Avian Influenza Virus and DIVA Strategies

Abstract

Vaccination is becoming a more acceptable option in the effort to eradicate avian influenza viruses (AIV) from commercial poultry, especially in countries where AIV is endemic. The main concern surrounding this option has been the inability of the conventional serological tests to differentiate antibodies produced due to vaccination from antibodies produced in response to virus infection. In attempts to address this issue, at least six strategies have been formulated, aiming to differentiate infected from vaccinated animals (DIVA), namely (i) sentinel birds, (ii) subunit vaccine, (iii) heterologous neuraminidase (NA), (iv) nonstructural 1 (NS1) protein, (v) matrix 2 ectodomain (M2e) protein, and (vi) haemagglutinin subunit 2 (HA2) glycoprotein. This short review briefly discusses the strengths and limitations of these DIVA strategies, together with the feasibility and practicality of the options as a part of the surveillance program directed toward the eventual eradication of AIV from poultry in countries where highly pathogenic avian influenza is endemic.

Avian Influenza Virus

Gene segments and proteins

Avian influenza viruses (AIV) are enveloped, segmented, negative-stranded RNA viruses, belonging to the family Orthomyxoviridae, genus Influenzavirus A (77,152). Influenza A virus (IAV) is composed of eight gene segments, and each gene segment codes for at least one protein. To date, IAV is known to code for 13 viral proteins (64,173). Some of the segments encoded more than one protein through mechanisms such as alternative reading frame (PB1-F2, PB1-N40, PA-X, and M2), and mRNA splicing (NS1/NEP) (31,64,75,78,172).

AIV is classified based on the antigenic variation displayed by the virus surface protein–hemagglutinin (HA) and neuraminidase (NA) (6). A total of 144 possible subtype combinations have been identified for AIV based on the 16 HA subtypes and 9 NA subtypes (46) found circulating in the aquatic bird population identified as the AIV natural reservoir, predominantly the Anseriformes (particularly ducks, geese, and swans) and Charadriiformes (particularly gulls, terns, and waders) (100,168). Two new HA subtypes (H17, H18) and NA subtypes (N10, N11) have recently been identified circulating in bats from Central America (Guatemala) and South America (Peru) (154,155).

AIV transmissibility

Observations indicated that movement of AIV from wild to domestic birds occurs relatively frequently due to shared ecosystem, where prolonged and repeated exposure of domestic birds to the virus facilitate adaptation of virus to a new host (140). However, virus adaptations for a new host is a complex and a rare event as majority of these transmissions will only cause transient virus infections with limited spread as observed in AIV poultry surveillance (2,132). However, it is important to note that some species such as domestic ducks and geese, turkeys, and Japanese quails are more susceptible to AIV infections and may have been the bridging species of wild birds AIV into chicken and other gallinaceous poultry (145).

AIV evolution

Continuous outbreaks of AIV infection are driven by two main evolutionary mechanisms used by the virus to evade host immune systems: antigenic drift and antigenic shift (103). Antigenic drift occurs in response to the host immune pressure when mutations accumulate in the surface glycoproteins HA and NA, causing minor changes to the antigenic structure of the virus (Nelson and Holmes, 2007). Antigenic shift results from reassortment of infecting virus subtypes that lead to introduction of strains with completely novel gene combination and often with improvements in the capacity for the production of more viable and fit virus progeny (58).

AIV pathogenicity

AIV is classified into low and highly pathogenic avian influenza virus (LPAIV and HPAIV, respectively) based on its lethality in chicken (Gallus gallus domesticus) (140,147). In domestic poultry, LPAIV generally causes subclinical infection with virus shedding in infected birds, if not mild respiratory disease. In contrast the HPAIV, also formerly known as the fowl plague, causes multiorgan systemic disease, with high percentage of morbidity and mortality in both domestic and wild birds (1,147).

The AIV pathogenicity generally relies on the cleavability of the HA0 subunit to HA1 and HA2 by the host cellular proteases (73,82,117), and HPAIV is characterized by the presence of polybasic amino acids at the HA0 cleavage site instead of a monobasic motif observed for LPAIV (15,60,119). The monobasic structure of the HA0 cleavage site is only cleavable by the trypsin-like enzymes, which are present at limited sites in the host, hence LPAIV infections are confined to respiratory or gastrointestinal tract (72,83,116). In contrast, the polybasic motif found in the HPAIV HA0 is cleaved by ubiquitous proteases present within cells of multiple organs throughout the body, such as furin and subtilisin-related proteases (proprotein convertase 6–PC6), causing fatal systemic infection (61,131).

