Marco Koppelman

Proliferation-dependent HIV-1 infection of monocytes occurs during differentiation into macrophages.

Requirements for the establishment of productive infection with the human immunodeficiency virus type 1 (HIV-1) in primary monocytes were investigated. In vitro, monocytes rendered susceptible for infection after at least a 2-d culture, but when cultured in the presence of differentiation-inducing agent IL-4, accelerated susceptibility was seen. Complete resistance to HIV-1 infection was observed in monocytes that had been treated for 5 d with rIL-4, and comparable results were obtained with other differentiation inducers such as dexamethasone or 1,25(OH)2 vitamin D3 (1,25(OH)2vitD3). The inhibition of productive infection was not caused by downregulation of CD4 expression or HIV-1 transcription, nor by intracellular accumulation of virions. Since treatment with rIL-4, dexamethasone, or 1,25(OH)2vitD3 also resulted in complete inhibition of monocyte proliferation, we studied whether establishment of productive infection in monocytes is proliferation dependent. Irradiation or mitomycin-C treatment within 24 h after inoculation prevented productive HIV-1 infection of monocytes, suggesting a proliferation-dependent step early in the virus replication cycle. Polymerase chain reaction (PCR) analysis revealed the presence of only incomplete proviral DNA species in non-proliferating monocytes, indicating restriction of viral replication at the level of reverse transcription. Thus, in analogy with HIV-1 infection of CD4+ T cells, proliferation of monocytes during differentiation into macrophages is a prerequisite for productive infection with HIV.

Seroprevalence of hepatitis E virus (HEV) and detection of HEV RNA with a transcription-mediated amplification assay in blood donors from Catalonia (Spain).

Hepatitis E virus (HEV) is an emerging threat to the safety of blood transfusion. The aim of this study was to determine HEV immunoglobulin (Ig)G and RNA prevalence in Catalan blood donors.

Multiple surface antigen mutations in five blood donors with occult hepatitis B virus infection

Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA while the HBV surface antigen (HBsAg) remains undetectable. The HBV genomes in five asymptomatic blood donors with occult HBV infection and low viremia (<10 to 1,000 HBV DNA copies/mL, genotype D) were studied. An unusually large number of amino acid mutations was present in the immunodominant a‐determinant of HBsAg (respectively 3, 6, 7, 10, and 10 mutations). Comparison of the HBV genomes in two donors to a consensus HBV genotype D sequence showed a most prominent hotspot of genetic variation in HBV nucleotides 480–570, encoding the HBsAg a‐determinant. The phylogenetic comparison of separate donor HBV genes to the HBV genes of 11 reference strains (genotypes A–H) showed the donor HBV surface genes to form an outgroup, while the HBV polymerase, core and X genes closely cluster with the HBV genotype D reference strain. Maybe the HBV strains in this study represent a natural end‐stage of seemingly cleared HBV infection, in which HBV maintains a low level of possibly non‐infectious replication, after sacrificing its immunologically offending surface antigen, thus avoiding final clearance by the immune system. J. Med. Virol. 80:1344–1349, 2008.

Efficacy of hepatitis B virus (HBV) DNA screening and characterization of acute and occult HBV infections among blood donors from Madrid, Spain

BACKGROUND: Screening of blood units for hepatitis B virus (HBV) DNA identifies donations collected during the window period (WP) of the acute infection and may improve viral safety of the blood supply. It also leads to the detection of occult hepatitis B infection (OBI).

Diversity and origin of hepatitis C virus infection among unpaid blood donors in the Netherlands

BACKGROUND: To improve transfusion policy and to increase understanding of the spread of hepatitis C virus (HCV) in the general population, HCV infections among voluntary Dutch blood donors were examined with molecular epidemiologic techniques.

Epidemiology of high-level parvovirus B19 viraemia among Dutch blood donors, 2003-2009

Background and Objectives  Plasma derivatives and blood components with low levels of parvovirus B19 (B19) seem not infectious, but recently infected, highly viraemic donors may transmit B19. We studied the incidence of high‐level B19 viraemia (B19 DNA > 106 IU/ml) in 6·5 million Dutch blood donations.

Use of competitive polymerase chain reaction to determine HIV-1 levels in response to antiviral treatments.

