Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response.

Influenza neuraminidase (NA) proteins expressed in TK- cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses

Serosurveillance for pandemic influenza A (H1N1) 2009 virus infection in domestic elephants, Thailand.

The present study conducted serosurveillance for the presence of antibody to pandemic influenza A (H1N1) 2009 virus (H1N1pdm virus) in archival serum samples collected between 2009 and 2013 from 317 domestic elephants living in 19 provinces situated in various parts of Thailand. To obtain the most accurate data, hemagglutination-inhibition (HI) assay was employed as the screening test; and sera with HI antibody titers ≥20 were further confirmed by other methods, including cytopathic effect/hemagglutination based-microneutralization (microNT) and Western blot (WB) assays using H1N1pdm matrix 1 (M1) or hemagglutinin (HA) recombinant protein as the test antigen. Conclusively, the appropriate assays using HI in conjunction with WB assays for HA antibody revealed an overall seropositive rate of 8.5% (27 of 317). The prevalence of antibody to H1N1pdm virus was 2% (4/172) in 2009, 32% (17/53) in 2010, 9% (2/22) in 2011, 12% (1/8) in 2012, and 5% (3/62) in 2013. Notably, these positive serum samples were collected from elephants living in 7 tourist provinces of Thailand. The highest seropositive rate was obtained from elephants in Phuket, a popular tourist beach city. Young elephants had higher seropositive rate than older elephants. The source of H1N1pdm viral infection in these elephants was not explored, but most likely came from close contact with the infected mahouts or from the infected tourists who engaged in activities such as elephant riding and feeding. Nevertheless, it could not be excluded that elephant-to-elephant transmission did occur

Molecular characterization and geographical distribution of Zika virus worldwide from 1947 to 2022

Objectives: We conducted molecular characterization, demonstrated the geographical distribution of Zika virus (ZIKV) circulating worldwide from 1947 to 2022 and explored the potential genetic recombination site in the Thailand ZIKV genomes. Methods: We constructed phylogenetic trees based on ZIKV coding sequences (CDS) and determined the geographical distribution of the representative viruses by genetic relationship and timeline. We determined genetic recombination among ZIKV and between ZIKV and other flaviviruses using similarity plot and bootscan analyzes, together with the phylogeny encompassing the CDS and eight subgenomic regions. Results: The phylogenetic trees comprising 717 CDS showed two distinct African and Asian lineages. ZIKV in the African lineage formed two sublineages, and ZIKV in the Asian lineage diversified into the Asian and American sublineages. The 1966 Malaysian isolate was designated the prototype of the Asian sublineage and formed a node of only one member, while the newer viruses formed a distinct node. We detected no genetic recombination in the Thailand ZIKV. Conclusion: Five Thailand isolates discovered in 2006 were the second oldest ZIKV after the Malaysian prototype. Our result suggested two independent routes of ZIKV spread from Southeast Asia to Micronesia in 2007 and French Polynesia in 2013 before further spreading to South American countries

Prevalence of viral load suppression and acquired drug resistance among people living with HIV in Nepal: a nationally representative surveillance study

ABSTRACT: Objectives: This is the first nationally representative study to estimate the prevalence of viral load (VL) suppression and acquired HIV drug resistance (ADR) among people living with HIV (PLHIV) in Nepal. Methods: A cross-sectional study recruited 1418 PLHIV from 20 ART centres in Nepal, using a two-stage cluster design. Participants were eligible if they were HIV-positive individuals on ART for 9–15 months or at least 48 months. Plasma specimens were collected and tested for the quantification of HIV-1 RNA. Specimens with a VL ≥1000 copies/mL were further processed for sequencing of PR and RT genes of HIV-1. The sequences were then analysed to detect mutations causing HIV drug resistance. Results: The prevalence of ADR was 3.7% (95% confidence interval [CI]: 1.8–7.6) and 3.0% (95% CI: 1.8–5.2) among PLHIV who received ART for 9–15 months and 48 months or more, respectively. The prevalence of VL suppression was 95.3% (95% CI: 91.7–97.4) among those on ART for 9–15 months, and 96.5% (95% CI: 94.7–97.7) among those on ART for at least 48 months. The prevalence of any detectable acquired resistance to antiretroviral drugs was 80.7% (95% CI: 58.6–92.5) among those on ART for 9–15 months with VL ≥1000 copies/mL and 81.6% (95% CI: 55.4–94.0) among those on ART for at least 48 months with VL ≥1000 copies/mL. Conclusion: This study suggests that improved accessibility to VL monitoring and timely assessment of drug resistance in routine HIV programs are crucial in Nepal to ensure access to HIV treatment for all in need

Molecular weights of NA proteins as demonstrated by WB assay.

<p>(A) The MW of rVac-H5N1 NA is compared with those NA synthesized in MDCK cells infected with wild type KAN-1 virus and the commercial recombinant H5N1 NA produced in mouse myeloma cells (NS0) (r-H5N1 NA). (B) The MW of rVac-H1N1 NA is compared with those NA synthesized in MDCK cells infected with wild type TH 104 virus and recombinant NA of A/California/04/2009 (H1N1) produced in insect cells (r-H1N1 NA).</p

Cellular localization of NA proteins.

<p>The NA protein expressed in TK⁻ cells infected with rVac-H5N1 NA or rVac-H1N1 NA are present in cytoplasm, and in particular, on the cytoplasmic membrane as demonstrated by IFA confocal microscopy. TK⁻ cells infected with rVac-pSC11 as the virus control did not have a fluorescent signal.</p

Phylogenetic tree of influenza virus <i>NA</i> constructed by neighbor-joining using MEGA 5 software.

<p>NA amino acid sequences of the H1N1pdm viruses and H5N1 viruses share the same node of origin, while H3N2 viruses are clustered in distinct node.</p

Binding efficiency of protein A/G-HRP to elephant serum Ig.

<p>(A) Under non-reducing conditions: protein A/G conjugate binds elephant IgG molecule at the MW of 150 kDa; (B) Under reducing conditions: protein A/G conjugate binds both H chain and L chain of elephant IgG with MW of 50 and 25 kDa, respectively (left panel). These MWs are identical to those of human IgG which reacts with HRP conjugated-goat anti-human IgG, IgM, IgA (H+L) and shows various bands of proteins with different MWs, suggesting of H chains and L chains of different Ig classes (right panel). The result also demonstrated that H chain, but not L chain of human Ig, binds protein A/G efficiently (middle panel).</p

Glycosylation of NA proteins.

<p>MWs of the cleavage products of NA protein after treatment with Peptide <i>N</i>-Glycosidase F (PNGase F) in comparison with the size of the untreated NA as demonstrated by WB assay. (A) H1N1pdm NA; (B) H5N1 NA.</p

Geographical distribution of domestic elephants infected with H1N1pdm virus in Thailand.

<p>Geographical distribution of domestic elephants infected with H1N1pdm virus in Thailand.</p