LPAIV and HPAIV in poultry

Any of the 16 HA subtypes circulating in wild birds reservoirs are considered as LPAIV, while all HPAIV are of H5 and H7 subtypes, although not all of these subtypes are HPAIV (3,147). Apart from the HPAIV H5N3 outbreak in common terns (Sterna hirundo) in South Africa in 1961 (11) and HPAIV H5N1 outbreak in wild waterfowl in two parks in Hong Kong in 2002 and bar-headed geese (Anser indicus) in western China in 2005 (29), HPAIV has been rarely isolated from wild bird populations (147). Due to the complex pathobiology of AIV, viruses that are highly pathogenic (HP) in domestic birds, generally do not necessarily cause diseases in Anseriformes birds (ducks and geese) in experimental condition (4,44). It is important to note that HPAIV usually occurs in domestic gallinaceous poultry (chickens, turkeys, quails, and guinea fowls) after exposure to and adaptation of LPAIV from wild birds (115,140). This is usually a unidirectional infection, where the domestic bird-adapted AIV rarely reinfects wild bird population (140), with the exception of the Asian lineage H5N1 HPAI virus (29,92).

Virulence shift of LPAIV to HPAIV

The LPAIV H5 and H7 subtypes can acquire virulence factors and become HPAIV through several mechanisms focused on the HA protein, which are (i) the substitution and insertion of basic amino acids (aa) in the HA cleavage site (62,139), (ii) loss of carbohydrate that covers the HA cleavage site through residue mutations (66), (iii) recombination of HA with other AIV viral genes such as nucleoprotein (NP) gene (136), matrix (M) protein gene (110), or 28S ribosomal RNA (67), and (iv) polymerase slippage that caused sequence duplication, and thus insertion in the HA gene (47,111). Nevertheless, it was suggested that a hidden virulence potential was readily embedded within the LPAIV strains capable of transformation to a HP strain, where the acquisition of polybasic cleavage site is the key activator for the virulence shift (14,130). This assumption is based on observations where alterations in other AIV viral proteins such as deletion of matrix 2 (M2) protein or NP cleavage site reduced AIV pathogenicity (178), while point mutation accumulation in the NA protein (36), amino acid deletion in the NA stalk (99), and amino acid substitution in the nonstructural protein 1 (NS1) (65) and polymerase proteins (basic polymerase 2, PB2 and acidic polymerase, PA) (35,54) promotes virulence of AIV.

Evolutionary pattern of H5N1

Within the AIV history, the pandemic potential of Asian lineage H5N1 virus is by far the most alarming due to the rate of its spread and the unusual evolutionary pattern showed by this particular subtype (45,167). Unlike the emergence of other HPAIV that occurs in chicken, the initial outbreak of H5N1 was recorded in domestic geese in Guangdong Province, China in 1996, which then became the primary precursor virus for the major outbreak in chicken farms in Hong Kong in 1997 (HK-97) (121,175). Although the HK-97 genotype had been eliminated through mass poultry depopulation in 1997, the genetic variants of the primary precursor virus (Goose/Gd-like) have continued to circulate exclusively in aquatic poultry until late 2000 (26,170), where the host range expanded to include terrestrial poultry in the following year, providing a larger pool of genetic material for reassortment (28,51).

The rapid rate of H5N1 evolution was later validated with the identification of six H5N1 reassortants in Hong Kong and mainland China in early 2001, immediately before the outbreak in Hong Kong, mid-May the same year (51,90,122). It was identified that this reassortant virus possessed a HA gene that originated from a Goose/GD/96-like virus, while the other seven internal genes were a result of reassortment from other non-H5 AIV (170). Although no infection with H5N1 was detected from July 2001 onward, Hong Kong experienced an outbreak caused by the HPAIV H5N1 again in February 2002 (90,122). Eight new H5N1 genotypes were isolated including genotype “Z”, which later become dominant in southern China (90). Characterized with the deletions of 20 aa in the NA stalk and 5 aa in the NS protein (51), genotype “Z” has been responsible for the emergence of the 2003 and 2004 H5N1 outbreaks, marking the first dissemination wave of H5N1 into eight countries in East and South East Asia, leading to establishment of endemicity in Vietnam and Indonesia (45,164).

Although the Asian lineage H5N1 virus was endemic in poultry since 1997, it had later spread and persisted in the wild bird population, evidenced by the H5N1 outbreak in the migratory waterfowl, the bar-headed geese (A. indicus) at Qinghai Lake in western China in 2005 (29,92). Subsequently, the virus rapidly spread across Asia, Europe, the Middle East, and Africa, marking the second wave of H5N1 dissemination, affecting wild migratory birds and poultry (49,164). The third wave of H5N1 dissemination to South East Asian countries followed immediately in late 2005. It was characterized by the emergence and predominance of the H5N1 Fujian-like viruses, replacing the multiple H5N1 sublineages in China, which were responsible for the previous disseminations (127). This event led to the panzootic of H5N1 in poultry, especially in the Asian continent where intermittent outbreaks have been reported, particularly in countries where H5N1 is endemic (China, Vietnam, Indonesia, and Bangladesh) (41,45).

