We have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus from cloned cDNA. In this plasmid-based expression system, viral cDNA is inserted between the ...RNA polymerase I (pol I) promoter and terminator sequences. This entire pol I transcription unit is flanked by an RNA polymerase II (pol II) promoter and a polyadenylation site. The orientation of the two transcription units allows the synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template. This pol I-pol II system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters, presumably in different compartments of the nucleus. The interaction of all molecules derived from the cellular and viral transcription and translation machinery results in the generation of infectious influenza A virus. The utility of this system is proved by the recovery of the two influenza A viruses: A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1). Seventy-two hours after the transfection of eight expression plasmids into cocultured 293T and MDCK cells, the virus yield in the supernatant of the transfected cells was between 2 × 105and 2 × 107infectious viruses per milliliter. We also used this eight-plasmid system for the generation of single and quadruple reassortant viruses between A/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1). Because the pol I-pol II system facilitates the design and recovery of both recombinant and reassortant influenza A viruses, it may also be applicable to the recovery of other RNA viruses entirely from cloned cDNA.
Respiratory infection with highly pathogenic influenza A viruses is characterized by the exuberant production of cytokines and chemokines and the enhanced recruitment of innate inflammatory cells. ...Here, we show that challenging mice with virulent influenza A viruses, including currently circulating H5N1 strains, causes the increased selective accumulation of a particular dendritic cell subset, the tipDCs, in the pneumonic airways. These tipDCs are required for the further proliferation of influenza-specific CD8⁺ T cells in the infected lung, because blocking their recruitment in CCR2⁻/⁻ mice decreases the numbers of CD8⁺ effectors and ultimately compromises virus clearance. However, diminution rather than total elimination of tipDC trafficking by treatment with the peroxisome proliferator-activated receptor-γ agonist pioglitazone moderates the potentially lethal consequences of excessive tipDC recruitment without abrogating CD8⁺ T cell expansion or compromising virus control. Targeting the tipDCs in this way thus offers possibilities for therapeutic intervention in the face of a catastrophic pandemic.
Can we beat influenza? Zhang, Wenqing; Webster, Robert G
Science (American Association for the Advancement of Science),
2017-Jul-14, 2017-07-14, 20170714, Volume:
357, Issue:
6347
Journal Article
Peer reviewed
Open access
For the past 65 years, the Global Influenza Surveillance and Response System (GISRS), coordinated by the World Health Organization (WHO), has engaged in open and efficient sharing of information, ...viruses, and responsibilities. The GISRS's extraordinary longevity can be attributed to several generations of dedicated scientists and to the engagement of over 100 countries, often with limited resources. Currently, only two influenza A viruses and two influenza B clades are circulating and causing disease in humans, but 16 additional subtypes of influenza A viruses are circulating in nature (14 in birds and two in bats). Of the latter, six occasionally infect humans, providing an ever-looming pandemic threat. However, there is still a lack of fundamental knowledge to predict if and when a particular viral subtype will acquire pandemic ability. We therefore still fail to predict influenza pandemics, and this must change.
The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to ...human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.
Toll-like receptors (TLRs) play key roles in innate immune recognition of pathogen-associated molecular patterns of invading microbes. Among the 10 TLR family members identified in humans, TLR10 ...remains an orphan receptor without known agonist or function. TLR10 is a pseudogene in mice and mouse models are noninformative in this regard. Using influenza virus infection in primary human peripheral blood monocyte-derived macrophages and a human monocytic cell line, we now provide previously unidentified evidence that TLR10 plays a role in innate immune responses following viral infection. Influenza virus infection increased TLR10 expression and TLR10 contributed to innate immune sensing of viral infection leading to cytokine induction, including proinflammatory cytokines and interferons. TLR10 induction is more pronounced following infection with highly pathogenic avian influenza H5N1 virus compared with a low pathogenic H1N1 virus. Induction of TLR10 by virus infection requires active virus replication and de novo protein synthesis. Culture supernatants of virus-infected cells modestly up-regulate TLR10 expression in nonvirus-infected cells. Signaling via TLR10 was activated by the functional RNAprotein complex of influenza virus leading to robust induction of cytokine expression. Taken together, our findings identify TLR10 as an important innate immune sensor of viral infection and its role in innate immune defense and immunopathology following viral and bacterial pathogens deserves attention.
