Appearance of reassortant European avian-origin H1 influenza A viruses of swine in Vietnam.

Three subtypes-H1N1, H1N2 and H3N2-of influenza A viruses of swine (IAVs-S) are currently endemic in swine worldwide, but there is considerable genotypic diversity among each subtype and limited geographical distribution. Through IAVs-S monitoring in Vietnam, two H1N2 influenza A viruses were isolated from healthy pigs in Ba Ria-Vung Tau Province, Southern Vietnam, on 2 December 2016. BLAST and phylogenetic analyses revealed that their HA and NA genes were derived from those of European avian-like H1N2 IAVs-S that contained avian-origin H1 and human-like N2 genes, and were particularly closely related to those of IAVs-S circulating in the Netherlands, Germany or Denmark. In addition, the internal genes of these Vietnamese isolates were derived from human A(H1N1)pdm09 viruses, suggesting that the Vietnamese H1N2 IAVs-S are reassortants between European H1N2 IAVs-S and human A(H1N1)pdm09v. The appearance of European avian-like H1N2 IAVs-S in Vietnam marks their first transmission outside Europe. Our results and statistical analyses of the number of live pigs imported into Vietnam suggest that the European avian-like H1N2 IAVs-S may have been introduced into Vietnam with their hosts through international trade. These findings highlight the importance of quarantining imported pigs to impede the introduction of new IAVs-S.

Binding of trans-acting factors to the double-stranded variant surface glycoprotein (VSG) expression site promoter of Trypanosoma brucei.

Trypanosoma brucei evades its host's immune response by utilizing the system of antigenic variation, whereby the organism sequentially expresses antigenically distinct variant surface glycoproteins (VSGs). Actively expressed VSG genes are found in VSG expression sites (ESs), and transcription of these ESs is directed by a small promoter composed of two essential cis-acting elements, the VSG ES promoter upstream element (VUE) and VSG ES promoter downstream element (VDE). Using electrophoretic mobility shift assays, we have identified double-stranded DNA binding activity in bloodstream-form trypanosome nuclear extracts. This activity, the VEP complex, is specific for the VSG ES promoter, and requires the intact sequences of the VUE and VDE in the appropriate spacing. These requirements of VEP Complex formation parallel the requirements for promoter function, suggesting that the VEP complex may be composed of functionally significant trans-acting factors. Furthermore, the requirement of both elements suggests that the binding of factors to the promoter may be cooperative. However, subtly different binding characteristics were observed when we used nuclear extracts derived from procyclic trypanosomes.

A detailed mutational analysis of the VSG gene expression site promoter.

The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. The parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than -67 bp 5' to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region -67/-60 and the region -35/-20, both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite.

RNA polymerase I can mediate expression of CAT and neo protein-coding genes in Trypanosoma brucei.

We show that the ribosomal RNA (rRNA) promoter can efficiently direct expression of protein-coding genes in the parasitic protozoan Trypanosoma brucei. The rRNA promoter was characterized by: (i) point mutations at the rRNA transcription initiation site which completely abolished its promoter function in transient CAT transformation assays; (ii) the alpha-amanitin resistance of transcription of rRNA promoter-neomycin phosphotransferase (neo) genes in stably transformed trypanosomes; and (iii) the nucleolar location of neo RNA, synthesized under the control of the rRNA promoter. The rRNA promoter-derived CAT mRNA required a 3' splice acceptor site and the neo mRNA was trans-spliced and polyadenylated. In situ hybridization revealed neo RNA at the nucleolus in stably transformed trypanosomes in which rRNA promoter-neo constructs were integrated either at a rRNA locus or at a locus for the procyclic acidic repetitive protein (PARP) coding genes. We postulate that trans-splicing, by uncoupling the requirement for transcription of protein-coding genes by RNA polymerase II, allows RNA polymerase I mediated protein-coding gene transcription, presumably because a 5' cap can be transferred to the pre-mRNA by trans-splicing.