AIV and Vaccination

Following the identification of wild birds as the agent of long distance virus transmission (5,108,153), and the possible transmission of the virus through domestic animals (162), culling of the infected birds and the flocks of birds with suspected exposure to the virus have been used as the primary control measures, especially in countries where disease has been recently introduced (133,134). However, in countries where infection was already widespread and endemic, and other methods were not likely to eradicate the infection, vaccination was chosen as the primary control tool (37,133,144).

To date, AIV vaccination using the inactivated vaccines, and to a smaller portion using the live recombinant vaccine (NDV-H5) has only been exercised as a control or a preventive measure to eradicate HPAI viruses in poultry, either in the event of epidemics, such as seen in Mexico (H5N2, 1994–1995, 1995–2001) (163), Italy (H7N1, 1999–2000; H7N3 and H5N2, 2003–2006), Hong Kong (H5N1, 2002–2003) (21,96,122,163), and others; or in countries where HPAIV are endemic, as is the case for HPAI H5N1 in China, Indonesia, Vietnam, and Egypt (27,41,96,97).

Vaccination helps to control the spread of infection as vaccinated birds will acquire an elevated level of resistance to infection, thus lower shedding and environmental contamination by virus (22,144). Nevertheless, to achieve disease eradication, it is important for a vaccination program to be implemented in conjunction with adequate biosecurity enforcement and continuous surveillance of infection in vaccinated bird population (22). Although vaccination is highly recommended as a control and preventive tool for AIV, silent spread of infection in vaccinated populations is a major concern, especially where AIV is endemic. This is due to inability of the available inactivated AIV vaccines to provide complete protection to a virulent field challenge, allowing a small number of birds to become infected and excrete the virus without apparent clinical manifestation of infection. Long-term circulation and establishment of AIV in vaccinated population have been reported to cause changes in the genetic and antigenic properties of the virus, producing escape mutants as reported in Mexico (85), China (127), and Egypt (50). Due to the inability of the available standard serological tests used in disease surveillance to differentiate antibodies produced by vaccination from those that arise by field virus infection, strategies have been developed to differentiate infected from vaccinated animals (DIVA).

Current Understanding of DIVA Strategies for AIV

Vaccine development work with the aim to enable DIVA application was first published by Van Oirschot, Rziha, Moonen, Pol, Van Zaane (158) for Aujeszky's disease virus in pigs; and this investigator later coined the acronym DIVA (157). In parallel growth with the use of vaccine against AIV, advances of DIVA strategies were focused on vaccine developments that are capable of DIVA while permitting the use of the available standard serological tests (DIVA-vaccine approach). Alternatively, DIVA-antigen approach focused more on the serological tests development while allowing the use of conventional vaccines (killed virus).

In this section, six DIVA strategies were discussed in terms of the vaccine format and the available complementary companion diagnostic tests: (i) sentinel birds, (ii) subunit vaccine, (iii) heterologous NA, (iv) nonstructural 1 (NS1) protein, (v) matrix 2 ectodomain (M2e) protein, and (iv) hemagglutinin subunit 2 (HA2) glycopolyprotein (gp) (12,16,23,56,81,133). Summary of these strategies can be seen in Table 1.

Table 1.

List of Available Strategies for Differentiating Infected Animals from Vaccinated Animals, with Some of Their Advantages and Limitations in General

Strategy	Sentinel bird	Recombinant subunit vaccines	Heterologous NA	Differential immune response against protein (NS1, M2, and HA2 gp)
Procedure and vaccine used	Naïve unvaccinated birds are marked and randomly spread in a vaccinated flock Sentinel birds are routinely tested for influenza virus exposure	Vaccine using a vector expressing HA and NA proteins Example: Fowlpox-vectored recombinant vaccine for the H5 subtype	Vaccines containing the same HA subtype as the field strain, but a different NA subtype. Example: If the field virus is H7N2, the vaccine is H7N3	Vaccination using whole-killed virus Observation of the differential immune responses to the targeted protein (NS1, M2, or HA2)
Available companion diagnostic test	Hemagglutinin Inhibition (HI) test Agar gel immunodiffusion Type A-specific ELISA (detect anti-NP antibodies)	Agar gel precipitin ELISA targeting the antibodies to matrix (M) protein or the nucleoprotein (NP) Fluorescence microsphere immunoassay (FMIA)	Neuraminidase Inhibition (NI) test Indirect immunofluorescence assay (iIFAT) FMIA Modified NI test	ELISA-based targeting the antibodies to specified proteins
Advantages	Low cost Readily applicable Sensitive procedure for monitoring in vaccinated flock	Efficacious in providing protection Commercially available Mass administration The standard diagnostic tests are applicable	Efficacious in providing protection Rapidly available through reverse genetics technology	Conventional inactivated virus can be used for vaccination Only a single diagnostic test needed
Limitations	Labor intensive Time consuming Naïve birds can potentially act as virus amplifiers and be the source of infection	Test sensitivity is yet to be determined	Prior knowledge on circulating strain Possible introduction of the same NA subtype field strain with the NA subtype used for vaccination Undetermined sensitivity of serologic testing Low-throughput screening capacity iIFAT–time consuming, laborious and the result interpretation is subjective	Risk of false-positive due to the presence of protein contaminant from nonpurified vaccine i.e NS1 protein Risk of false-negative in surinfected host due to the inability of host to seroconvert HA2 gp approach–need more studies

