 varied greatly between groups receiving different vaccines. Because of the variability of immune response and the duration of the antibody response, the NS1 protein may not provide the ideal DIVA strategy in chickens. 39

48 Introduction Influenza viruses are segmented, negative strand RNA viruses classified in the family Orthomyxoviridae which include several genera including Influenza virus A, B and C, Thogotovirus, and Isavirus (1). All three Types of influenza are found in humans, but only Type A viruses are found in birds and are referred to as avian influenza viruses. Type A influenza viruses are divided into subtypes based on the antigenic properties of the major surface glycoproteins: the hemagglutinin (HA) and neuraminidase (NA) proteins. A total of 16 HA subtypes (H1 H16) and 9 neuraminidase subtypes (N1 N9) are described currently (1). AI viruses can be further divided into two distinct virulence groups based on the severity of the disease that they cause in chickens in standard pathotyping studies (1). Highly Pathogenic Avian Influenza (HPAI) results in mortality of 75% or greater in chickens after intravenous inoculation and in the field typically results in flock mortality in susceptible species that may be as high as 100% (2). Only the H5 and H7 subtypes are known to have the highly pathogenic phenotype, but most viruses of these subtypes are not highly pathogenic (2). Low Pathogenic Avian Influenza (LPAI) viruses typically cause no disease or mortality in experimentally infected chickens and include the large majority of AI viruses isolated from poultry. Some of these viruses in the field however may cause a mild to severe respiratory disease, depression, and reproductive disease in a variety of domestic species, particularly when complicated by concurrent infection or poor environmental conditions (1). Although AI vaccines can be successful in controlling the clinical signs of the disease, they do not prevent infection of vaccinated flocks if they are exposed to high 40

49 levels of virus (3). Vaccination has not been commonly used as part of programs for the control and eradication of LPAI or HPAI because the AI vaccines used world-wide are mainly killed, whole virus vaccines, containing nucleoprotein, hemagglutinin, neuraminidase and other viral antigens. Therefore, they interfere with the traditional serologic tests used for surveillance of influenza infection such as the AGP, HI and nucleoprotein- specific ELISA (4-7). Thus, the current serological surveillance tests are unable to differentiate whether antibodies are from infection or vaccination. Vaccination is growing in acceptance as an alternative to the stamping out approach (5-8) where all infected and exposed flocks are euthanized because it is viewed as a less costly control strategy. However, vaccination may have a negative impact on international trade because of the limitations of the current serologic tests. Therefore, efforts have focused on improving surveillance and diagnostic methods including developing a DIVA strategy to not only discriminate between infected and vaccinated birds, but also to identify vaccinated birds that become infected with AIV. Several different DIVA strategies have been proposed (6), some of which require the use of appropriate vaccines and specific serological discriminatory tests (4, 9-12). Such a strategy is important for detecting any outbreak of AIV in vaccinated birds, and also for trade purposes as it enables restrictions on vaccinated poultry meat to be lifted once the flocks can be clearly shown to be free of past infection (4, 8, 13). The DIVA strategy has been accepted by some countries as a way to monitor and provide assurance on the infection free status of vaccinated poultry (5, 6). One potential target for the DIVA strategy for avian influenza is the non-structural protein (NS1). The NS1 protein is encoded by the smallest gene segment of influenza virus, segment 8. The NS1 protein is 41

50 a conserved protein amongst type A influenza viruses, but it exists as two major subtypes, A and B, that differ by about 35% in nucleotide sequence (14). The NS1 protein is only produced after influenza virus infects the cells (12, 15), but it is not packaged into the infectious viral particle. Because most AIV vaccines are primarily chemically inactivated whole virus products, vaccinated animals theoretically should not have an antibody response to the NS1 protein, where those chickens infected should have antibodies against NS1 protein. This DIVA approach was first proposed for equine influenza viruses, and purified NS1 protein was used to detect antibodies to NS1 in serum samples from ponies and horses experimentally infected with influenza virus (15, 16). Serological studies on chickens and turkeys using an ELISA with AIV NS1 protein produced in E. coli as antigen indicated that infected birds show higher levels of NS1 antibody as compared to vaccinated birds (12, 17). Since available data on base line production of NS1 antibodies after natural infection with influenza virus is very limited, this work was undertaken to determine the kinetics of the antibodies against NS1 protein after infection with LPAI virus, and to determine whether a baculovirus -derived NS1 ELISA approach is a reliable DIVA diagnostic test. Materials and Methods Viruses The HPAI virus A/CK/Queretaro/ /95 (H5N2) (18) and the low pathogenic avian influenza viruses A/CK/Pennsylvania/13609/93 (H5N2), A/CK/California/K Ct/03 (H6N2), A/TK/Oregon/1971 (H7N3), and A/TK/Wisconsin/68 (H5N9) used in this study were obtained from the virus repository at 42

