In December 2006, three human specimens were received that were suspected positive for influenza A(H5N1). The specimens were tested using real time PCR. And the presence of A(H5N1) virus was confirmed in 2 patients (16F and 26M), The NA sequence from A(H5N1) positive specimens collected before and after antiviral therapy revealed a mutation (N294S) (N295S according to N1 numbering), previously associated with resistance to oseltamivir. When tested with NA inhibition assays, the two N294S viruses from Egypt exhibited from 57 to 138-fold reduction in susceptibility to oseltamivir, depending on the assay. To our knowledge, this is the first time oseltamivir resistance has been detected in A(H5N1) infecting a human prior to treatment.

A cell-based screening assay was performed to identify compounds that inhibited the postintegration stage of the human immunodeficiency virus (HIV) life cycle. This assay utilized a cell line that contains the HIV gag and pol genes expressed in a Rev-dependent fashion. The cell line produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. Any compound that inhibits a postintegration step in the HIV life cycle scores in this assay by decreasing particle production. Forty thousand compounds were screened, and 192 compounds were selected from the original screen because they showed more than 50% inhibition at a 10 M concentration. The cumulative evidence presented in this study strongly suggests that 2 of the 192 compounds work as inhibitors of HIV Rev function. This was determined by a variety of cell-based assays, although the compounds do not interfere with Rev-RRE (Rev response element) binding in vitro. Both compounds inhibit replication of the lab isolate NL4-3 as well as an HIV primary isolate from Brazil (93BR021) and thus are promising leads as therapeutic candidates that target HIV replication through inhibition of Rev function.Most of the current drugs in use for the treatment of AIDS work by targeting the enzymatic activities of the human immunodeficiency virus (HIV) reverse transcriptase or protease, although entry (7) and integrase (13) inhibitors are starting to be used, and presently there is also promising development of other novel targets (51, 59). However, because of the emergence of drug-resistant virus that commonly occurs as the result of treatment, there remains a great need to continue the search for alternative therapies that target other essential viral activities.The Rev protein is absolutely essential for HIV replication (for a review see reference 49). Proviral clones lacking a functional rev gene have no replicative ability, even in established tissue culture cell lines or peripheral blood mononuclear cells (PBMCs). In the absence of Rev, genomic RNA and several other HIV mRNAs cannot exit the nucleus (22,30,42). Thus, viral structural proteins are not made and the infectious cycle cannot continue. It is thus clear that modalities inhibiting the function of Rev could form the basis for therapy against HIV infection and AIDS.Although the Rev/RRE (Rev response element) export pathway is still not fully understood, several important steps have been identified (see reference 49). The pathway starts with the import of Rev into the nucleus (34, 58). Rev then binds specifically to RNA containing the RRE (17,28,30,42,53) and multimerizes on the RRE in a process believed to involve protein-protein as well as protein-RNA interactions (12,14,16,32,36,43,67). The Rev-RRE complex is then recognized by Crm1 (exportin 1; official gene symbol, XPO1) and RAN-GTP (1), which initiates the export process and eventually targets the complex to the nuclear pore, where it interacts with nucleoporins (1,4,25,70). This results in translocation of the com...

Numerous pathogens cause respiratory infections with similar symptoms. Routine diagnostics detect only a limited number of pathogens, leaving a gap in respiratory illness etiology surveillance. This study evaluated next-generation sequencing for unbiased pathogen identification. Respiratory samples collected in Thailand, Philippines, Bhutan, and Nepal, that were negative by several molecular and immunofluorescence assays, underwent viral cultivation. Samples which demonstrated cytopathic effect in culture (N = 121) were extracted and tested by Luminex xTAG respiratory viral panel (RVP) assay and deep sequencing by Roche 454 FLX Titanium system. Using RVP assay, 52 (43%) samples were positive for enterovirus or rhinovirus and another three were positive for respiratory syncytial virus B, parainfluenza 4, and adenovirus. Deep sequencing confirmed the Luminex assay results and identified additional viral pathogens. Human enteroviruses, including Enterovirus A type 71 and 12 types of Enterovirus B (EV-B) were identified from a hospital in Bangkok. Phylogenetic and recombination analysis showed high correlation of VP1 gene-based phylogeny with genome-wide phylogeny and the frequent genetic exchange among EV-B viruses. The high number and diversity of enteroviruses in the hospital in Bangkok suggests prevalent existence. The metagenomic approach used in our study enabled comprehensive diagnoses of respiratory viruses.

Previously we described the identification of two compounds (3-amino-5-ethyl-4,6-dimethylthieno[2,3-b] pyridine-2-carboxamide [103833] and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile[104366]) that interfered with HIV replication through the inhibition of Rev function. We now describe resistant viral variants that arose after drug selection, using virus derived from two different HIV proviral clones, NL4-3 and R7/3. With HIV NL4-3 , each compound selected a different single point mutation in the Rev response element (RRE) at the bottom of stem-loop IIC. Either mutation led to the lengthening of the stem-loop IIC stem by an additional base pair, creating an RRE that was more responsive to lower concentrations of Rev than the wild type. Surprisingly, wild-type HIV R7/3 was also found to be inhibited when tested with these compounds, in spite of the fact this virus already has an RNA stem-loop IIC similar to the one in the resistant NL4-3 variant. When drug resistance was selected in HIV R7/3 , a virus arose with two nucleotide changes that mapped to the envelope region outside the RRE. One of these nucleotide changes was synonymous with respect to env, and one was not. The combination of both nucleotide changes appeared to be necessary for the resistance phenotype as the individual point mutations by themselves did not convey resistance. Thus, although drug-resistant variants can be generated with both viral strains, the underlying mechanism is clearly different. These results highlight that minor nucleotide changes in HIV RNA, outside the primary Rev binding site, can significantly alter the efficiency of the Rev/RRE pathway. Cells infected with human immunodeficiency virus (HIV)produce stable species of unspliced and incompletely spliced mRNAs that require the Rev protein for their nuclear-cytoplasmic export and expression (for a review, see reference 29). Rev is a small basic protein of about 116 amino acids that is encoded in the HIV genome (for a review, see reference 44). One well-defined function of Rev in HIV infection is to bind (7,10,11,30,31,41,53) to the Rev response element (RRE) (15,26,45) that is found in these unspliced and incompletely spliced mRNAs and promote their nucleo-cytoplasmic export (21,28,40) by interacting with the Crm1 cellular export receptor and Ran-GTP (1).Many previous studies identified stem-loop IIB of the RRE as the primary binding site for a Rev protein monomer and suggested that 6 to 10 subunits of Rev can subsequently bind to the RRE (2, 9, 32, 33, 36, 50). Oligomerization of Rev requires the Rev multimerization domain that has also been shown to be necessary for Rev function (12,34,39,42,54). Additionally, the molecular details of the interaction of the Rev monomer and its primary binding site have been well characterized through the use of an isolated Rev peptide containing only the arginine-rich binding motif (ARM) and the stem IIB RNA hairpin (3,4,48,49).Recently, Rev has been shown to form a cooperative highaffinity oligomeric complex with the RRE (13)....

(12), uncoating as it is transported to the host cell nucleus (13). The viral RNA genome undergoes reverse transcription (14) to produce DNA that is integrated into the host cell genome (15). At this point HIV exists as a provirus.