Sentinel birds

The most basic strategy used for detection of live virus infection in a vaccinated flock is the employment of sentinel birds, where approximately 1% of the birds in the monitored farm are left unvaccinated and routinely tested serologically to detect flock exposure to live virus (133,134). This strategy offers a sensitive measure of any rising infection within the vaccinated flocks, and monitoring can be done using the available diagnostic tests such as the hemagglutination inhibition (HI) test and the ELISA test detecting NP or HA antibodies. This strategy was successfully employed alongside the heterologous NA emergency vaccination during the HPAI H7N1 outbreak in Italy in 2000 to monitor the field situation (22).

Recombinant subunit vaccines

As described earlier, HA gene is a structural virus protein with important functions for immunity and is one of the key determinants of AIV antigenic properties (73,82). Although optimum protection is achieved through the use of vaccination with whole inactivated virus homologous to the circulating strain, studies have indicated that the presence of HA alone in vaccine elicits protective immune response against viral infection (114,169). In the subunit vaccine strategy, the AIV HA gene is expressed in bacteria, viruses, or yeast system before being purified and prepared for use as a vaccine (32,34,118). A variety of different AIV viral vectors have been studied, where protective immunity was demonstrated upon experimental challenges (Table 2).

Table 2.

List of Selected Studies on the Development of DIVA Strategies for Influenza A Virus and the Summary of Their Findings Within the Last Decade

Strategy	Vaccination	Challenge virus subtype	Animal model	Companion diagnostic test/DIVA test tool	Protection	DIVA	Comments	Reference
DIVA vaccines: Recombinant vaccine	Newcastle Disease (NDV) virus expressing HA protein (H5) Herpesvirus of turkey (HVT) expressing HA (H7) Virus-like particle (VLP) expressing HA and M1 (H9) Fowlpox (FP) recombinant expressing HA (H7 and H5) Infectious laryngotracheitis (ILT) virus expressing HA (H7) H5N1/PR8-519 (S2 glycoprotein of murine hepatitis virus-MHV replacing NA stalk region)	H5N1 H5N2 H7N1 H9N2 H5N8 H5N3 H5N2 H5N9	Chicken, mice	NP-GST fusion based ELISA NP-ELISA (IDEXX Laboratories, Inc., Westbrook, ME)	+/−	+	Reduced protection shown by FP-HA (H7) in chicken previously vaccinated or infected with fowlpox	(18,48,71,88,89,91,94,141,142,160,161)
DIVA-vaccine: Heterologous NA	Inactivated wild-type: H7N3 H5N2 H7N2 H9N8 H9N2 H7N1 Inactivated reassortant: H5N1 H7N8 H9N8 H5N8 H3N4 Inactivated reassortant with truncated NS: H3N3 Commercial H5N9	H7N1 H5N2 H7N2 H9N2 H7N3 H3N2	Chicken, turkey	Indirect immunofluorescent antibody test (iIFAT) expressing N1, N2 or N3 protein Micro-NI test with N1, N2 and N8 antigen Modified micro-NI test with N2, N8 and N9 antigen N2-ELISA	+	+/−	Micro-NI test is time consuming Modified version of the micro-NI test provide a more rapid option ELISA-based test offers a relatively easier and rapid application overall	(8,23 –25,74,84,165)
DIVA test: Recombinant NS1	Inactivated wild-type: H3N2 H3N8 H7N7 H5N9 H7N2 H5N1 H9N2 Commercial: H7N2 Live virus: H5N9 H7N1 H7N2	H3N8 H3N2 H7N2 H9N2	Horse, mice, chicken	NS1-ELISA Agar gel precipitin	+	+/−	Nonspecific reactions in NS1-ELISA reported is speculated due to the nonpurified vaccine used Incorporation of truncated NS1 in vaccine strain is recommended to address NS1 protein contamination issue Differences in NS1 immune response between strains and species have been demonstrated, that is, in turkeys and chicken	(38,109,128,156,177)
DIVA test: Truncated NS1	Live virus with truncated NS1: H7N3 H3N2	H7N2 H3N2	Chicken, turkey	NS1-ELISA Fluorescence microsphere immunoassays (FMIA) targeting recombinant NS1	+	+/−	Low seroconversion in vaccinated-and-challenged turkeys limited replication site by LPAIV lead to low titer of AIV despite infection (in comparison to HPAIV infection) could vary between bird species	(165,166)
DIVA-test: Matrix 2 protein	Inactivated wild-type: H5N9 H7N1 H5N1 Commercial: H9N2	H7N7 H5N1 H9N2	Chickens	M2e-peptide-ELISA Recombinant M2e-ELISA Tetrameric-recombinant M2e ELISA	+	+/−	Recombinant M2e-ELISA is more cost effective than synthetic peptide-based ELISA Development of tetramer-M2e as antigen has increased the sensitivity of this strategy, compared with the monomer-M2e-based ELISA systems	(53,56,70,81)