51 Southeast Poultry Research Laboratory (SEPRL). The egg infectious dose 50% (EID 50 ) of all the viruses used in this study was calculated using the method of Reed and Muench (19). Briefly, 10-day-old embryonating chicken eggs (ECE) were infected with 100 µl of 10-fold dilutions in brain heart infusion medium (BHI; Difco - Detroit, MI) from stock virus. Eggs were candled daily for 7 days. Deaths detected before 24 hours were discarded and after that period deaths were chilled overnight at 4 C before harvesting allantoic fluid and testing for hemagglutination. Incubation of chicken red blood cells with control serum or PBS served as negative controls. Recombinant vaccine viruses RNA from the isolates used for the vaccine preparation was extracted with the RNeasy mini kit (Qiagen, Valencia, CA) from infected allantoic fluid. Then, HA genes from A/CK/PA/13609/93 (H5) virus, NA gene A/DK/NY/ /02 (N8), and NS1 gene from A/TK/OR/71-SEPRL viruses were amplified by one-step RT PCR with HA, NA and NS1 segment-specific primers as described previously (20). The RT PCR products were re-amplified by PCR with primers containing BsmBI sites, digested with BsmBI restriction enzyme, and cloned into phh21 vector between the promoter and terminator sequences of RNA polymerase I. Primer sequences will be provided upon request. RNA polymerase I constructs that contain the other 5 internal genes of A/WSN/33 virus and four expression plasmids (pcaggs-wsn-np, pcdna774-pb1, pcdna762-pb2, and pcdna787-pa) were provided by Dr. Yoshihiro Kawaoka at the University of Wisconsin (21). Reassortant viruses were generated by DNA transfection as described previously. Briefly, 293T cells were transfected with 12 plasmids with the use of Lipofectamine 2000 reagent (Invitrogen, San Diego, CA). Supernatant was 43

52 collected after 72 h of transfection and subsequently inoculated into 9- to 10-day-old ECE. After 48 h of incubation, allantoic fluid containing the reassortant virus was harvested and stored at 70 C for additional experiments. Vaccine preparation The virus generated by reverse genetics was grown in 10-day-old ECE and the allantoic fluid was pooled and stored at 70 C until inactivation. Infectious titers were determined prior to inactivation with 0.1% betapropiolactone (Sigma, St. Louis, MO). Oil-emulsion vaccines were as previously described (22). Briefly, one part aqueous antigen was emulsified in four parts oil phase (Drakeol 6 VR mineral oil, Penreco, Burler, PA) containing 7.5% sorbitan monooleate (Arlacel 80, ICI United States, Inc., Wilmington, DE), and 2.5% polysorbate 80 (Tween 80, ICI United States, Inc.). The inactivated vaccine prepared with H5 reassortant for this study was named as rh5n8- SEPNS1 inactivated vaccine. Infection and Vaccination Studies For the kinetics study of antibodies against NS1, 38-day-old chickens were infected with low pathogenic avian influenza virus and housed in biosafety level 3 Ag facilities. Groups of eight 38-day-old birds were infected by the intranasal route with CK/CA/03 (H6N2) LPAI virus or TK/WI/68 (H5N9) LPAI virus at 10 5 EID 50 and 10 6 EID 50 /0.2ml, respectively. Serum samples were taken for serological analysis before the infection and every week after the infection for five consecutive weeks. Food and water were provided at libitum in all the experiments and management was provided as required by the Institutional Animal Care and Use Committee. 44

53 In two different experiments, groups of ten 2-week-old birds were immunized subcutaneously with the equivalent of EID 50 /0.5 ml of rh5n8-sepns1 inactivated vaccine or with 0.5 ml commercial avian influenza vaccine subtype H5N9. In each experiment a sham vaccinated group was included as a challenge control, as well as a group kept as negative controls. Chickens were challenged intranasally via the choanal cleft 2 weeks after vaccination with 10 5 EID 50 /0.2mL of A/CK/PA (H5N2) LPAI virus (experiment 1) or 10 5 EID 50 /0.2mL of A/CK/Queretaro (H5N2) HPAI virus (experiment 2) (18). Morbidity and mortality were monitored daily. In both experiments, blood samples were obtained via the brachial vein right before challenge and every week after challenge for four weeks. Serum were harvested from each sample and stored in -20C for serological testing. Tracheal or oropharyngeal swabs were taken from infected birds at days 3 and 5 days after infection, suspended in 1mL of BHI broth, and stored at 70 C. Total RNA was extracted by using an RNeasy mini kit (Qiagen), and quantitative realtime RT-PCR was performed with primers and probe specific for type A AI virus matrix RNA using a Smart Cycler II (Cepheid Sunnyvale, CA) real-time PCR machine (23). The quantitative virus shedding (number of copies of viral RNA recovered from swab/ml of BHI) was interpolated from the sample cycle thresholds by using a standard curve generated from known amounts of control A/CK/Queretaro/95 RNA (titers were EID 50 /ml) (23). Generation of recombinant baculovirus encoding viral NS1 Two recombinant NS1 proteins were generated using the Bac-to-Bac Baculovirus Expression System (Invitrogen, Carlsbad, California). Viral RNA was extracted from allantoic fluid of ECE infected with A/TK/Oregon/71-SEPRL using Trizol LS 45

54 (Invitrogen) as described by the manufacturer. The NS1 gene was amplified by one-step RT-PCR as described previously (20). RT-PCR products were cloned into the pcr 2.1- TOPO (Invitrogen, Carlsbad, California), and transformed into the competent DH5α E. coli cells (Invitrogen). Prepared plasmids DNA from selected colonies were screened by PCR for the presence of the NS1 genes with NS1 specific primers. DNA extracted from a confirmed positive colony was digested with BamHI and Hind III (NS1-Amino terminal) or with EcoRI and Hind III (NS1-Carboxy terminal) and cloned into the corresponding sites of the plasmids pfastbac HT A or pfastbac Dual, respectively. To confirm the appropriate DNA fragments sequence analysis was performed. The generation of recombinant baculoviruses was performed as described in the manufacturer s protocols. Spodoptera frugiperda (Sf9) cells (Invitrogen) were cultivated in all experiments with serum free medium (SF900 II, Invitrogen). The viral titers (TCID 50 ) of the obtained supernatants were determined by immunofluorescence using the baculovirus specific monoclonal antibody (mab) AcV5 (Sigma, Saint Louis, Missouri) with tenfold diluted virus in SF900 II medium and 100 μl of each dilution was fourfold transferred to a 96 well tissue culture plate. 100 μl of Sf9 cells, at a density of 3 x 105cells /ml were added to each well. The cells were incubated for 72 hours and after removal of the medium the cells were fixed with ice cold ethanol for 10 minutes. Immunofluorescence was performed following standard procedures using the mab AcV5 and goat anti-mouse FITC conjugated antibodies (Jackson Immunoresearch, West Grove, PA). Production and purification of recombinant NS1 Sf9 cells were infected with recombinant baculovirus at multiplicity of infection (MOI) of at least 5 and harvested 3 days pos-infection. by centrifugation at 700 x g for 10 46

55 min. The NS1 proteins were purified using a TALON resin (Clontech) essentially as described by Letzel et al. (24). Briefly, pelleted cells of one T150 tissue culture flask were suspended in 5 ml lysis buffer containing 1x protease inhibitor Complete EDTAfree (Roche) and 100 mg IGEPAL CA-630 (SIGMA). After 30 min incubation on ice insoluble proteins were sedimented by centrifugation at x g for 45 min at 4 C using the table-top ultracentrifuge Optima TLX (Beckman). The obtained supernatants were used for the purification as described previously (24). The recombinant proteins were eluted using 3 ml of elution buffer containing 300 mm Imidazol (BD-Biosciences). The eluated molecules were collected in fractions of 500µl and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by either Coomassie brilliant blue staining or Western blot analysis. Fractions containing the highest amount of eluted NS1 were pooled and the protein concentration was determined using the Micro BCA Protein Assay Kit ((Pierce, Rockford Illinois). Hemagglutination Inhibition (HI) Test All serum samples were tested for antibodies against hemagglutinin by HI test using homologous antigens. For that purpose the following virus: A/turkey/Wisconsin/68 (H5N9), A/CK/California/K Ct/03 (H6N2), and the experimental rh5n8- SEPNS1 were inactivated with 0.1% β-propiolactone (BPL) and used for the HI test as previously described (19). All HI titers were reported both as Log 2 titers and as geometric mean titers (GMT) with 8 being the minimum positive titer. ELISA test development The optimum dilution of each NS1 Protein for the ELISA test was obtained by checkerboard titration on a 96-well ELISA plate against known positive and negative 47

56 samples. The optimum dilution considered was the highest dilution of antigen that still saturated the plate and gave maximum contrast in terms of optical density (OD) between known positive and known negative sera. A dilution of 1:50 for the experimental serum samples was determined to produce an optimal level of sensitivity for detection of positive samples with low background in negative samples. A cut-off point of represents the mean of the optical density (OD) of 120 negative samples tested at the 1:50 dilution plus 3 standard deviations. Indirect ELISA was performed using the following procedure. Ninety-six well ELISA plates (Nunc) were coated with ng/well purified NS1-his tag protein diluted in carbonate buffer, ph 9.6, in a 50 μl volume and incubated overnight at 4 C. All washing steps were performed using an automated ELISA washer (Biotek, Vermont, USA) three times with 1X Wash Solution (KPL Gaithersburg, Maryland). Incubation of serum samples and conjugate were performed at 37 C for one hour. After overnight incubation the plates were washed and the wells blocked with 100 µl/well of 1xBSA/Blocking solution and incubated for 45 min at 37 C, followed by another washing step. Each test sample was diluted 1:50 in Sample Dilution Buffer (Synbiotics, San Diego, California) and 50 μl were added to the ELISA plate. Horseradish peroxidase-conjugated goat anti-chicken (KPL) antibodies were diluted 1:2000 in Sample Dilution Buffer and 50 μl added to each well. After a last washing step, 50 μl of ABTS (2, 2 - Azino Diethhylbenzothiazoline Sulfuric Acid) 2-Component Micro-well Peroxidase Substrate (KPL) were added followed by 15 minutes of incubation time at room temperature, and the reaction was stopped by the addition ABTS Peroxidase Stop 48

57 Solution (KPL). The optical density (OD) of each well was read at 405 nm using a microplate reader (Biotek, Vermont, USA). Positive and Negative serum sample controls for NS1-ELISA Positive control was obtained from a non-vaccinated bird that survived a challenge with HPAI Ck/Chile/02 (H7N3) in an on going experiment at SEPRL. Negative control used was normal chicken serum obtained from Bethyl Laboratories, Montgomery, TX. The initial ODs of for the positive for negative were determined by NS1 ELISA at a dilution of 1:50. Results Purification of the NS1 protein The identity of the purified N-terminal NS1 protein was confirmed by direct protein sequencing using MALDI-TOF MS/MS analysis (data not shown) using a protein sample separated on a 12% SDS-PAGE and processed for trypsin cleavage (Proteomics Resource Facility and Integrated Biotechnology Laboratories at the University of Georgia, Athens, Georgia). Confirmation of results with NS1-Carboxy terminal The N-terminal location of the 6xHis tag in the NS1-Amino terminal protein may have some influence on the NS1 ELISA results. To test this possibility, a baculovirus encoding a 6xHis at the C terminus of the NS1 protein (NS1-Carboxy terminal) was generated and recombinant protein was purified similar to the N-terminal NS1 protein (Figure 3.1). A comparison of different ELISA tests with NS1- Carboxy and NS1- Amino terminal proteins were performed with positive control serum samples. The location of 49

