Identification and characterization of immunoglobulin tau (IgT) in Asian Seabass (Lates calcarifer) and mucosal immune response to nervous necrosis virus.

Mucosal immunity plays a critical role in the protection of teleost fish against infection, but mucosal immunoglobulin of important aquaculture species unique to Southeast Asia remained greatly understudied. In this study, the sequence of immunoglobulin T (IgT) from Asian sea bass (ASB) is described for the first time. IgT of ASB possesses the characteristic structure of immunoglobulin with a variable heavy chain and four CH4 domains. The CH2-CH4 domains and full-length IgT were expressed and CH2-CH4 specific antibody was validated against full-length IgT expressed in Sf9 III cells. Subsequent use of the anti-CH2-CH4 antibody in immunofluorescence staining confirmed the presence of IgT-positive cells in the ASB gill and intestine. The constitutive expression of ASB IgT was characterized in different tissues and in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. The highest basal expression of secretory IgT (sIgT) was observed in the mucosal and lymphoid tissues such as the gills, intestine and head kidney. Following NNV infection, IgT expression was upregulated in the head kidney and mucosal tissues. Moreover, a significant increase in localized IgT was found in gills and intestines of infected fish on day 14 post-infection. Interestingly, a significant increase in NNV-specific IgT secretion was only observed in the gills of the infected group. Our results suggest that ASB IgT may play an important role in the adaptive mucosal immune responses against viral infection and could potentially be adapted as a tool for the evaluation of prospective mucosal vaccines and adjuvants for the species.

Vaccines against Major Poultry Viral Diseases: Strategies to Improve the Breadth and Protective Efficacy.

The poultry industry is the largest source of meat and eggs for human consumption worldwide. However, viral outbreaks in farmed stock are a common occurrence and a major source of concern for the industry. Mortality and morbidity resulting from an outbreak can cause significant economic losses with subsequent detrimental impacts on the global food supply chain. Mass vaccination is one of the main strategies for controlling and preventing viral infection in poultry. The development of broadly protective vaccines against avian viral diseases will alleviate selection pressure on field virus strains and simplify vaccination regimens for commercial farms with overall savings in husbandry costs. With the increasing number of emerging and re-emerging viral infectious diseases in the poultry industry, there is an urgent need to understand the strategies for broadening the protective efficacy of the vaccines against distinct viral strains. The current review provides an overview of viral vaccines and vaccination regimens available for common avian viral infections, and strategies for developing safer and more efficacious viral vaccines for poultry.

Development and translation of a paper-based top readout vertical flow assay for SARS-CoV-2 surveillance.

Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.

Characterization of a Broadly Neutralizing Monoclonal Antibody against SARS-CoV-2 Variants.

The constant mutation of SARS-CoV-2 has led to the emergence of new variants, which call for urgent effective therapeutic interventions. The trimeric spike (S) protein of SARS-CoV-2 is highly immunogenic with the receptor-binding domain (RBD) that binds first to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and is therefore the target of many neutralizing antibodies. In this study, we characterized a broadly neutralizing monoclonal antibody (mAb) 9G8, which shows potent neutralization against the authentic SARS-CoV-2 wild-type (WT), Alpha (B.1.1.7), and Delta (1.617.2) viruses. Furthermore, mAb 9G8 also displayed a prominent neutralizing efficacy in the SARS-CoV-2 surrogate virus neutralization test (sVNT) against the Epsilon (B.1.429/7), Kappa (B.1.617.1), Gamma (P.1), Beta (B.1.351), and Delta Plus (1.617.2.1) RBD variants in addition to the variants mentioned above. Based on our in vitro escape mutant studies, we proved that the mutations V483F and Y489H within the RBD were involved in ACE2 binding and caused the neutralizing evasion of the virus from mAb 9G8. The development of such a cross-reactive neutralizing antibody against majority of the SARS-CoV-2 variants provides an important insight into pursuing future therapeutic agents for the prevention and treatment of COVID-19.

Modification of neutralizing epitopes of hemagglutinin for the development of broadly protective H9N2 vaccine.

The H9N2 avian influenza viruses cause significant economic losses in poultry worldwide and could potentially cause human pandemic. Currently, the available vaccines have limited efficacy due to antigenic drift of H9N2. To improve vaccine efficacy, we developed monovalent vaccine strain via the modification of neutralizing epitopes on hemagglutinin (HA) to broaden the protection against H9N2 viruses. In this study, single and multiple mutation were introduced to amino acid at position 148, 150 (site I) and 183, 186, 188 (site II) on the full-length HA gene of H9N2 strain (A/Hong Kong/33982/2009). These mutant HA constructs were displayed on the baculovirus surface (BacH9), and evaluated for their cross-protective efficacy against H9N2 viruses in a mouse model. Our findings indicate that mice immunized with multiple BacH9 mutant constructs (148-150 183 and 186) induced cross-protective immunity against circulating H9N2 in the viral challenge study and prove to be a promising vaccine candidate for H9N2.