“+” indicates presence of protection by the vaccines or the strategy successfully demonstrated DIVA ability; “−” indicates negative protection by vaccines or unsuccessful DIVA ability; “+/−” indicates partial protection against challenge infection by vaccine or evidence of nonspecific reaction for DIVA test results.

Apart from being efficacious and safe for application, the recombinant subunit vectored-virus vaccines offer immunity through a single vaccination, with the option of vaccination against multiple diseases and the availability of mass vaccine administration (91,148). Works on recombinant subunit vaccines have expanded significantly following the advances of reverse genetic technology (104), where it allows rapid regeneration of reassortant viruses, and thus reduces vaccine production time by approximately 2 months (57). However, most importantly, the subunit vaccines allow a clear distinction between antibodies produced by vaccination or wild-type AIV infection, which is crucial for DIVA surveillance purposes using the standard diagnostic tools. In theory, the vaccinated birds will only produce antibody against the expressed HA protein, but none for internal proteins such as NP and M proteins. Since the vaccinated birds will remain naïve to the internal proteins, infected birds can be identified if antibodies against these proteins are present (91). Standard diagnostics test available are the agar gel immunodiffusion, which detects the anti-NP and anti-M antibodies (106); and the commercially available enzyme-linked immunosorbent assay (ELISA) kit such the AIV FlockChek ELISA kit (IDEXX labs) (91), specifically designed for detecting anti-NP antibodies. To date, the recombinant fowlpox-influenza H5 vaccine is licensed and available in El Salvador, Guatemala, Mexico, China, and USA (143), while recombinant herpesvirus turkey (rHVT) is licensed in Egypt and USA, with recombinant duck enteritis virus (rDEV) being licensed in China (106,146).

Heterologous NA vaccine

The heterologous NA vaccine strategy employs an inactivated AIV containing similar HA subtype but different NA subtype to the outbreak strain (23). Vaccinated birds are protected against live virus infection by development of anti-HA antibodies, and they can be differentiated from infected birds through detection of antibodies against the NA subtype. This strategy allows the use of standard killed vaccines and screening can be done against anti-NA antibodies using an indirect immunofluorescence assay (23), in place of the conventional neuraminidase inhibition (NI) test (9).

There are only three known applications of the heterologous NA vaccine. It was first introduced as a measure to differentiate between vaccinated and infected birds during the 1999–2000 H7N1 HPAIV outbreak in Italy (21). The vaccine was prepared using inactivated H7N3 virus, and infected birds were detected by an indirect immunofluorescent antibody test (iIFAT) specifically developed for anti-N1 antibody (23). Similar strategy was implemented during the outbreak of LPAI H7N3 in Italy in 2002–2003, where inactivated H7N1 was used for vaccination, and during the outbreak of HPAI H5N1 in Hong Kong in 2002, inactivated H5N2 virus was used for vaccination (20).

AIV nonstructural 1 protein: differential immune response

The NS1 protein is a multifunctional protein that regulates viral RNA polymerase activities and viral mRNA translation (40,76,120). It is a nonstructural protein that is only detectable in infected cells, but not in packaged virions (123). Based on this observation, a DIVA-antigen approach has been suggested, which allows the use of conventional whole-killed virus for vaccination (109). A diagnostic ELISA that targets NS1 antibodies is a simple screening test, as had been previously recognized for foot and mouth disease virus (102). The first successful demonstration of this strategy for AIV was reported for the equine IAV (12), where NS1 antibodies were identified only in infected ponies but not in the vaccinated ones. Most works on the development of NS1 protein as antigen for DIVA have expressed recombinant NS1 protein in vectors such as pMAL and pET (17,156,177).