58 the 6x His tag had no influence on the outcome of the results. There were not significant changes in the level of NS1 antibody detected (data not shown). The NS1-Amino terminal protein was chosen as antigen for the ELISA test for analysis of serum samples in subsequent experiments. Antibody response to NS1 protein produced in birds after infection with low pathogenic influenza virus. To test the hypothesis that infection with AIV in chickens produces a humoral immune response to the NS1 protein, groups of 8 birds at 38 days of age were inoculated with two different low pathogenic avian influenza viruses, CK/CA/03 and TK/WI/68. The serological response to these viruses is summarized in Table 3.1. Infection with 10 5 EID 50 of CK/CA/03 induced detectable levels of NS1 antibody in 25% of birds, but 75% of the birds failed to seroconvert throughout the duration of the experiment. The level of antibodies as measured by the NS1-ELISA in the serum of the 2 positive birds was highest at 3 weeks post-infection, but began to decline rapidly although antibodies against NS1 were still detectable at 5 weeks which was the end of the experiment (Figure 3.2). The HI antibody response to infection with CK/CA/03 was high with a geometric mean titer (GMT) of 194 at week 1 after infection, reaching a peak at week 3 with a GMT of 891. No NS1 antibodies were detected in the birds inoculated with TK/WI/68. The HI antibody level in this group was lower than in the previous group and only 50% of the birds seroconverted during the challenge, indicating an incomplete challenge. To analyze the level of NS1 antibodies present in the seroconverted birds, 3 weeks postinfection samples from the group infected with the CK/CA/03 AIV were selected for further analysis. Series of two-fold dilutions beginning at 1:5 were tested on ELISA 50

59 revealed (Figure 3.3) a titration curve for the two positive samples similar to the titration curve obtained with the known positive serum sample suggesting high level of antibodies to NS1 while the negative samples showed a titration curve similar to the negative control (Figure 3.3). Western blot analysis The sensitivity of the NS1 ELISA may not be able to detect low levels of antibodies. To test this possibility, Western blot analysis was performed using serum samples from NS1 ELISA positive and negative birds, tested at two different dilutions (1:50 and 1:10). Lane bands of the predicted size for the NS1 protein were considered positive for NS1 antibody. Results from western blot analysis corroborate the results from the NS1 ELISA. Only serum samples which previously had NS1 antibody using ELISA (Table 3.4) were also positives in the western blot analysis at both dilutions (Figure 3.1). NS1 antibody detection in birds immunized with H5 subtype oil emulsion vaccine and challenge with homologous LPAI virus. To determine if the antibody response to the NS1 protein after infection with LPAI could be detected in birds vaccinated with a whole virus killed oil adjuvanted vaccine, groups of 10 birds were vaccinated with inactivated rh5n8-sepns1 (H5N8) vaccine or inactivated commercial H5N9 vaccine and challenged with the LPAI virus A/CK/PA/93 (H5N2) at a low challenge dose (10 5 EID 50 /bird) to simulate a typical infection seen in the field. NS1 ELISA results showed that one bird developed a detectable NS1 antibody response two weeks after challenge in vaccinated birds with inactivated rh5n8-sepns1 (Table 51

60 3.2). This bird was positive from week 1 until week 3 after challenge but by week 4 the OD values had fallen below the cut-off point (0.223). Interestingly, in the sham vaccinated group no NS1 antibody response was detected after challenge. The HI test in this group was positive only in 20% of the birds starting at 2 weeks post-infection, indicating a suboptimal challenge. Oropharyngeal virus shedding was detected at 3 and 5 days after challenge from the sham vaccinated group in the same 2 birds (out of 10) which were positive for NS1 (Table 3.2). In the group vaccinated with rh5n8-sepns1 3/10 birds were positive for virus shedding at 3 days after challenge and 2/10 at 5 days after challenge. No positives samples for virus shedding were detected in the group vaccinated with the commercial vaccine. NS1 antibody detection in birds immunized with H5 subtype oil emulsion vaccine and challenge with HPAI virus. To determine if vaccinated birds would develop a detectable NS1 antibody response after challenge with HPAI H5 virus, chickens were vaccinated with inactivated rh5n8-sepns1 or commercial vaccine H5N9 and challenged with 10 5 EID 50 /bird of the HPAI virus CK/Que/95. The NS1-ELISA results before challenge (2 weeks after vaccination) were negative with the exception of a single positive serum sample with borderline OD values (0.237) in the group vaccinated with the commercial vaccine (Table 3.3). All the sham vaccinated birds died within the first week after challenge. In contrast, vaccination completely prevented virus-associated mortality. In the group vaccinated with rh5n8-sepns1, NS1 antibodies were first detected 3 weeks after challenge from 1 of 10 birds (OD 0.377). The same bird was positive at week 4 showing lower level of NS1 activity (OD 0.266), where a total of 2/10 birds were positive. In the 52