ObjectiveTo develop a competitive polymerase chain reaction technique with which to evaluate the usefulness of HIV-1 level as a marker of response to antiviral treatment. DesignHIV-1 sequences were assessed by competitive polymerase chain reaction in four subjects participating in a double-blind study of monotherapy versus combination therapy with nucleoside analogues. MethodsWe inserted a mutant construct of the HIV-1 pol sequence into a commercial vector, enabling us to generate known amounts of mutant DNA and RNA for competitive polymerase chain reaction. To measure HIV-1 DNA copies in cells, the mutant DNA fragments were allowed to compete in a 10-fold dilution series with a constant amount of nucleic acid from the subject. To measure HIV-1 RNA copies in plasma, in vitro synthesized mutant RNA was added in a 10-fold dilutation series to a constant amount of subject RNA and copy DNA was synthesized. DNA and copy DNA were used as the input for nested pol polymerase chain reaction. Mutant and wild-type amplimers were discriminated by size. ResultsThe competitive polymerase chain reaction technique has been validated in model experiments and can be used over a broad range (at least 6 logs) of levels. Three of the four subjects showed a decline of 1 log in proviral DNA levels in cells after beginning antiviral treatment. All four showed a decline of at least 1 log in viral RNA levels in plasma, but this decline was transient in one subject. ConclusionThe HIV-1 sequence level is a useful marker in antiviral treatment studies.

Acute and Chronic Hepatitis E Virus Infection in Human Immunodeficiency Virus‐Infected U.S. Women

Exposure to hepatitis E virus (HEV) is common in the United States, but there are few data on prevalence of HEV/human immunodeficiency virus (HIV) coinfection in U.S. populations. We tested 2,919 plasma samples collected from HIV‐infected (HIV+) women and men enrolled in U.S. cohort studies for HEV viremia using a high‐throughput nucleic acid testing (NAT) platform. NAT+ samples were confirmed by real‐time polymerase chain reaction. Samples were selected for testing primarily on the basis of biomarkers of liver disease and immune suppression. Prevalence of HEV viremia was 3 of 2,606 and 0 of 313 in tested plasma samples collected from HIV+ women and men, respectively. All HEV isolates were genotype 3a. Based on follow‐up testing of stored samples, 1 woman had chronic HEV infection for >4 years whereas 2 women had acute HEV detectable at only a single study visit. Conclusions: To our knowledge, this is the first reported case of chronic HEV infection in an HIV+ U.S. individual. We also confirm that chronic HEV infection can persist despite a CD4+ count >200 cells/mm3. Overall, though, these data suggest that HEV infection is rare in the HIV+ U.S. population. (Hepatology 2016;63:712–720)

Multicenter evaluation of a commercial multiplex polymerase chain reaction test for screening plasma donations for parvovirus B19 DNA and hepatitis A virus RNA

BACKGROUND: Three European laboratories evaluated the TaqScreen DPX test (DPX test), a multiplex nucleic acid test assay for the simultaneous detection and quantitation of parvovirus B19 (B19V) DNA and the detection of hepatitis A virus (HAV) RNA.

Mechanism of anti-HIV activity of succinylated human serum albumin

In the present study, we described the interaction of succinylated human serum albumin (Suc-HSA), a negatively charged anti-HIV-1 active protein, with HIV-1 gp120 and in detail with the third variable domain of gp120 (V3 loop). To this end, different assay formats were tested in which gp120- and V3-related peptides were presented in various configurations in order to investigate the effect of the conformational structure of the V3 loop on the interaction with negatively charged albumins. When gp120 presented via a lectin was used, it was observed that Suc-HSA bound to native gp120. The binding site appeared to be located at or near the thrombin digestion site (GPGRAF sequence) in the V3 loop of gp120, since the cleavage of the loop resulted in decreased binding of Suc-HSA. In addition, Suc-HSA was able to protect the V3 region of gp120 from cleaving with thrombin. In contrast, significant binding of Suc-HSA to V3 loop or gp120 peptides was not observed when both were presented in a fluid phase system, suggesting the involvement of a monovalent-low affinity binding of Suc-HSA. Using overlapping peptides delineating the whole V3 loop immobilized to CNBr-Sepharose, we noticed that the interaction of the V3 loop with Suc-HSA was predominantly induced by electrostatic interactions between positively charged linearized peptide fragments and Suc-HSA and was positively influenced by the presence of hydrophobic amino in the V3 loop fragments as well. Moreover, the highest affinity site was located at sites near the GPGRAF sequence. These observations add to the evidence, collected earlier, that Suc-HSA interferes at the level of virus entry, independent of interaction with the CD4 receptor. Since the recently discovered chemokine receptors are negatively charged, we can hypothesize that Suc-HSA is able to prevent the positively charged V3 loop from interacting with these types of receptors, thereby inhibiting virus entry.