M2e protein: highly conserved protein

M2e protein is the external part of a homotetrameric transmembrane protein encoded by segment 7 of the IAV through an alternative reading frame (+1) mechanism (59,79). This protein forms ion channels on the AIV surface that are crucial for the release of viral genome into the host cell cytoplasm during virus entry (80,98), and serves as a pH regulator for the Golgi apparatus, which is essential for HA glycoprotein maturation (137). Two factors have led to the recommendation of M2e protein as DIVA antigen: (i) the relatively invariable nature of M2e protein across AIV strains (63,69), where its small size and low abundance in comparison to the other two surface glycoproteins (HA and NA) have allowed M2e protein to escape immune selection pressure and antigenic drift (43); and (ii) the abundance of the M2e protein on the surface of infected cells despite being low in copy number in a mature virion (∼3% of the surface glycoprotein population) (13,176). Both of these characteristics have suggested that M2e protein could be a sensitive, specific, and universal DIVA antigen. The earliest report on the application of M2e as DIVA antigen in poultry has demonstrated a sensitive M2e peptide-based ELISA for detection of M2e antibodies following infection with HPAIV strains H5 and H7 (81). Similar sensitivity of M2e protein as DIVA antigen has also been demonstrated in a challenge study using LPAIV H9N2 (70), and against multiple AIV reference antisera (56).

Hemagglutinin subunit 2 glycoprotein: highly conserved epitope

HA2 glycoprotein (gp) is the C-terminal fragment of the cleaved form HA protein (125,171). It is considerably the more conserved region out of the two HA cleavage products (HA1 and HA2), especially at its N-terminal end, known as the fusion peptide (first 11 residues), which is involved in the fusion activity of IAV (33,125). The HA2 gp has been suggested as another potential target for DIVA tool based on two key criteria. First, HA2 is highly conserved throughout the 16 HA subtypes of IAV (46,105,107), with only two known epitope variants corresponding to the classical phylogenetic grouping of AIV HA protein (138). Four antigenic sites have been identified from HA2, namely site I (aa 1–38, the N-terminal), sites II and IV (aa 125–175), which exhibit different reactivity among IAV subtypes, and site III (aa 38–112) (159). As observed with the M2e protein approach, detection of antibodies against the highly conserved HA2 gp would theoretically enable a universal detection of all IAV subtypes. Second, this conserved region is only accessible to immune recognition following virus infection. It has long been noted that HA0 cleavability is essential for IAV infectivity (73,82), where the cleavage of HA0 to form HA1 and HA2 subunits is a prerequisite for membrane binding and virus entry to the host cell (95,124). HA2 gp is not accessible in the HA0 native form as it is buried in the pocket formed by the stalk of the HA stem trimer (126,159). However, once the HA0 is cleaved, the HA2 gp will be exposed and inserted into the target membrane to allow the conformational change, which will lead to membrane fusion and virus entry (19,30). Considering these findings, it is reasonable to assume that the presence of antibodies against discrete epitopes on HA2 gp would also be indicative of virus infection.

DIVA Strategies Applicability and Developments

An ideal surveillance tool is required to be (i) cost effective, (ii) rapid and easily manageable, and (iii) possess a high sensitivity and specificity in discriminating between naïve-infected host from a vaccinated-only host, and a vaccinated-infected host.

Although the sentinel bird strategy is simple to employ, there are concerns that the naïve birds may increase the infection risk for the vaccinated flock following repeated and lengthy exposure to the high load shedding of the virus by the sentinels (133). Acquiring a new infection is still possible in the vaccinated flock due to the continuously evolving nature of AIV, and technical vaccination issues, such as ineffective application or insufficient coverage, with poor antigenic match of the vaccine with the field strains (85). Furthermore, this strategy is only capable of detecting virus infection in a naïve host placed in a vaccinated flock, with no direct indication of live virus infection in the vaccinated host itself. This decisively dismisses it from being an option for a long-term application for surveillance purposes.

DIVA vaccine-based strategies: recombinant subunit and heterologous NA

For DIVA vaccines approach, multiple studies have demonstrated the effectiveness of recombinant vaccine strategies in providing the necessary protection against clinical signs, and fulfilling its role for DIVA purposes (Table 2). However, the fowlpox-HA (H7) vaccine was found to show a reduced protection in chicken that have been previously vaccinated or infected with fowlpox virus (18). Host range restriction may also apply for a particular virus vector such as observed for the infectious laryngotracheitis virus (ILTV) as it replicates poorly in turkeys (106). Nevertheless, mass administration and multiple diseases vaccination options offered by the recombinant vaccines highlight the feasible application of recombinant vaccines, as evidenced by the continuous development and application of this particular strategy.

Following the introduction of heterologous NA vaccination application in Italy (23), various combinations of HA and NA proteins have been tested and recommended, including the use of rare NA subtypes for vaccine development such as N5 and N8 (Table 2) (10,22). Introduction of the eight-plasmid reverse genetics system, which allows rapid de novo generation of reassortant live virus, has made it possible for the rapid availability of a heterologous vaccine once the NA subtype of the wild-type circulating virus is known (10,86). Nevertheless, a collection of vaccine with various combinations is necessary to ensure swift implementation in case of outbreak where multiple virus subtypes are present in a single host or population (144).

Since the conventional diagnostic tests are not applicable for the heterologous NA approach, companion tests specific for this strategy, iIFAT have been developed (23). Although the test is highly specific and sensitive for application (24), iIFAT is also time-consuming and a labor intensive assay, as it is with the classical NI test (9,23). It has been suggested that these NA-based tests be replaced with a faster, simpler and higher throughput ELISA-based screening system, such as the N2-specific ELISA-based test (74) and truncated-N1-specific ELISA (174). Alternatively, a modified version of the NI test is made available where MUN (2′-[4-methylumbelliferyl]-α-D-N-acetylneuraminic acid sodium salt hydrate) was used as the NA substrate in place of the traditional fetuin-based NI test, providing a more rapid analysis and quantitative results where the antibody responses can be measured over time (8). Recent developments have revealed a range of refinements on the available known tests (NI and ELISA) (8,165). However, due to the need for the production of both vaccine and its tailor-made companion test for an optimized performance, limited availability of facilities and resources are the major drawbacks for this particular strategy. Most importantly, in dealing with H5N1 endemic countries, homologous strain is a much preferred option for vaccination as heterologous NA is not an ideal strategy to apply given the diverse genetic variants of H5N1 (27,50,52).

DIVA test-based strategies: NS1, M2e, and HA2 proteins

DIVA tests based on NS1, M2e, and HA2 proteins are viewed more favorably in terms of their practicality (Table 1). These strategies offer a more straightforward approach in comparison to the subunit and the heterologous NA vaccination strategies, where the DIVA test strategy complements the conventional homologous inactivated vaccine administration. Although studies have shown that the presence of HA protein in a vaccine is enough to provide good protection against live virus infection, in most cases it only reduces the clinical signs, and AIV is still shed in the feces of infected birds (141,142). Virus shedding could be in low amount, but the silent spread (asymptomatic) of viral infection is still possible due to the generation of escape mutants in response to vaccination pressure (84). Taken together, homologous strain vaccination still by far provides the most optimum protection against virus infection, as antigenic relatedness is a significant factor in determining the level of protection induced by vaccination (87,142).

NS1 protein is highly conserved among AIV subtypes, which is a highly favorable diagnostic property (156,165,177). However, several studies have identified that the NS1 protein also exists in truncated forms in nature (39,93,135), giving rise to concerns that this could affect the overall accuracy of NS1 DIVA test. Also, different level of species susceptibility to AIV infection should be taken into consideration before NS1 DIVA test is adopted for routine use. A study in turkey showed that the NS1 antibodies were only present for a short time following infection (10 days postchallenged). AIV with a low replication capability in a specific host, either due to low virus adaptability or due to host vaccinal immunity will not be able to produce detectable level of NS1 antibodies despite infection (7,38,128,149). Similar observation can also be resulted due to the poor immunogenicity of NS1 protein as reported in a challenge study in chicken (7).

This strategy also suffers from decreasing specificity with increasing number of vaccination. Low amount of NS1 antibodies were detected in chicken after three times of vaccination with the killed virus contributing to nonspecific reactions in the tests, thought to be due to antibody response against leftover NS1 proteins present in the unpurified vaccine (128,156,177). This shortcoming, however, suggested to be eliminated through the use of vaccination virus with truncated NS1, which remove the possibility of NS1 antibodies detection in vaccinated hosts (150,156). Studies on the truncated NS1 protein (10 nucleotides deletion in the middle of the NS1 protein-coding sequence) demonstrated its capability of providing protective host immunity after influenza virus challenge in mouse, pig, and horse models (112,113,166). This has raised the possibility of developing live attenuated virus as vaccine while retaining the capacity of NS1 protein as DIVA marker, although the reversion of the live-attenuated virus to virulent virus is a concern (166). This was later vindicated by a study on live mutant NS1 AIV showing its reversion to virulence after five back passages in chicken, thus suggesting that a killed vaccine made from a mutant virus with shorter NS1 gene is much safer and practical for DIVA application (17). Following the occasional detection of NS1 protein antibodies in vaccinated chicken, the NS1-ELISA was suggested to be more suitable for flock monitoring rather than individual birds diagnosis (149,165).

