Discrimination of the H1N1 and H5N2 Variants of Influenza A Virus Using an Isomeric Sialic Acid-Conjugated Graphene Field-Effect Transistor.

There has been a continuous effort to fabricate a fast, sensitive, and inexpensive system for influenza virus detection to meet the demand for effective screening in point-of-care testing. Herein, we report a sialic acid (SA)-conjugated graphene field-effect transistor (SA-GFET) sensor designed using α2,3-linked sialic acid (3'-SA) and α2,6-linked sialic acid (6'-SA) for the detection and discrimination of the hemagglutinin (HA) protein of the H5N2 and H1N1 viruses. 3'-SA and 6'-SA specific for H5 and H1 influenza were used in the SA-GFET to capture the HA protein of the influenza virus. The net charge of the captured viral sample led to a change in the electrical current of the SA-GFET platform, which could be correlated to the concentration of the viral sample. This SA-GFET platform exhibited a highly sensitive response in the range of 101-106 pfu mL-1, with a limit of detection (LOD) of 101 pfu mL-1 in buffer solution and a response time of approximately 10 s. The selectivity of the SA-GFET platform for the H1N1 and H5N2 influenza viruses was verified by testing analogous respiratory viruses, i.e., influenza B and the spike protein of SARS-CoV-2 and MERS-CoV, on the SA-GFET. Overall, the results demonstrate that the developed dual-channel SA-GFET platform can potentially serve as a highly efficient and sensitive sensing platform for the rapid detection of infectious diseases.

Human genes with codon usage bias similar to that of the nonstructural protein 1 gene of influenza A viruses are conjointly involved in the infectious pathogenesis of influenza A viruses.

Molecular mechanisms of the non-structural protein 1 (NS1) in influenza A-induced pathological changes remain ambiguous. This study explored the pathogenesis of human infection by influenza A viruses (IAVs) through identifying human genes with codon usage bias (CUB) similar to NS1 gene of these viruses based on the relative synonymous codon usage (RSCU). CUB of the IAV subtypes H1N1, H3N2, H3N8, H5N1, H5N2, H5N8, H7N9 and H9N2 was analyzed and the correlation of RSCU values of NS1 sequences with those of the human genes was calculated. The CUB of NS1 was uneven and codons ending with A/U were preferred. The ENC-GC3 and neutrality plots suggested natural selection as the main determinant for CUB. The RCDI, CAI and SiD values showed that the viruses had a high degree of adaptability to human. A total of 2155 human genes showed significant RSCU-based correlation (p < 0.05 and r > 0.5) with NS1 coding sequences and was considered as human genes with CUB similar to NS1 gene of IAV subtypes. Differences and similarities in the subtype-specific human protein-protein interaction (PPI) networks and their functions were recorded among IAVs subtypes, indicating that NS1 of each IAV subtype has a specific pathogenic mechanism. Processes and pathways involved in influenza, transcription, immune response and cell cycle were enriched in human gene sets retrieved based on the CUB of NS1 gene of IAV subtypes. The present work may advance our understanding on the mechanism of NS1 in human infections of IAV subtypes and shed light on the therapeutic options.

Recombinant hemagglutinin glycoproteins provide insight into binding to host cells by H5 influenza viruses in wild and domestic birds.

Clade 2.3.4.4, H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have caused devastating effects across wild and domestic bird populations. We investigated differences in the intensity and distribution of the hemagglutinin (HA) glycoprotein binding of a clade 2.3.4.4 H5 HPAIV compared to a H5 low pathogenic avian influenza virus (LPAIV). Recombinant HA from gene sequences from a HPAIV, A/Northern pintail/Washington/40964/2014(H5N2) and a LPAIV, A/mallard/MN/410/2000(H5N2) were generated and, via protein histochemistry, HA binding in respiratory, intestinal and cloacal bursal tissue was quantified as median area of binding (MAB). Poultry species, shorebirds, ducks and terrestrial birds were used. Differences in MAB were observed between the HPAIV and LPAIV H5 HAs. We demonstrate that clade 2.3.4.4 HPAIV H5 HA has a broader host cell binding across a variety of bird species compared to the LPAIV H5 HA. These findings support published results from experimental trials, and outcomes of natural disease outbreaks with these viruses.

Serial passage in ducks of a low-pathogenic avian influenza virus isolated from a chicken reveals a high mutation rate in the hemagglutinin that is likely due to selection in the host.

A comparative study of the ability of three low-pathogenic avian influenza virus (LPAIV) isolates to be transmitted from duck to duck was performed. Pekin ducks were inoculated with two LPAIV isolates from chickens (A/Ck/PA/13609/93 [H5N2], H5N2-Ck; A/Ck/TX/167280-4/02 [H5N3], H5N3-Ck) and one isolate from a wild bird (A/Mute Swan/ MI/451072/06 [H5N1], H5N1-WB). During the establishment of the passage model, only two viruses (H5N1, H5N2) were able to be transmitted from duck to duck. Transmission of these isolates was dependent on the inoculation dose and route of infection. Analysis of swab samples taken from ducks revealed that the wild-bird isolate, H5N1-WB, was primarily shed via the cloacal route. The chicken isolate, H5N2-Ck, was isolated from cloacal as well as oro-pharyngeal swabs. Analysis of the amino acid sequences of the viral surface glycoproteins showed that the hemagglutinin (HA) of the H5N2-Ck isolate was under a stronger evolutionary pressure than the HA of the H5N1-WB isolate, as indicated by the presence of a larger number of amino acid changes observed during passage. The neuraminidase (NA) of both viruses showed either no (in the case of H5N1-WB) or very few amino acid changes.