Incorporating G-C Pair-Recognizing Guanidinium into PNAs for Sequence and Structure Specific Recognition of dsRNAs over dsDNAs and ssRNAs.

Recognition of RNAs under physiological conditions is important for the development of chemical probes and therapeutic ligands. Nucleobase-modified dsRNA-binding PNAs (dbPNAs) are promising for the recognition of dsRNAs in a sequence and structure specific manner under near-physiological conditions. Guanidinium is often present in proteins and small molecules for the recognition of G bases in nucleic acids, in cell-penetrating carriers, and in bioactive drug molecules, which might be due to the fact that guanidinium is amphiphilic and has unique hydrogen bonding and stacking properties. We hypothesized that a simple guanidinium moiety can be directly incorporated into PNAs to facilitate enhanced molecular recognition of G-C pairs in dsRNAs and improved bioactivity. We grafted a guanidinium moiety directly into a PNA monomer (designated as R) using a two-carbon linker as guided by computational modeling studies. The synthetic scheme of the PNA R monomer is relatively simple compared to that of the previously reported L monomer. We incorporated the R residue into various dbPNAs for binding studies. dbPNAs incorporated with R residues are excellent in sequence specifically recognizing G-C pairs in dsRNAs over dsDNA and ssRNAs. We demonstrated that the R residue is compatible with unmodified T and C and previously developed modified L and Q residues in dbPNAs for targeting model dsRNAs, the influenza A viral panhandle duplex structure, and the HIV-1 frameshift site RNA hairpin. Furthermore, R residues enhance the cellular uptake of PNAs.

Sequence- And Structure-Specific Probing of RNAs by Short Nucleobase-Modified dsRNA-Binding PNAs Incorporating a Fluorescent Light-up Uracil Analog.

RNAs are emerging as important biomarkers and therapeutic targets. The strategy of directly targeting double-stranded RNA (dsRNA) by triplex-formation is relatively underexplored mainly due to the weak binding at physiological conditions for the traditional triplex-forming oligonucleotides (TFOs). Compared to DNA and RNA, peptide nucleic acids (PNAs) are chemically stable and have a neutral peptide-like backbone, and thus, they show significantly enhanced binding to natural nucleic acids. We have successfully developed nucleobase-modified dsRNA-binding PNAs (dbPNAs) to facilitate structure-specific and selective recognition of dsRNA over single-stranded RNA (ssRNA) and dsDNA regions at near-physiological conditions. The triplex formation strategy facilitates the targeting of not only the sequence but also the secondary structure of RNA. Here, we report the development of novel dbPNA-based fluorescent light-up probes through the incorporation of A-U pair-recognizing 5-benzothiophene uracil (btU). The incorporation of btU into dbPNAs does not affect the binding affinity toward dsRNAs significantly, in most cases, as evidenced by our nondenaturing gel shift assay data. The blue fluorescence emission intensity of btU-modified dbPNAs is sequence- and structure-specifically enhanced by dsRNAs, including the influenza viral RNA panhandle duplex and HIV-1-1 ribosomal frameshift-inducing RNA hairpin, but not ssRNAs or DNAs, at 200 mM NaCl, pH 7.5. Thus, dbPNAs incorporating btU-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.

A Short Chemically Modified dsRNA-Binding PNA (dbPNA) Inhibits Influenza Viral Replication by Targeting Viral RNA Panhandle Structure.