61 group vaccinated with the commercial H5N9 vaccine, NS1 antibodies after challenge were detected at week 2 in 1 of 10 chickens (OD 0.330), at week 3 in 2 of 10 birds (OD 0.332; OD 0.220), and at week 4 in 3 of 10 chickens (OD 0.290; OD 0.385; OD 0.333). Two birds were positive during 2 consecutives weeks while one bird only appeared positive at 4 weeks after challenge. The HI test results using homologous antigen to each vaccine were positive in the groups immunized with rh5n8-sepns1 and commercial H5N9 vaccines showing a GMT of 42 and 416 respectively. Both groups experienced an increase in the HI titers until week 3 (GMT 2521 and respectively) after challenge. At week 4 the HI antibodies began to decline in both groups. Similar to the previous experiment (Table 3.2), the commercial H5N9 produced higher HI levels than those vaccinated with rh5n8- SEPNS1 vaccine. The virus shedding data showed a total of 3 of 10 birds positive at 3 days after challenge and 1 of 10 birds positive at 5 days after challenge. As with the previous experiment, the commercial vaccine completely prevented detectable viral shedding. Discussion The main objective was to determine whether or not the infection of chickens with AIV induced a detectable NS1 immune response capable of establishing a different serological status of naturally infected birds within a vaccinated population. The kinetics of the antibody response to the NS1 protein after infection of naive chickens with AIV virus demonstrate a low number of responders (2/8) in the group infected with 53

62 CK/CA/03 LPAI. In contrast, the HI antibody titer from these same birds was detected in 100% of the birds, indicating a poor diagnostic correlation between these two tests. These results were confirmed by Western blot (Figure 3.4) showing the sensitivity of the NS1-ELISA test to be at least comparable to that of the Western blot analysis when samples are tested at low dilutions (1:10). Therefore, the low detection of the antibody to NS1 after infection of naïve birds with AIV in this study is more likely due to the poor immune response to the NS1 protein rather than to the lack of sensitivity of the NS1- ELISA test. Previous work on NS1 based DIVA strategy development for influenza virus in horses showed the same pattern of response (15). The infection with AIV did not produce antibodies to NS1 in all infected horses as detected by NS1-ELISA. Although a similar challenge dose was used for two different LPAI viruses in the present studies, the CK/CA/03 virus produced a greater antibody response than TK/WI/68 based on the number of HI positive birds and the levels of HI antibody titer. This data supports that the CK/CA/03 virus was better adapted to chickens than TK/WI/68, which could be anticipated because the CK/CA lineage of virus circulated for several years in chicken flocks in California, but the TK/WI/68 virus appeared to be a recent introduction into turkeys (25) The immune response to the NS1 protein appears to develop later than the response to HA and showed a rapid decrease after the initial detection (Figure 3.2). These results are in disagreement with previous reports (17) where positive correlation was obtained in measuring the immune response with HI test and NS1-ELISA after infection with AIV in chickens. The challenge model used in the previously mentioned studies differed from the present study. For example, the previous study used 10 month old 54

63 chickens and challenged them 2 times with 10 6 dose of LPAI by subcutaneous and intramuscular routes. In the present study, a lower challenge dose and the mucosal route of inoculation were used, to simulate the natural exposure to AIV. The immune response observed to the NS1 protein during AIV challenge in this work was unexpected, and appeared to vary from bird to bird. These results might be related to complex interactions of each individual birds immune response and the specific strain of virus whose NS1 protein is designed to evade the host innate immune response (26, 27). Differences with regard to the immune response to non-structural proteins from individual animals and species have been reported with other viruses (28-30). For example, researchers have tried to develop a DIVA test based on NS proteins as antigen for foot and mouth disease (FMD) virus in cattle. Several NS proteins including Lb, 2C, 3A, 3D, 3AB and 3ABC have been used as antigens in serological assays for FMD. In one study (29) and based on virus isolation data experimental animals were classified as: naive, infected and eliminating the virus (convalescent), infected and persistently infected with FMD virus (carriers), vaccinated alone, vaccinated and either convalescent or carrier. However when they evaluated the immune response using an ELISA, the variation of the results was so high that convalescent animals could not be differentiated from carrier animals on the basis of their antibody response to any of the NS proteins examined. Currently, the 3ABC based ELISA test appears to successfully detect the majority of infected animals within a vaccinated population but doesn t detect the carrier state which has to be demonstrated by virus isolation or nucleic acid detection (31). In pigs, however, the ELISA test based on the nonstructural protein 3AB of FMD virus, has allowed the differentiation of vaccinated from infected pigs. The application of this assay 55

