A reporter system for assaying influenza virus RNP functionality based on secreted Gaussia luciferase activity

<p>Abstract</p> <p>Background</p> <p>Influenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. Viral ribonucleoprotein (vRNP) was involved in genome replication, transcription and host adaptation. Currently, firefly luciferase (Fluc) reporter system was used in vRNP functional assay. However, its limitation for the testing by virus infection resulted in an increased need for rapid, sensitive, and biosafe techniques. Here, an influenza A virus UTR-driven gene reporter for vRNP assay based on secreted <it>Gaussia </it>luciferase (Gluc) activity was evaluated.</p> <p>Results</p> <p>By measuring Gluc levels in supernatants, reporter gene activity could be detected and quantitated after either reconstitution of influenza A virus polymerase complex or viral infection of 293T and A549 cells, respectively. As compared with Fluc reporter, Gluc-based reporter was heat-tolerant (65°C for 30 min) and produced 50-fold higher bioluminescent activity at 24 h posttransfection. Signals generated by Gluc reporter gene could be detected as early as 6 h post-infection and accumulated with time. Testing by viral infection, stronger signals were detected by Gluc reporter at a MOI of 0.001 than that of 1 and the effects of PB2-627K/E or amantadine on influenza vRNP activity were elucidated more effectively by the Gluc reporter system.</p> <p>Conclusions</p> <p>This approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.</p

Mutations in Polymerase Genes Enhanced the Virulence of 2009 Pandemic H1N1 Influenza Virus in Mice

Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis

International laboratory comparison of influenza microneutralization assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) influenza viruses by CONSISE

The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HAMNassay protocols to enable better correlation of these assays in the future

A Mechanical Model of Gas Drainage Borehole Clogging under Confining Pressure and Its Application

Drilling in a coal seam that has gas and coal outburst activities is closely related to the discharge of drill cuttings into a borehole. Due to the low effectiveness of slagging, there is a risk that the drilling equipment will be lost if the borehole contains too many drill cuttings, especially when drilling in soft coal seams that suffer from borehole deformation and instability problems. In order to investigate the mechanisms underlying clogged boreholes, a mechanical model is established that considers the confining pressure pi that surrounds a borehole. The characteristics of clogged boreholes, which are affected by parameters such as the clogging segment&rsquo;s length L, the drilling angle &theta; and confining pressure pi, were analyzed. The results show that the dredging pressure has nearly exponential growth as the clogging segment&rsquo;s length L increases and the gravity of the clogging segment reduces the demand for dredging pressure, especially in upward drilling. In downward drilling, the blowing-through pressure increases as the absolute value of the drilling angle increases and will reach a maximum value when the drilling angle &theta;D is in the range of &minus;&pi;/2~0. At the same time, the borehole&rsquo;s confining pressure pi is the dominant factor in borehole clogging. Meanwhile, boreholes with a high confining pressure pi, especially in soft coal seams and coal seams with a coal outburst, constitute a significant risk. Finally, an actual drilling field construction was evaluated and optimized by applying the clogging segment mechanical model. The results show that the drilling depth was improved by 18.5% on average, and the drilling efficiency was improved by 39.7%, in comparison to drilling activities without optimization

Database search for SC_PB2-H357N or PA-A36T mutation in virus isolates from nature.

<p>Polymorphisms of PA-36 and PB2-357 in influenza virus isolates were assessed and compared with our findings. The data shown are the number of sequences of different influenza subtypes in the NCBI database, the number of sequences of identical same amino acid composition as SC_WT and SC_M, and the number of sequences with a different amino acid at the same position (with the amino acids shown in parentheses). SC_M, SC_PB2-H357N/PA-A36T; Others*, subtypes which were not listed above; —, not applicable.</p

Growth properties of recombinant viruses in human (A, B), porcine (C, D) and murine cells (E, F).

<p>Confluent monolayer of A549, PK15 and LA-4 cell lines were inoculated with SC_WT, SC_PA-A36T or SC_PB2-H357N virus at MOI of 0.0001. Culture supernatants were harvested at 12, 24, 48, 60, 72 and 96 hpi at 35(<b>A, C, E</b>) or 39°C (<b>B, D, F</b>), respectively. Virus titers were determined by TCID₅₀ assay using MDCK cells. Results are presented as mean ± SEM and are representative of three determinations. *, °, <i>p</i><0.05, when comparing SC_PA-A36T and SC_PB2-H357N with SC_WT respectively, as determined by a <i>t</i>-test of TCID₅₀ values. **, °°, <i>p</i><0.001, as determined by <i>t</i>-test.</p

Viral RNA polymerase activity of SC_WT, SC_PA-A36T and SC_PB2-H357N in 293T cells cultured at different temperatures.

<p>Luciferase-based minigenome reporter assays were used to measure polymerase activity in 293T cells at 33, 37, or 39°C. Cells were co-transfected with Gluc reporter plasmid and expression plasmids PB1 and NP, PA, and PB2 (WT or PA-A36T, PB2-H357N mutants) to generate different viral RNPs. After culturing at 33, 37, or 39°C for 24 h, <i>Gaussia</i> luciferase production was measured. Results are presented as mean ± SEM and are representative of three determinations. **, <i>p</i><0.001, as determined by <i>t</i>-test.</p

Analysis of viral replication efficiency in the respiratory tracts of mice.

<p>Six-week-old female BALB/c mice (<i>n</i> = 3/group/time-point) were inoculated intranasally with 50 µl containing 10⁴ TCID₅₀ of SC_WT, SC_PA-A36T, and SC_PB2-H357N. Animals were euthanized at 12, 24, 48 and 72 hpi. The right lung of each animal was homogenized in PBS (1 ml) and then centrifuged. Viral titers in the supernatant from lung homogenates were determined by TCID₅₀ assay. Results are presented as mean ± SEM and are representative of three determinations. *, <i>p</i><0.05 and **, <i>p</i><0.001, as determined by <i>t</i>-test.</p