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As guppies are one of the commercially important fish in freshwater ornamental aquaculture industry, it is important to gain an understanding of guppy immune response for infectious disease control. Till now, the number of study that examined the immune response of guppies is limited and effective tools for monitoring guppy antibody have not been reported. In this study, we successfully isolated guppy IgM using mannan-binding protein (MBP) affinity chromatography and produced specific polyclonal antibodies against guppy IgM heavy and light chains, that showed a molecular weight of approximately 74 and 23 kDa respectively. The produced polyclonal antibodies were used to develop an enzyme-linked immunosorbent assay (ELISA) and have demonstrated to be an effective tool for the detection and quantification of antigen-specific antibody of guppies immunized with Pseudomonas fluorescens. In conclusion, the produced anti-guppy IgM polyclonal antibodies should prove its future implications for immunology and epidemiology studies in guppies.

Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype has caused devastating damage to poultry flocks and sporadic human H5N1 infections. There is concern that this virus subtype may gain transmissibility and become pandemic. Rapid diagnosis and surveillance for H5N1 subtype viruses are critical for the control of H5N1 infection. In this study, we report a robust antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) based on H5- and N1-specific monoclonal antibodies (MAbs) for the rapid detection of H5N1 subtype viruses. The H5 hemagglutinin (HA)-specific MAb (2D9) targets a conformational epitope which recognized multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8. The N1 neuraminidase (NA)-specific MAb (8H12) recognized a linear epitope comprising the sequence AELPF. This epitope was 99% conserved in the NA of 708 analyzed H5N1 viruses, while the epitope was absent in NAs of subtypes N2 through N9. The specificity of the AC-ELISA was examined by using 41 H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. The estimated detection limit was 1 to 2 HA titers. It is concluded that this H5N1 AC-ELISA can simultaneously detect H5 and N1 subtype antigens, eliminating the need for secondary testing for the NA subtype. Implementation of this assay in ELISA-like formats suitable for field use, such as dot ELISA, immunofiltration, or electrochemical biosensor technologies, would provide dual on-site detection of H5 and N1 in clinical or environmental specimens.

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.

Neuraminidase (NA) inhibitors are a class of antivirals designed to target the conserved residues of the influenza NA active site. While there are many conserved residues in the NA active site that are involved in NA inhibitor binding, only a few have been demonstrated to confer resistance. As such, little is known regarding the potential of the other conserved residues in the NA active site to cause NA inhibitor resistance. Two conserved residues (E227 and E276) of an N1 NA that have not previously been associated with resistance to NA inhibitors were investigated. Site-directed mutagenesis was used to generate three alternative amino acids at each residue. Reverse genetics was used to generate recombinant mutant viruses which were characterized for growth, NA activity and NA inhibitor sensitivity. Of the six recombinant viruses expressing NA with mutations at either E227 or E276, only the E227D and E276D viruses were able to grow without supplementary NA activity, and all mutant viruses had a significant reduction in NA activity. The E227D virus demonstrated significantly reduced sensitivity to zanamivir while the E276D virus did not demonstrate any significant changes in NA inhibitor sensitivity. Interestingly, the resistance profiles of E227D and E276D in N1 NA were significantly different from these sites that have been reported for N2 NA. This study confirmed the essential role of NA active site residues in viral fitness, and identified clear differences in the role of residues E227 and E276 in NA inhibitor resistance with N1 and N2 neuraminidases.

BackgroundRecent outbreaks of highly pathogenic H5N1 viruses in humans indicate that no endogenous protection exists in the general population. Vaccination programmes against this new pathogen require synthesis of endogenous antibodies and cannot provide any immediate protection in the event of a pandemic. Passive immunization with humanized neutralizing monoclonal antibodies can prove to be promising in preventing a catastrophic pandemic.MethodsA murine monoclonal antibody (mAb) 3B1 of immunoglobulin M isotype was switched to a chimeric immunoglobulin G1. BALB/c mice were used to study the protective efficacy of the chimeric mAbs against a lethal H5N1 virus challenge with strains from clades 1 and 2.1. Kinetics of the viral load were determined during the course of the treatment.ResultsThe chimeric mAb, in passive administration, was able to protect 100% of the mice when challenged with H5N1 strains from clades 1 or 2.1. Prophylaxis at 1 day prior to challenge and treatment at 1 day after challenge with this mAb resulted in the clearance of the virus from the lungs of the infected mice within 6 days post-viral challenge.ConclusionsPassive immunotherapy using chimeric mAb 3B1 can be an effective tool in both the prophylaxis and treatment of highly pathogenic H5N1 infection, providing the immediate immunity needed to contain a future influenza pandemic.

Background Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. Methods/Principal Findings We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. Conclusions/Significance Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.

Background Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available. Methodology/Principal Findings Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274–281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitope-blocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization. Conclusions/Significance The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera.

Development of effective drugs for the treatment or prevention of epidemic and pandemic influenza is important in order to reduce its impact. Adamantanes and neuraminidase inhibitors are two classes of anti-influenza drugs available for influenza therapy currently. However, emergence of resistance to these drugs has been detected, which raises concerns regarding their widespread use. In this review, resistance to the adamantanes and neuraminidase inhibitors will be discussed in relation to both epidemic and pandemic influenza viruses.

BACKGROUND: Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met. METHODOLOGY/PRINCIPAL FINDINGS: Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA. CONCLUSION/SIGNIFICANCE: The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.