Recognition of dual targets by a molecular beacon-based sensor: subtyping of influenza A virus.

A molecular beacon (MB)-based sensor to offer a decisive answer in combination with information originated from dual-target inputs is designed. The system harnesses an assistant strand and thermodynamically favored designation of unpaired nucleotides (UNs) to process the binary targets in "AND-gate" format and report fluorescence in "off-on" mechanism via a formation of a DNA four-way junction (4WJ). By manipulating composition of the UNs, the dynamic fluorescence difference between the binary targets-coexisting circumstance and any other scenario was maximized. Characteristic equilibrium constant (K), change of entropy (ΔS), and association rate constant (k) between the association ("on") and dissociation ("off") states of the 4WJ were evaluated to understand unfolding behavior of MB in connection to its sensing capability. Favorable MB and UNs were furthermore designed toward analysis of genuine genetic sequences of hemagglutinin (HA) and neuraminidase (NA) in an influenza A H5N2 isolate. The MB-based sensor was demonstrated to yield a linear calibration range from 1.2 to 240 nM and detection limit of 120 pM. Furthermore, high-fidelity subtyping of influenza virus was implemented in a sample of unpurified amplicons. The strategy opens an alternative avenue of MB-based sensors for dual targets toward applications in clinical diagnosis.

An RNA aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells.

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. In the present study, we isolated an RNA aptamer specific to the glycosylated receptor-binding domain of the HA protein (gHA1) after 12 cycles of the systematic evolution of ligands by exponential enrichment procedure (SELEX), and we then investigated if the selected aptamer suppresses viral infection in host cells. Nitrocellulose filter binding and enzyme-linked immunosorbent assay (ELISA) experiments revealed that 1 RNA aptamer, HA12-16, bound specifically to the gHA1 protein. Cell viability assay showed that the HA12-16 RNA aptamer suppressed viral infection in host cells by enhancing cell viability. Immunofluorescence microscopic analysis further demonstrated that the HA12-16 RNA aptamer suppresses viral attachment to host cells by neutralizing the receptor-binding site of influenza virus HA. These results indicate that the isolated RNA aptamer can be developed as an antiviral reagent against influenza through appropriate therapeutic formulation.

Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

[Effect of antiviral drugs with various mechanisms of action on morphogenesis of infection caused by extremely pathogenic influenza virus strains in animals].

The effect of meglumine salt of acridonoacetic acid (cycloferon) on the in vivo morphogenesis of influenza infection caused by viruses of different origin (avian, swine and human) and variable susceptibility to antivirals (rimantadine and oseltamivir) has been studied. The administration of cycloferon results in stimulation of the immune response, restriction of the foci of post-influenza pneumonia, and normalization of the structure of respiratory zones independently of the susceptibility or resistance of infectious virus to the drugs. Among virions formed in the lungs of cycloferon-treated mice, prevalence of irregular-shaped virions with defects of surface glycoproteins was observed. The data obtained suggest that cycloferon is a drug with the complex mechanism of activity.

Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain.

Highly pathogenic avian influenza viruses (HPAIV) differ from all other strains by a polybasic cleavage site in their hemagglutinin. All these HPAIV share the H5 or H7 subtype. In order to investigate whether the acquisition of a polybasic cleavage site by an avirulent avian influenza virus strain with a hemagglutinin other than H5 or H7 is sufficient for immediate transformation into an HPAIV, we adapted the hemagglutinin cleavage site of A/Duck/Ukraine/1/1963 (H3N8) to that of the HPAIV A/Chicken/Italy/8/98 (H5N2), A/Chicken/HongKong/220/97 (H5N1), or A/Chicken/Germany/R28/03 (H7N7) and generated the recombinant wild-type and cleavage site mutants. In contrast to the wild type, multicycle replication of these mutants in tissue culture was demonstrated by positive plaque assays and viral multiplication in the absence of exogenous trypsin. Therefore, in vitro all cleavage site mutants resemble an HPAIV. However, in chicken they did not exhibit high pathogenicity, although they could be reisolated from cloacal swabs to some extent, indicating enhanced replication in vivo. These results demonstrate that beyond the polybasic hemagglutinin cleavage site, the virulence of HPAIV in chicken is based on additional pathogenicity determinants within the hemagglutinin itself or in the other viral proteins. Taken together, these observations support the notion that acquisition of a polybasic hemagglutinin cleavage site by an avirulent strain with a non-H5/H7 subtype is only one among several alterations necessary for evolution into an HPAIV.