Systemic virus distribution and host responses in brain and intestine of chickens infected with low pathogenic or high pathogenic avian influenza virus

<p>Abstract</p> <p>Background</p> <p>Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic (HP), based on virulence in chickens.</p> <p>Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses.</p> <p>Methods</p> <p>To study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains.</p> <p>Results</p> <p>Both H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection.</p> <p>Conclusions</p> <p>Although both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.</p

Substrate specificity of vaccinia virus thymidylate kinase.

International audienceAnti-poxvirus therapies are currently limited to cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], but drug-resistant strains have already been characterized. In the aim of finding a new target, the thymidylate (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed in Escherichia coli after cloning the gene (A48R). Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42% identity with human TMP kinase, the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer, like all known TMP kinases. Initial velocity studies showed that the Km for ATP-Mg2+ and dTMP were 0.15 mm and 20 microM, respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP, dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5-Halogenated dUMP such as 5-iodo-2'-deoxyuridine 5'-monophosphate (5I-dUMP) and 5-bromo-2'-deoxyuridine 5'-monophosphate (5Br-dUMP) were also efficient alternative substrates. Using thymidine-5'-(4-N'-methylanthraniloyl-aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP-induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase