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H-NS is an abundant nucleoid-associated protein in bacteria that globally silences genes, including horizontally-acquired genes related to pathogenesis. Although it has been shown that H-NS has multiple modes of DNA-binding, which mode is employed in gene silencing is still unclear. Here, we report that in H-NS mutants that are unable to silence genes, are unable to form a rigid H-NS nucleoprotein filament. These results indicate that the H-NS nucleoprotein filament is crucial for its gene silencing function and serves as the fundamental structural basis for gene silencing by H-NS and likely other H-NS-like bacterial proteins.

The aim of this study was to verify the influence of the dosage of the gene Ns (simplex or duplex), derived from S. tuberosum subsp. Andigena, on potato resistance to PVS and to evaluate changes in the response of potatoes depending on the method of inoculation used and different cultivation temperatures. The analyses carried out made it possible to distinguish 42 clones in duplex and 8 in simplex form. The analysis showed that the Ns gene, even in simplex form, confers full resistance to PVS. Our results also suggest that an increase in temperature may weaken the resistance response of host plants carrying the Ns gene. In our research, PVS overcame the resistance conferred by the Ns gene at higher temperatures in three tetraploid clones: Ns-II-3, Ns-II-4 and Ns-II-81. These clones were classified as ‘temperature-dependent’ (TD). For clones Ns-II-6, Ns-II-10, Ns-II-40 and Ns-II-43, the increase in temperature had no effect on the resistance response of host plants carrying the Ns gene. These clones were classified as ‘resistant’ (R).

Little is known about the processes that enable influenza A viruses to jump into new host species. Here we show that the non-structural protein1 nucleotide substitution, A374G, encoding the D125G(GAT→GGT) mutation, which evolved during the adaptation of a human virus within a mouse host, activates a novel donor splice site in the non-structural gene, hence producing a novel influenza A viral protein, NS3. Using synonymous 125G mutations that do not activate the novel donor splice site, NS3 was shown to provide replicative gain-of-function. The protein sequence of NS3 is similar to NS1 protein but with an internal deletion of a motif comprised of three antiparallel β-strands spanning codons 126 to 168 in NS1. The NS1-125G(GGT) codon was also found in 33 natural influenza A viruses that were strongly associated with switching from avian to mammalian hosts, including human, swine and canine populations. In addition to the experimental human to mouse switch, the NS1-125G(GGT) codon was selected on avian to human transmission of the 1997 H5N1 and 1999 H9N2 lineages, as well as the avian to swine jump of 1979 H1N1 Eurasian swine influenza viruses, linking the NS1 125G(GGT) codon with host adaptation and switching among multiple species.

Background Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. Results After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/μl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A. , S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. Conclusions We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.

RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82–148) NS RNA region influence NS1 protein expression.

The N gene, a member of the Toll-IL-1 homology region-nucleotide binding site-leucine-rich repeat region (LRR) class of plant resistance genes, encodes two transcripts, NSand NL, via alternative splicing of the alternative exon present in the intron III. The NStranscript, predicted to encode the full-length N protein containing the Toll-IL-1 homology region, nucleotide binding site, and LRR, is more prevalent before and for 3 hr after tobacco mosaic virus (TMV) infection. The NLtranscript, predicted to encode a truncated N protein (Ntr) lacking 13 of the 14 repeats of the LRR, is more prevalent 4-8 hr after TMV infection. Plants harboring a cDNA-NStransgene, capable of encoding an N protein but not an Ntrprotein, fail to exhibit complete resistance to TMV. Transgenic plants containing a cDNA-NS-bearing intron III and containing 3′N-genomic sequences, encoding both NSand NLtranscripts, exhibit complete resistance to TMV. These results suggest that both N transcripts and presumably their encoded protein products are necessary to confer complete resistance to TMV.

Background Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in Muscovy ducks, with the pathogenesis of the virus still unknown in many respects. Specific MDPV detection is often rife with false positive results because of high identity at the genomic nucleotide level and antigenic similarity with goose parvovirus (GPV). The objective of this study was to develop a sensitive, highly specific, and repeatable TaqMan-based real-time PCR (qPCR) assay for facilitating the molecular detection of MDPV. Results The specific primers and probe were designed based on the conserved regions within MDPVs, but there was a variation in GPVs of the nonstructural (NS) genes after genetic comparison. After the optimization of qPCR conditions, the detection limit of this qPCR assay was 29.7 copies/μl. The assay was highly specific for the detection of MDPV, and no cross-reactivity was observed with other non-targeted duck-derived pathogens. Intra- and inter-assay variability was less than 2.21%, means a high degree of repeatability. The diagnostic applicability of the qPCR assay was proven that MDPV-positive can be found in cloacal swabs samples, Muscovy duck embryos and newly hatched Muscovy ducklings. Conclusions Our data provided incidents that MDPV could be possible vertically transmitted from breeder Muscovy ducks to Muscovy ducklings. The developed qPCR assay in the study could be a reliable and specific tool for epidemiological surveillance and pathogenesis studies of MDPV.

From a stock of A/turkey/Oregon/71-delNS1 (H7N3) virus, which has a 10 nucleotide deletion in the coding region of the NS1 gene, we found that several variants with different sizes of NS genes could be produced by passaging the virus in 10- and 14-day-old embryonating chicken eggs (ECE), but not in 7-day-old ECE or Vero cells. We were able to rescue the reassortant virus that has different sizes of the NS genes and confirmed that those NS genes are genetically stable. By conducting in vivo studies in 2-week-old chickens, we found two plaque purified variants (D-del pc3 and pc4) which can be used as a potential live-attenuated vaccine. The variants were highly attenuated in chickens and did not transmit the virus from infected chickens to uninoculated cage mates. At the same time, the variants induced relatively high antibody titers which conferred good protection against a high dose heterologous virus challenge. Our study indicates that naturally selected NS1 deletion variants might be useful in the development of live-attenuated influenza vaccines in poultry. Furthermore, deletion in the NS1 protein can be potentially useful as a negative marker for a differentiating infected from vaccinated animals (DIVA) approach.

More and more H5N1 subtype avian influenza viruses possessing a 15-nucleotide (15-nt) deletion in the viral nonstructural protein (NS) gene from position 263 to 277 have emerged since 2000. In order to investigate the biological significance of this deletion, two pairs of H5N1 reassortants designated as rWSN-SD versus rWSN-mSD and rWSN-YZ versus rWSN-mYZ were generated by reverse genetics technique. These recombinant viruses shared the same inner genes of PB1, PB2, PA, NP, and M from strain A/WSN/33(H1N1) and outer genes of HA and NA from strain A/Duck/Shandong/093/2004 (H5N1) (A/D/SD/04), whereas they bore different NS gene. Recombinant rWSN-SD carried the full sequence NS gene from A/D/SD/04 in the natural state without deletion, whereas rWSN-mSD carried the same NS gene, but with an artificial 15-nt deletion from position 263 to 277. On the other hand, rWSN-YZ contained the NS gene in the natural state with a deletion from A/Duck/Yangzhou/232/2004 (H5N1) (A/D/YZ/04), while rWSN-mYZ bore the same NS gene but with an artificial insertion of 15-nt in site 263-277. All the four reassortants grew well in embryonated chicken eggs with similar mean death time (MDT) and viral titer of EID₅₀ or HA. However, the virulence of these reassortant viruses in chickens and mice was different. Reassortant viruses with deletion in their NS gene (rWSN-mSD and rWSN-YZ) had much higher intraveneous pathogenicity index (IVPI) in chickens and lower MLD₅₀ in mice than their counterparts without the deletion (rWSN-SD and rWSN-mYZ). Furthermore, rWSN-mSD and rWSN-YZ caused significantly more deaths in infected chickens and higher virus titers in tissues of inoculated mice than did rWSN-SD and rWSN-mYZ respectively. Sequence analysis also showed that H5N1 viruses carrying the 15-nt deletion in the NS gene invariably had the D92E shift in their NS1 protein. The results indicated that the 15-nucleotide deletion of NS gene from site 263 to 277 associated with D92E shift in NS1 protein contributes to the virulence increase of H5N1 viruses in chickens and mice.

Background Influenza A virus (IAV) is a segmented negative-sense RNA virus that causes seasonal epidemics and periodic pandemics in humans. Two regions (nucleotide positions 82–148 and 497–564) in the positive-sense RNA of the NS segment fold into a multi-branch loop or hairpin structures. Results We studied 25,384 NS segment positive-sense RNA unique sequences of human and non-human IAVs in order to predict secondary RNA structures of the 82–148 and 497–564 regions using RNAfold software, and determined their host- and lineage-specific distributions. Hairpins prevailed in avian and avian-origin human IAVs, including H1N1 pdm1918 and H5N1. In human and swine IAV hairpins distribution varied between evolutionary lineages. Conclusions These results suggest a possible functional role for these RNA secondary structures and the need for experimental evaluation of these structures in the influenza life cycle.