M2e DIVA strategy on the other hand has issues on its specificity and immunogenicity of the M2e antigen. Nonspecificity in the recombinant M2e-ELISA was identified to be caused by test serum reactions against the carrier protein used in the M2e expression system (56). Although this was not observed in the ELISA system employing synthetic M2e-peptide, the use of recombinant-M2e protein is much preferred as the latter offers a much lower cost for higher output, with continuous access for use in large-scale screening (56).

Concerns have also been raised where undetectable levels of seroconversion in infected animals may lead to false negative results in M2e-based ELISA. Previous findings indicated that M2e is a weak immunogen (101), where AIV infections (H1N1 and H3N2, respectively) in mice and humans have engendered poor M2e-specific antibody responses (42). A low M2e-antibody response was also observed after a primary infection in pigs with H3N2 or H1N1, but it was significantly increased following challenge infection using H1N1 (55). This is hypothesized to be contributed by the small size of the M2e antigenic determinant, which limits the number of M2e-reactive B cells for antibody secretion. This is further exacerbated by the antigenic competition posed by the much higher population of HA and NA proteins on the virus surface particle (42).

However, in a challenged duck study by Lambrecht, Steensels, Van Borm, Meulemans, van den Berg (81), a decreasing trend of M2e antibodies level was reported with the increasing number of vaccinations. Increased immunity established by vaccination was assumed to reduce efficient virus replication, hence influencing development of M2e antibody, which in turn affected test sensitivity. False negative results have been observed by Kim, Choi, Kwon, Kang, Paek, Jeong, Kwon, Lee (70) where low level of M2e-antibodies was detected despite a H9N2 challenge in chicken vaccinated twice.

Nevertheless, attempts to address these issues have been demonstrated through the improvement in the M2e-ELISA detection efficiency by incorporation of multiple repeats of the M2e protein in the recombinant-M2e-ELISA system (53,151). Otherwise, DIVA application based on M2e protein is proven to have a wide range of reactivity against other IAV subtypes in chicken (56).

HA2 peptides were first demonstrated as antigen for H5N1 serodiagnosis using ELISA by Khurana, Sasono, Fox, Nguyen, Le, Pham, Nguyen, Nguyen, Horby, Golding (68) following identification of one immunodominant epitope through a complete antibody repertoire characterization of H5N1 infection in humans (69). Although HA2-specific antibodies have been reported in natural infection in both humans and mice, HA2 is a weak natural immunogen (129). As observed for the M2e protein DIVA strategy, this factor may also lead to false negative results for the HA2 gp-based antibody detection due to low seroconversion in infected hosts. However, this approach warrants further study to validate this assumption and to overcome this limitation, as otherwise it offers specificity and universality for surveillance purposes.

Recommendations for DIVA Programs

For AIV successful monitoring program, DIVA vaccine needs to be (i) effective, (ii) readily distinguishable from the wild-type virus, (iii) rapidly available, (iv) cost effective, and ideally (v) applicable by mass administration (by spraying or drinking water); along with companion diagnostic tests or DIVA test that are (i) simple and rapid, (ii) suitable for mass screening, (iii) highly sensitive and specific, and (iv) low cost.

In general, DIVA vaccines (subunit, recombinant, and heterologous vaccines), which have been described in the previous section, showed high efficiency in providing optimal protection against AIV infection and capable of DIVA application. Factors affecting vaccine effectiveness such as vaccine strain and target species have to be critically considered to ensure maximum vaccine coverage. Close monitoring of field virus is vital especially where AIV is endemic as continuous infection and circulation of virus promotes immune pressure, thus drifting off the field virus from vaccine seed virus (143). Availability of vaccine supply particularly in AIV endemic countries should be well managed and maintained as vaccine production is a time consuming process despite its relatively short shelf life (about 2 years) (96). AIV endemic countries usually possess high poultry density, thus cost effectiveness is a critical factor in decision making, which is why advanced vaccines with mass applicability have highly favorable features.

By far, ELISA–based diagnostic test is highly recommended for surveillance and monitoring purposes. However, to ensure the robustness of a DIVA test, field trials using both LPAIV and HPAIV challenge strains still need to be explored in various poultry species model since previous findings have demonstrated that test sensitivity varies between challenge strain and bird species used. Epitope mapping of the DIVA antigens will be an interesting venue to explore as this may aid in scoring a highly sensitive and specific DIVA tool.

Footnotes

Acknowledgments

The authors would like to thank the School of Animal Veterinary Sciences, The University of Adelaide (SAVS-UoA), South Australia, the Australian Centre for International Agricultural Research (ACIAR), the Ministry of Education Malaysia (MoE-Malaysia), and the Institute for Tropical Biology & Conservation, Universiti Malaysia Sabah (ITBC-UMS), Sabah for providing resources and facilities.

Author Disclosure Statement

No competing financial interests exist.

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