64 to serological surveillance in FMD eradication program in Taiwan showed that the positive reactors steadily have decreased over overtime (32). Overall, these data illustrate how the variable immune responses against non-structural proteins create different degrees of difficulties in the development and implantation of DIVA strategies for the eradication of FMD virus in two different animal species. The NS1-ELISA based DIVA strategy in the present research detected infection better after HPAI challenge, but not after LPAI challenge. However, the duration of the immunity appears to be brief and may not be practical for field use. In vaccinated protected animals the replication of a challenge virus is expected to be lower than unvaccinated controls, and there is concern about whether enough viral replication occurs to stimulate an antibody response to the NS1 protein. A closely matched vaccine and challenge strain should provide the best protection from virus shedding, and a poor vaccine match may provide increased virus replication that could correlate to the induction of higher NS1 antibody levels (33, 34). Therefore, the low detection of the NS1 after challenge with the homologous strains used in this study maybe due to the effective neutralization of the virus by the antibodies produced for the already prime immune system of the vaccinated birds. Data obtained in the present study doesn t support the ability of the NS1 protein to serve as a reliable DIVA indicator for chickens which disagrees with previous reports (12, 17). As previously mentioned this study used different tests and challenge models than previous studies, including the use of NS1 antigen expressed and purified in a baculovirus system where the earlier studies used NS1 antigen expressed in E.coli (12, 17). Furthermore, Tumpey et al. reported that by diluting the experimentally sera 1:200, 56

65 the low antibody activity to NS1 due to vaccination was diluted and a distinction, could be observed between vaccinated and infected chickens and turkeys (12). In this work the birds were vaccinated just one time and under those conditions the avian unpurified vaccines didn t seem to produce a detectable immune response to the NS1 protein. However, under the conditions of the present research diluting the serum samples 1:200 would likely compromise the detection of antibodies against NS1 (Figure 3.3) in the responders. Previous data (12, 17) show successful discrimination of AIV infected from vaccinated turkeys using NS1-ELISA. Although we did not attempt studies in turkeys, recent studies (35) indicate that the immune response to AIV NS1 differs between turkeys and chickens under experimental infections with a H9N2 subtype virus. No antibodies to a peptide corresponding to the C-terminus of the NS1 protein were detected in chickens by ELISA, whereas the opposite was true in turkeys as early as day 3 post infection (35). The observations of these studies, compared with works previously published (12, 35) suggest that the response to the NS1 protein after infection with AIV in chickens is different from turkeys, and each species must be evaluated separately to determine the immune response. The data of the present study do not favor the development of a DIVA assay for AIV in chickens based of the immune response to NS1. References 1. Wright, P., G. Neumann, and Y. Kawaoka Orthomyxovirus, Fith edition ed. Lippincott Williams & Wilkins (LWW), Philadelphia, PA Swayne, D. E., and D. L. Suarez Highly pathogenic avian influenza. Rev Sci Tech 19:

66 3. Swayne, D. E. Principles for vaccine protection in chickens and domestic waterfowl against avian influenza: emphasis on Asian H5N1 high pathogenicity avian influenza. Annals of the New York Academy of Sciences 1081: Capua, I., G. Cattoli, and S. Marangon DIVA--a vaccination strategy enabling the detection of field exposure to avian influenza. Dev Biol (Basel) 119: Capua, I., and S. Marangon Vaccination policy applied for the control of avian influenza in Italy. Dev Biol (Basel) 114: Suarez, D. L. Overview of avian influenza DIVA test strategies. Biologicals 33: Suarez, D. L., C. W. Lee, and D. E. Swayne Avian influenza vaccination in North America: strategies and difficulties. Dev Biol (Basel) 124: Capua, I., and S. Marangon The use of vaccination to combat multiple introductions of Notifiable Avian Influenza viruses of the H5 and H7 subtypes between 2000 and 2006 in Italy. Vaccine 25: Capua, I., C. Terregino, G. Cattoli, F. Mutinelli, and J. F. Rodriguez Development of a DIVA (Differentiating Infected from Vaccinated Animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza. Avian Pathol 32: Cattoli, G., A. Milani, F. Bettini, M. Serena Beato, M. Mancin, C. Terregino, and I. Capua Development and validation of an anti-n3 indirect immunofluorescent antibody test to be used as a companion diagnostic test in the framework of a "DIVA" vaccination strategy for avian influenza infections in poultry. Avian Pathol 35: Lee, C. W., D. A. Senne, and D. L. Suarez Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza. Vaccine 22: Tumpey, T. M., R. Alvarez, D. E. Swayne, and D. L. Suarez Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus. J Clin Microbiol 43: Capua, I., and S. Marangon The challenge of controlling notifiable avian influenza by means of vaccination. Avian Dis 51: Suarez, D. L., and M. L. Perdue Multiple alignment comparison of the nonstructural genes of influenza A viruses. Virus Res 54:

67 15. Birch-Machin, I., A. Rowan, J. Pick, J. Mumford, and M. Binns Expression of the nonstructural protein NS1 of equine influenza A virus: detection of anti-ns1 antibody in post infection equine sera. Journal of virological methods 65: Ozaki, H., T. Sugiura, S. Sugita, H. Imagawa, and H. Kida Detection of antibodies to the nonstructural protein (NS1) of influenza A virus allows distinction between vaccinated and infected horses. Vet Microbiol 82: Zhao, S., M. Jin, H. Li, Y. Tan, G. Wang, R. Zhang, and H. Chen Detection of antibodies to the nonstructural protein (NS1) of avian influenza viruses allows distinction between vaccinated and infected chickens. Avian Dis 49: Garcia, A., H. Johnson, D. K. Srivastava, D. A. Jayawardene, D. R. Wehr, and R. G. Webster Efficacy of inactivated H5N2 influenza vaccines against lethal A/Chicken/Queretaro/19/95 infection. Avian Dis 42: Swayne DE, S. D., Beard CW A laboratory manual for the isolation of avian pathogens. Kennet Square, PA Suarez, D. L., M. L. Perdue, N. Cox, T. Rowe, C. Bender, J. Huang, and D. E. Swayne Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong. J Virol 72: Neumann, G., T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, M. Hughes, D. R. Perez, R. Donis, E. Hoffmann, G. Hobom, and Y. Kawaoka Generation of influenza A viruses entirely from cloned cdnas. Proceedings of the National Academy of Sciences of the United States of America 96: Stone, H. D. The preparation and efficacy of manually emulsified Newcastle disease oil-emulsion vaccines. Avian Dis 35: Lee, C. W., and D. L. Suarez Application of real-time RT-PCR for the quantitation and competitive replication study of H5 and H7 subtype avian influenza virus. Journal of virological methods 119: Letzel, T., F. Coulibaly, F. A. Rey, B. Delmas, E. Jagt, A. A. van Loon, and E. Mundt Molecular and Structural Bases for the Antigenicity of VP2 of Infectious Bursal Disease Virus. J Virol 81: Cardona, C. J., B. R. Charlton, and P. R. Woolcock Persistence of immunity in commercial egg-laying hens following vaccination with a killed H6N2 avian influenza vaccine. Avian Dis 50: Kochs, G., A. Garcia-Sastre, and L. Martinez-Sobrido Multiple anti-interferon actions of the influenza A virus NS1 protein. J Virol 81:

68 27. Kochs, G., I. Koerner, L. Thiel, S. Kothlow, B. Kaspers, N. Ruggli, A. Summerfield, J. Pavlovic, J. Stech, and P. Staeheli Properties of H7N7 influenza A virus strain SC35M lacking interferon antagonist NS1 in mice and chickens. J Gen Virol 88: Mackay, D. K. Differentiating infection from vaccination in foot-and-mouth disease. The Veterinary quarterly 20 Suppl 2:S Mackay, D. K., M. A. Forsyth, P. R. Davies, A. Berlinzani, G. J. Belsham, M. Flint, and M. D. Ryan Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA. Vaccine 16: Bergmann, I. E., V. Malirat, E. Neitzert, and E. Correa Melo Vaccines and companion diagnostic tests for foot-and-mouth disease virus. An overview of the experience in South America. Dev Biol (Basel) 114: Pasick, J. Application of DIVA vaccines and their companion diagnostic tests to foreign animal disease eradication. Anim Health Res Rev 5: Chung, W. B., K. J. Sorensen, P. C. Liao, P. C. Yang, and M. H. Jong Differentiation of foot-and-mouth disease virus-infected from vaccinated pigs by enzyme-linked immunosorbent assay using nonstructural protein 3AB as the antigen and application to an eradication program. J Clin Microbiol 40: Swayne, D. E., M. Garcia, J. R. Beck, N. Kinney, and D. L. Suarez Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 18: Swayne, D. E., G. Avellaneda, T. R. Mickle, N. Pritchard, J. Cruz, and M. Bublot Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-h5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals. Avian Dis 51: Dundon, W. G., S. Maniero, A. Toffan, I. Capua, and G. Cattoli Appearance of serum antibodies against the avian influenza nonstructural 1 protein in experimentally infected chickens and turkeys. Avian Dis 51:

69 Table 3.1 Antibody response to NS1 protein as detected by an indirect NS1-ELISA after infection with LPAI viruses during a period of 5 weeks Weeks after infection Virus HI ELISA ± HI ELISA HI ELISA HI ELISA HI ELISA HI ELISA TK/WI/68 (H5N9) 0/8 0/8 2/8 4.5±0.71 (22) 0/8 4/8 5.1±1.5 (56) 0/8 4/8 6±1.8 (64) 0/8 4/8 6±1.8 (64) 0/8 4/8 6±1.8 (64) 0/8 CK/CA/03 (H6N2) 0/8 0/8 5/8 5.6±1.8 (194) 0/8 7/8 8±2.4 (512) 0/8 8/8 9.8±1.4 (891) 2/8 8/8 9.8±1.4 (891) 2/8 8/8 9.8±1.4 (891) 2/8 SPF chickens were inoculated in the choanal cleft with 10 5 EID 50 A/chicken/CA/K Ct/03 Ck/CA/K (H6N2); 10 6 EID 50 A/Turkey/Wisconsin/68 (H5N9) HI serological response was measured using homologous BPL-inactivated AI virus strains for HI antigen. The results represent total number of positives/total number tested, mean±standard deviation log 2 HI activity, (GMT). ±NS1-ELISA results represent number of samples with Optical Density higher that (405 nm)/total of serum samples tested (OD average of positive samples) 61

