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Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.Suppressive combination highly active antiretroviral therapy (HAART) has improved the health and life span of human immunodeficiency virus type 1 (HIV-1)-infected children (34) and adults (23). The long-term efficacy of HAART, however, has been limited in many individuals (11, 16), often due to the selection of drug-resistant viral mutants (2, 48). Transient lowlevel viremias (LLV), with plasma HIV-1 RNA levels in the range of 50 to 400 copies (c)/ml, have been reported to occur in 25 to 40% of adults in whom viral replication appeared to have been suppressed by HAART (21,24,45) and up to 90% of subjects with very frequent determinations of plasma HIV-1 RNA levels (35). LLV have been associated with selection of drug-resistant virus in several (8, 21, 22), but not all, studies (25,35,45).LLV appeared to be clinically insignificant in two studies, as subjects with and without LLV had similar rates of virologic failure of HAART (24,45). Detailed studies of LLV have demonstrated viral sequences similar to that found early in the course of infection (25,35,38). These observations support the hypothesis that activation of latently infected cells is the source of LLV (7,17,25,38). A modification of this hypothesis proposes that there is continuous low-level expression of virus from a stable proviral reservoir, with detectable virus occurring when random biological pr...

To the best of our knowledge, this is the first report of a horizontally transferable macrolide resistance mechanism in thermophilic Campylobacter. Surveillance of erm(B) and its associated MDRGI in both C. coli and C. jejuni is urgently warranted.

Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal β-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, an HP14 substrate similar to Drosophila Snake. The recombinant proHP21 is a 51.1 kDa glycoprotein with an amino-terminal clip domain, a linker region, and a carboxyl-terminal serine proteinase domain. HP14, generated by incubating proHP14 with β-1,3-glucan and β-1,3-glucan recognition protein-2, activated proHP21 by limited proteolysis between Leu 152 and Ile 153 . Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 to be Arg 355 and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in PO activity. HP21 cleaved proPO activating proteinase-2 precursor (proPAP-2) after Lys 153 and generated an amidase activity which activated proPO in the presence of serine proteinase homolog-1 and 2. In summary, we have discovered and reconstituted a branch of the proPO activation cascade in vitro: β-1,3-glucan recognition -proHP14 autoactivation -proHP21 cleavage -PAP-2 generation -proPO activation -melanin formation.

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