The rapid spread of highly pathogenic avian influenza (HPAI) A (H5N1) viruses in Southeast Asia in 2004 prompted the New Zealand Ministry for Primary Industries to expand its avian influenza ...surveillance in wild birds. A total of 18,693 birds were sampled between 2004 and 2020, including migratory shorebirds (in 2004-2009), other coastal species (in 2009-2010), and resident waterfowl (in 2004-2020). No avian influenza viruses (AIVs) were isolated from cloacal or oropharyngeal samples from migratory shorebirds or resident coastal species. Two samples from red knots (Calidris canutus) tested positive by influenza A RT-qPCR, but virus could not be isolated and no further characterization could be undertaken. In contrast, 6179 samples from 15,740 mallards (Anas platyrhynchos) tested positive by influenza A RT-qPCR. Of these, 344 were positive for H5 and 51 for H7. All H5 and H7 viruses detected were of low pathogenicity confirmed by a lack of multiple basic amino acids at the hemagglutinin (HA) cleavage site. Twenty H5 viruses (six different neuraminidase NA subtypes) and 10 H7 viruses (two different NA subtypes) were propagated and characterized genetically. From H5- or H7-negative samples that tested positive by influenza A RT-qPCR, 326 AIVs were isolated, representing 41 HA/NA combinations. The most frequently isolated subtypes were H4N6, H3N8, H3N2, and H10N3. Multivariable logistic regression analysis of the relations between the location and year of sampling, and presence of AIV in individual waterfowl showed that the AIV risk at a given location varied from year to year. The H5 and H7 isolates both formed monophyletic HA groups. The H5 viruses were most closely related to North American lineages, whereas the H7 viruses formed a sister cluster relationship with wild bird viruses of the Eurasian and Australian lineages. Bayesian analysis indicates that the H5 and H7 viruses have circulated in resident mallards in New Zealand for some time. Correspondingly, we found limited evidence of influenza viruses in the major migratory bird populations visiting New Zealand. Findings suggest a low probability of introduction of HPAI viruses via long-distance bird migration and a unique epidemiology of AIV in New Zealand.
Millennia of human conflict with wildlife have built a culture of intolerance toward wildlife among some stakeholders. We explored 2 key obstacles to improved human–wildlife coexistence: coexistence ...inequality (how the costs and benefits of coexisting with wildlife are unequally shared) and intolerance. The costs of coexisting with wildlife are often disproportionately borne by the so‐called global south and rural communities, and the benefits often flow to the global north and urban dwellers. Attitudes and behaviors toward wildlife (tolerance versus intolerance) vary with social and cultural norms. We suggest more empathetic advocacy is needed that, for example, promotes conservation while appropriately considering those who bear the costs of conflict with wildlife. To achieve more equitable cost‐sharing, we suggest limiting the costs incurred by those most affected or by sharing those costs more widely. For example, we advocate for the development of improved wildlife compensation schemes, increasing the scale of rewilding efforts, and preventing wildlife‐derived revenue leaching out of the local communities bearing the costs of coexistence.
Soluciones para la Desigualdad y la Intolerancia en la Coexistencia Humano – Fauna
Resumen
Los milenios de conflicto entre los humanos y la fauna han construido una cultura de intolerancia hacia la fauna entre algunos actores. Exploramos dos obstáculos importantes para la mejora de la coexistencia humano – fauna: la desigualdad de coexistencia (cómo los costos y los beneficios de la coexistencia con la fauna están compartidos de una manera desigual) y la intolerancia. Los costos de coexistir con la fauna generalmente están asumidos de manera desproporcional por las llamadas comunidades del sur global o rurales, y los beneficios de convivir con la fauna generalmente fluyen hacia el norte mundial y hacia los habitantes de zonas urbanas. Las actitudes y comportamientos hacia la fauna (tolerancia versus intolerancia) varían con las normas culturales y sociales. Sugerimos la necesidad de una defensa más empática que, por ejemplo, promueva la conservación a la vez que considera de manera apropiada a aquellos que asumen los costos del conflicto con la fauna. Para lograr costos compartidos más equitativos sugerimos limitar los costos incurridos por aquellos más afectados o compartir los costos de manera más amplia. Por ejemplo, abogamos por el desarrollo de esquemas mejorados de compensación de fauna, el incremento de la escala de los esfuerzos por el retorno a la vida silvestre y la prevención del secuestro de ingresos derivados de la fauna fuera de las comunidades locales que asumen los costos de la coexistencia.
Article Impact Statement: Reduce coexistence inequality and intolerance by prioritising local economies, smarter compensation schemes, rewilding and empathetic advocacy.
We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased ...nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.
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