70 Table 3.2 Summary of experimental data obtained in vaccinated birds with oil emulsion inactivated vaccines after homologous challenge with LPAI virus Time After Challenge Week 0 Week 1 Week 2 Week 3 Week 4 Virus Shedding Inactivated vaccines 3 days 5 days HI Ŧ ELISA HI ELISA HI ELISA HI ELISA HI ELISA Sham 2/10 2/10 0/10 0/10 0/10 0/10 2/10 2.5±0.7 (22.6) 0/10 2/10 4.5±2.1 (90.5) 0/10 2/10 5±1.4 (128) 0/10 H5N8- SEPNS1 3/10 2/10 10/10 4.1±2.5 (68.6) 0/10 10/10 6±2.5 (256) 0/10 10/10 7.4±2.1 (675) 1/10 (0.339) 10/10 6±1.1 (256) 1/10 (0.451) 10/10 5.6±1.3 (294) 1/10 (0.236) Commercial H5N9 Negative Control 0/10 0/10 10/10 8.8±1.3 (1782.9) 0/10 10/10 9.6±2.2 (3104.2) 9/9 11.1±0.9 (5420) 0/10 10/ ±0.6 (8780) 0/10 10/ ±0.5 (13307) 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/10 SPF birds were vaccinated at weeks of age with rh5n8 or H5N9 commercial oil emulsion vaccines. Birds received intra-choanal cleft homologous challenge dose of 10 5 LPAI A/Chicken/PA/13609/93 (H5N2) two weeks after vaccination. Tracheal swabs were collected from each bird 3 and 5 days after challenge, RNA extracted and quantitative viral shedding was assayed using real time RT-PCR, ŦHI serological response was measured using homologous BPL-inactivated AI virus strains for HI antigen. The results represent total number of positives/total number tested,mean±standard deviation log2 HI activity, (GMT). NS1-ELISA numbers represent: number of positive samples (OD higher that 0.223)/Total of serum samples tested. (OD average of positive samples) 62

71 Table 3.3. Summary of experimental data obtained in vaccinated birds with oil emulsion inactivated vaccines after homologous challenge with HPAI virus Inactivated vaccines Time after Challenge Week 0 Week 1 Week 2 Week 3 Week 4 Virus shedding Ŧ 5 3 days days HI ELISA HI ELISA HI ELISA HI ELISA HI ELISA Sham 0/10 0/10 0/10 0/10 H5N8- SEPNS1 3/10 1/10 9/10 3.8± 0.8 (42) 0/10 10/10 8.7±1.3 (415.9) 0/10 10/10 9.0±1.6 (2048) 0/10 10/10 9.3±1.1 (2521) 1/10 (0.330) 10/10 8.9±1.1 (1910.9) 2/10 (0.252) Commercial H5N9 0/10 0/10 10/10 6±1.6 (256) 1/10 10/ ±1.8 (1448.2) 0/10 10/ ±0.6 (7131.6) 1/10 (0.333) 10/ ±0.7 (10809) 2/10 (0.276) 10/ ±0.7 (7131.6) 3/10 (0.336) Negative 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 SPF birds were vaccinated at weeks of age with rh5n8 or H5N9 commercial oil emulsion vaccines. Birds received intra-choanal cleft homologous challenge dose of 10 5 HPAI A/chicken/Queretaro/ /95(H5N2) two weeks after vaccination. ŦTracheal swabs were collected from each bird 3 and 5 days after challenge, RNA extracted and quantitative viral shedding was assayed using real time RT-PCR, HI serological response was measured using homologous BPL-inactivated AI virus strains for HI antigen. The results represent total number of positives/total number tested, mean±standard deviation log2 HI activity, (GMT). NS1-ELISA numbers represent: number of positive samples (OD higher that 0.223)/Total of serum samples tested. (OD average of positive samples) 63

72 Figure 3.1 (A) (B) Panel (A) SDS-PAGE gel (15% agarose gel) showing purification steps of AIV NS1 proteins from recombinant baculovirus infected Sf9 cells. ). Lines 1 and 5 = Protein marker; Lines 2, 3, and 4 = lysate, supernatant, and eluate NS1-C-terminal; Lines 6, 7, and 9 = lysate, supernatant, and eluate NS1-N-terminal Panel (B) Western blot analysis. Lines 2, 3, and 4 = lysate, supernatant, and eluate NS1-C-terminal; Lines 6, 7, and 9 = lysate, supernatant, and eluate NS1-N-terminal. Detection of the proteins was carried out with anti-his peroxidase. 64

73 Figure HI titer (GMT) ELISA OD 405nm 0 W - 0 W - 1 W - 2 W - 3 W - 4 W Weeks after infection HI (GMT) ELISA (OD) Antibody response to HA and to NS1 proteins after infection with Ch/Ca/03 LPAI virus. GMT on the average of the HI titers obtained from all the birds (n=8) on each testing time. The ELISA value corresponds to the average OD reading of the same two birds that show response to the NS1 protein. 65

74 Figure ELISA OD 405 nm :5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 Serum dilutions Neg Pos Plots showing the titration of antibodies to NS1 in individual chicken serum samples taken three weeks after infection with Ch/Ca/03. Serum samples were diluted from 1/5 in a twofold series against constant NS1 protein concentration. 66

75 Figure 3.4 Table Weeks Post-Infection Bird ID HI* NS1-ELISA > > > Figure 3.4 Western blot analysis of serum samples from birds infected CK/CA/03 (H6N2) LPAI taken at 3 week after infection was performed to test the sensitivity of NS1- ELISA NS1. Lane bands specific for the NS1 protein were considered positive for NS1 antibody. The numbers on each line correspond to the bird ID shown in Table 3.4. Positive and Negative control samples are the same used for NS1-ELISA as stated in Materials and Methods. Table 3.4 *HI test was performed with homologous antigen prepared with A/CK/California/K Ct/03 (H6N2) virus inactivated with 0.1% β-propiolactone (BPL). The numbers represent Log 2 HI titers of individual samples. NS1-ELISA was performed on serum samples diluted at 1:50. Optical density was read at 405 nm wave length. The cut-off point as indicated in Materials and Methods was