RNAs play critical roles in diverse catalytic and regulatory biological processes and are emerging as important disease biomarkers and therapeutic targets. Thus, developing chemical compounds for targeting any desired RNA structures has great potential in biomedical applications. The viral and cellular RNA sequence and structure databases lay the groundwork for developing RNA-binding chemical ligands through the recognition of both RNA sequence and RNA structure. Influenza A virion consists of eight segments of negative-strand viral RNA (vRNA), all of which contain a highly conserved panhandle duplex structure formed between the first 13 nucleotides at the 5' end and the last 12 nucleotides at the 3' end. Here, we report our binding and cell culture anti-influenza assays of a short 10-mer chemically modified double-stranded RNA (dsRNA)-binding peptide nucleic acid (PNA) designed to bind to the panhandle duplex structure through novel major-groove PNA·RNA2 triplex formation. We demonstrated that incorporation of chemically modified PNA residues thio-pseudoisocytosine (L) and guanidine-modified 5-methyl cytosine (Q) previously developed by us facilitates the sequence-specific recognition of Watson-Crick G-C and C-G pairs, respectively, at physiologically relevant conditions. Significantly, the chemically modified dsRNA-binding PNA (dbPNA) shows selective binding to the dsRNA region in panhandle structure over a single-stranded RNA (ssRNA) and a dsDNA containing the same sequence. The panhandle structure is not accessible to traditional antisense DNA or RNA with a similar length. Conjugation of the dbPNA with an aminosugar neamine enhances the cellular uptake. We observed that 2-5 µM dbPNA-neamine conjugate results in a significant reduction of viral replication. In addition, the 10-mer dbPNA inhibits innate immune receptor RIG-I binding to panhandle structure and thus RIG-I ATPase activity. These findings would provide the foundation for developing novel dbPNAs for the detection of influenza viral RNAs and therapeutics with optimal antiviral and immunomodulatory activities.

Baculovirus Surface Display of Immunogenic Proteins for Vaccine Development.

Vaccination is an efficient way to prevent the occurrence of many infectious diseases in humans. To date, several viral vectors have been utilized for the generation of vaccines. Among them, baculovirus-categorized as a nonhuman viral vector-has been used in wider applications. Its versatile features, like large cloning capacity, nonreplicative nature in mammalian cells, and broad tissue tropism, hold it at an excellent position among vaccine vectors. In addition to ease and safety during swift production, recent key improvements to existing baculovirus vectors (such as inclusion of hybrid promoters, immunostimulatory elements, etc.) have led to significant improvements in immunogenicity and efficacy of surface-displayed antigens. Furthermore, some promising preclinical results have been reported that mirror the scope and practicality of baculovirus as a vaccine vector for human applications in the near future. Herein, this review provides an overview of the induced immune responses by baculovirus surface-displayed vaccines against influenza and other infectious diseases in animal models, and highlights the strategies applied to enhance the protective immune responses against the displayed antigens.

Saffold virus, an emerging human cardiovirus.

Saffold virus (SAFV) is an emerging human cardiovirus that has been shown to be ubiquitous. Initial studies of SAFV focused on respiratory and gastrointestinal infection; however, it has also recently been associated with diverse clinical symptoms including the endocrine, cardiovascular, and neurological systems. Given the systemic nature of SAFV, and its high prevalence, understanding its pathogenicity and clinical impact is of utmost importance. This comprehensive review highlights and discusses recent developments in epidemiology, human pathogenicity, animal, and molecular studies related to SAFV. It also provides detailed insights into the neuropathogenicity of SAFV. We argue that human studies have been confounded by coinfections and therefore require support from robust molecular and animal research. Thereby, we aim to provide foresight into further research to better understand this emerging virus.

Broadening the H5N3 Vaccine Immunogenicity against H5N1 Virus by Modification of Neutralizing Epitopes.

The highly pathogenic avian influenza (HPAI) H5N1 virus remains to be one of the world's largest pandemic threats due to the emergence of new variants. The rapid evolution of new sub-lineages is currently the greatest challenge in vaccine development. In this study, we developed an epitope modified non-pathogenic H5N3 (A/duck/Singapore/97) vaccine for broad protection against influenza H5 subtype. H5N3 hemagglutinin (HA) mutant reassortant viruses with A/Puerto Rico/8/34 (PR8) backbone were generated by mutating amino acids at the 140th loop and 190th α-helix of hemagglutinin. The cross-neutralizing efficacy of reverse genetics-derived H5N3HA (RG-H5N3HA) mutants was confirmed by testing reactivity with reference chicken anti-H5N1 clade 2 virus sera. Furthermore, RG-H5N3HA mutant immunized mice induced cross-neutralizing antibodies and cross-protection against distinct H5N1 viral infection. Our findings suggest that the use of non-pathogenic H5 viruses antigenically related to HPAI-H5N1 allows for the development of broadly protective vaccines and reduces the need for biosafety level 3 (BSL3) containment facilities.

Immunohistochemical insights into Saffold virus infection of the brain of juvenile AG129 mice.

BACKGROUND: Saffold Virus (SAFV) is a human cardiovirus that is suspected of causing infection of the central nervous system (CNS) in children. While recent animal studies have started to elucidate the pathogenesis of SAFV, very little is known about the mechanisms behind it. METHOD: In this study, we attempted to elucidate some of the mechanisms of the pathogenesis of SAFV in the brain of a juvenile mouse model by using immunohistochemical methods. RESULTS: We first showed that SAFV is able to infect both neuronal and glial cells in the brain of 2 week-old AG129 mice. We then showed that SAFV is able to induce apoptosis in both neuronal and glial cells in the brain. Lastly, we showed that SAFV infection does not show any signs of gross demyelination in the brain. CONCLUSION: Overall, our results provide important insights into the mechanisms of SAFV in the brain.

Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells.

The Saffold virus (SAFV) genome is translated as a single long polyprotein precursor and co-translationally cleaved to yield 12 separate viral proteins. Little is known about the activities of SAFV proteins although their homologs in other picornaviruses have already been described. To further support research on functions and activities of respective viral proteins, we investigated the spatio-temporal distribution of SAFV proteins in Vero and HEp-2 cells that had been either transfected with plasmids that express individual viral proteins or infected with live SAFV. Our results revealed that, with the exception of the Leader (L) protein, all viral proteins were localized in the cytoplasm at all the time points assayed. The L protein was found in the cytoplasm at an early time point but was subsequently translocated to the nucleus of HEp-2, but not Vero, cells. This was observed in both transfected and infected cells. Further mutational analysis of L protein revealed that Threonine 58 of the Ser/Thr-rich domain of L protein is crucial for protein trafficking between the cytoplasm and nucleus in HEp-2 cells. These findings contribute to a deeper understanding and stimulate investigation of the differetial cellular responses of HEp-2 cells in comparison to other mammalian cell lines during SAFV infection.

The Pathogenesis of Saffold Virus in AG129 Mice and the Effects of Its Truncated L Protein in the Central Nervous System.

Saffold Virus (SAFV) is a human cardiovirus that has been suggested to cause severe infection of the central nervous system (CNS). Compared to a similar virus, Theiler's murine encephalomyelitis virus (TMEV), SAFV has a truncated Leader (L) protein, a protein essential in the establishment of persistent CNS infections. In this study, we generated a chimeric SAFV by replacing the L protein of SAFV with that of TMEV. We then compared the replication in cell cultures and pathogenesis in a mouse model. We showed that both SAFV and chimeric SAFV are able to infect Vero and Neuro2a cells well, but only chimeric SAFV was able to infect RAW264.7. We then showed that mice lacking IFN-α/ß and IFN-γ receptors provide a good animal model for SAFV infection, and further identified the locality of the infection to the ventral horn of the spine and several locations in the brain. Lastly, we showed that neither SAFV nor chimeric SAFV causes persistence in this model. Overall, our results provide a strong basis on which the mechanisms underlying Saffold virus induced neuropathogenesis can be further studied and, hence, facilitating new information about its pathogenesis.

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge.

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.

Progress toward a universal H5N1 vaccine: a recombinant modified vaccinia virus Ankara-expressing trivalent hemagglutinin vaccine.

BACKGROUND: The rapid evolution of new sublineages of H5N1 influenza poses the greatest challenge in control of H5N1 infection by currently existing vaccines. To overcome this, an MVAtor vector expressing three H5HA antigens A/Vietnam/1203/04, A/Indonesia/669/06 and A/Anhui/01/05 (MVAtor-tri-HA vector) was developed to elicit broad cross-protection against diverse clades by covering amino acid variations in the major neutralizing epitopes of HA among H5N1 subtypes. METHODS: BALB/c mice and guinea pigs were immunized i.m. with 8×107 TCID50/animal of MVAtor-tri-HA vector. The immunogenicity and cross-protective immunity of the MVAtor-tri-HA vector was evaluated against diverse clades of H5N1 strains. RESULTS: The results showed that mice immunized with MVAtor-tri-HA vector induced robust cross-neutralizing immunity to diverse H5N1 clades. In addition, the MVAtor-tri-HA vector completely protected against 10 MLD50 of a divergent clade of H5N1 infection (clade 7). Importantly, the serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector was able to elicit strong cross-clade neutralizing immunity against twenty different H5N1 strains from six clades that emerged between 1997 and 2012. CONCLUSIONS: The present findings revealed that incorporation of carefully selected HA genes from divergent H5N1 strains within a single vector could be an effective approach in developing a vaccine with broad coverage to prevent infection during a pandemic situation.

Cross-protective efficacy of baculovirus displayed hemagglutinin against highly pathogenic influenza H7 subtypes.

The outbreak of human infections with avian-origin H7N9 influenza has raised global concerns about a potential human pandemic. Therefore, the generation of simple and reliable newer vaccines is high priority for pandemic preparedness. In this study, we aimed to develop a recombinant vaccine by expressing HA of H7N9 (A/Shanghai/2/2013) on the surface of baculovirus (BacHA). Further, live or inactive form of BacHA (H7N9) vaccine was immunized twice either intranasally or subcutaneously into mice. The immunogenicity and cross-protective efficacy of the BacHA (H7N9) vaccine was assessed against H7N9 or H7N7 subtype challenge. The results showed that mice immunized subcutaneously with adjuvanted inactive BacHA (H7N9) induced robust cross-neutralizing antibody responses against H7 subtypes (H7N9, H7N7 and H7N3) compared to subcutaneous or intranasal immunization of live BacHA. In contrast, mice immunized intranasally with live BacHA stimulated higher HA-specific mucosal IgA levels in the upper airways, the port of virus entry. Also, intranasal immunization of BacHA of either H7N9 or H7N7 completely protected against 5 MLD50 of both H7N9 and H7N7 infections. An overall study revealed that intranasal administration of HA expressed on the baculovirus envelope is alternative way to prime the immune system against influenza infection during a pandemic situation.

Monovalent H5 vaccine based on epitope-chimeric HA provides broad cross-clade protection against variant H5N1 viruses in mice.

H5N1 HPAI virus continues to be a severe threat for public health, as well as for the poultry industry, due to its high mortality and antigenic drift rate. There is no monovalent vaccine available which provides broad protection against those major circulating strains. In the present study, a monovalent H5 vaccine strain was developed with antigenic sequence analysis and epitope mutations. H5 from Indonesia strain (A/Indonesia/CDC669/2006) was used as backbone sequence. Three amino acids were mutated to express immunogenic epitopes from other circulating H5N1s in the backbone. RG influenza virus expressing the epitope-chimeric H5 can react in HI with multiple H5 monoclonal antibodies which fail to neutralize wild type CDC669. High titers in HI and virus neutralization against different clades H5N1s (clade 1, 2, 4 and 7) were detected using sera from mice immunized with the epitope-chimeric H5N1. The monovalent vaccine with RG-epitope-chimeric H5N1 protected mice from lethal challenge with H5N1s of different clades, including clade 1.0, 2.1, 2.2 and 2.3. This study indicates that the broad immune response elicited by this single H5N1 virus allows it to be a promising candidate for a monovalent H5 universal vaccine.

Recombinant baculovirus displayed vaccine: a novel tool for the development of a cross-protective influenza H5N1 vaccine.

The rapid evolution of new sublineages of H5N1 influenza in Asia poses the greatest challenge in vaccine development for pre-pandemic preparedness. To overcome the antigenic diversity of H5N1 strains, multiple vaccine strains can be designed based on the distribution of neutralizing epitopes in the globular head of H5 hemagglutinin (HA). Recently, we selected two different HAs of H5N1 strains based on the neutralizing epitopes and reactivity with different neutralizing antibodies. The HAs of selected vaccine strains were individually expressed on the baculovirus envelope (bivalent-BacHA) with its native antigenic configuration. Further, oral delivery of live bivalent-BacHA elicited broadly reactive humoral, mucosal and cell-mediated immune responses and showed complete protection against antigenically distinct H5N1 strains in mice. The strategy for the vaccine strain selection, vaccine design and route of administration will provide an idea for development of a widely protective vaccine against highly pathogenic H5N1 for pre-pandemic preparedness.

Development of dual-function ELISA for effective antigen and antibody detection against H7 avian influenza virus.

BACKGROUND: Outbreaks in poultry involving influenza virus from H7 subtype have resulted in human infections, thus causing a major concern for public health, as well as for the poultry industry. Currently, no efficient rapid test is available for large-scale detection of either antigen or antibody of H7 avian influenza viruses. RESULTS: In the present study, a dual function ELISA was developed for the effective detection of antigen and antibody against H7 AIVs. The test was established based on antigen-capture-ELISA and epitope blocking ELISA. The two Mabs 62 and 98 which were exploited in the assay were identified to recognize two conformational neutralizing epitopes on H7 HA1. Both of the epitopes exist in all of the human H7 strains, including the recent H7N9 strain from China and > 96.6% of avian H7 strains. The dual ELISA was able to detect all of the five H7 antigens tested without any cross reaction to other influenza subtypes. The antigen detection limit was less than 1 HA unit of H7. For antibody detection, the sensitivity and specificity of the dual ELISA was evaluated and compared to HI and microneutralization using immunized animal sera to different H7 strains and different subtypes of AIVs. Results indicated that antibodies to H7 were readily detected in immunized animal sera by the dual ELISA whereas specimens with antibodies to other AIVs yielded negative results. CONCLUSIONS: This is the first dual-function ELISA reported for either antigen or antibody detection against H7 AIVs. The assay was highly sensitive and 100% specific in both functions rendering it effective for H7 diagnosis.