Detection of Hybrid Fusion Transcripts, Aberrant Transcript Expression, and Specific Single Nucleotide Variants in Acute Leukemia and Myeloid Disorders with Recurrent Gene Rearrangements.

INTRODUCTION: A variety of gene rearrangements and molecular alterations are key drivers in the pathobiology of acute leukemia and myeloid disorders; current classification systems increasingly incorporate these findings in diagnostic algorithms. Therefore, clinical laboratories require versatile tools, which can detect an increasing number and variety of molecular and cytogenetic alterations of clinical significance. METHODS: We validated an RNA-based next-generation sequencing (NGS) assay that enables the detection of: (i) numerous hybrid fusion transcripts (including rare/novel gene partners), (ii) aberrantly expressed EVI1 (MECOM) and IKZF1 (Del exons 4-7) transcripts, and (iii) hotspot variants in KIT, ABL1, NPM1 (relevant in the context of gene rearrangement status). RESULTS: For hybrid fusion transcripts, the assay showed 98-100% concordance for known positive and negative samples, with an analytical sensitivity (i.e., limit of detection) of approximately 0.8% cells. Samples with underlying EVI1 (MECOM) translocations demonstrated increased EVI1 (MECOM) expression. Aberrant IKZF1 (Del exons 4-7) transcripts detectable with the assay were also present on orthogonal reverse transcription PCR. Specific hotspot mutations in KIT, ABL1, and NPM1 detected with the assay showed 100% concordance with orthogonal testing. Lastly, several illustrative samples are included to highlight the assay's clinically relevant contributions to patient workup. CONCLUSION: Through its ability to simultaneously detect various gene rearrangements, aberrantly expressed transcripts, and hotspot mutations, this RNA-based NGS assay is a valuable tool for clinical laboratories to supplement other molecular and cytogenetic methods used in the diagnostic workup and in clinical research for patients with acute leukemia and myeloid disorders.

Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site.

UNLABELLED: Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011. During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition. IMPORTANCE: Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for severe outbreaks in both commercial and backyard poultry, causing considerable economic losses and regular zoonotic transmissions to humans. Vaccination is used increasingly to reduce the burden of HPAI H5N1 virus in poultry. Influenza viruses can escape from recognition by antibodies induced upon vaccination or infection through genetic changes in the hemagglutinin protein. The evolutionary patterns and molecular basis of antigenic change in HPAI H5N1 viruses are poorly understood, hampering formulation of optimal vaccination strategies. We have shown here that HPAI H5N1 viruses in Indonesia diversified into multiple antigenic variants, that antigenic differences were due to one or a very few substitutions near the receptor binding site, and that the molecular basis for antigenic change was remarkably similar to that for seasonal human influenza viruses. These findings have consequences for future vaccination and surveillance considerations and contribute to the understanding of the antigenic evolution of influenza viruses.

MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies.

Recent gain-of-function studies in influenza A virus H5N1 strains revealed that as few as three-amino-acid changes in the hemagglutinin protein confer the capacity for viral transmission between ferrets. As transmission between ferrets is considered a surrogate indicator of transmissibility between humans, these studies raised concerns about the risks of gain-of-function influenza A virus research. Here we present an approach to strengthen the biosafety of gain-of-function influenza experiments. We exploit species-specific endogenous small RNAs to restrict influenza A virus tropism. In particular, we found that the microRNA miR-192 was expressed in primary human respiratory tract epithelial cells as well as in mouse lungs but absent from the ferret respiratory tract. Incorporation of miR-192 target sites into influenza A virus did not prevent influenza replication and transmissibility in ferrets, but did attenuate influenza pathogenicity in mice. This molecular biocontainment approach should be applicable beyond influenza A virus to minimize the risk of experiments involving other pathogenic viruses.

Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses.

Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza.

Partial and full PCR-based reverse genetics strategy for influenza viruses.

Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus de novo. To overcome some of these shortcomings we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice is assembled into a RG competent unit by virtue of overlapping PCR reactions containing a cDNA copy of the viral gene segment under the control of RNA polymerase I promoter (pol1) and termination (t1) signals - herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids.

Isolation and characterization of an H9N2 influenza virus isolated in Argentina.

As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) - herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale.

Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters.

Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses.

Variations in the hemagglutinin of the 2009 H1N1 pandemic virus: potential for strains with altered virulence phenotype?

A novel, swine-origin influenza H1N1 virus (H1N1pdm) caused the first pandemic of the 21st century. This pandemic, although efficient in transmission, is mild in virulence. This atypical mild pandemic season has raised concerns regarding the potential of this virus to acquire additional virulence markers either through further adaptation or possibly by immune pressure in the human host. Using the mouse model we generated, within a single round of infection with A/California/04/09/H1N1 (Ca/04), a virus lethal in mice--herein referred to as mouse-adapted Ca/04 (ma-Ca/04). Five amino acid substitutions were found in the genome of ma-Ca/04: 3 in HA (D131E, S186P and A198E), 1 in PA (E298K) and 1 in NP (D101G). Reverse genetics analyses of these mutations indicate that all five mutations from ma-Ca/04 contributed to the lethal phenotype; however, the D131E and S186P mutations--which are also found in the 1918 and seasonal H1N1 viruses-in HA alone were sufficient to confer virulence of Ca/04 in mice. HI assays against H1N1pdm demonstrate that the D131E and S186P mutations caused minor antigenic changes and, likely, affected receptor binding. The rapid selection of ma-Ca/04 in mice suggests that a virus containing this constellation of amino acids might have already been present in Ca/04, likely as minor quasispecies.

Intranasal delivery of an IgA monoclonal antibody effective against sublethal H5N1 influenza virus infection in mice.

Highly pathogenic avian H5N1 influenza viruses are endemic in poultry in Asia and pose a pandemic threat to humans. Since the deployment of vaccines against a pandemic strain may take several months, adequate antiviral alternatives are needed to minimize the effects and the spread of the disease. Passive immunotherapy is regarded as a viable alternative. Here, we show the development of an IgA monoclonal antibody (DPJY01 MAb) specific to H5 hemagglutinin. The DPJY01 MAb showed a broad hemagglutination inhibition (HI) profile against Asian H5N1 viruses of clades 0, 1.0, 2.1, 2.2, and 2.3 and also against H5 wild bird influenza viruses of the North American and Eurasian lineages. DPJY01 MAb displayed also high neutralization activity in vitro and in vivo. In mice, DPJY01 MAb provided protection via a single dose administered intranasally before or after inoculation with a sublethal dose of H5N1 viruses of clades 1.0 and 2.2. Pretreatment with 50 mg of DPJY01 MAb kg of body weight at either 24, 48, or 72 h before highly pathogenic H5N1 virus (A/Vietnam/1203/2004 [H5N1]) inoculation resulted in complete protection. Treatment with 50 mg/kg at either at 24, 48, or 72 h after H5N1 inoculation provided 100%, 80%, and 60% protection, respectively. These studies highlight the potential use of DPJY01 MAb as an intranasal antiviral treatment for H5N1 influenza virus infections.