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Objectives: Viral hepatitis co‐infection among people living with HIV is known to accelerate the progression of liver disease and AIDS. An increased prevalence and incidence of hepatitis B virus (HBV) infection among people living with HIV demands continuous monitoring to adapt targeted prevention strategies to reach the global goals of eliminating viral hepatitis as a public health threat. Methods: We determined the prevalence and incidence of HBV for the years 1996–2019 from yearly blood sample testing and questionnaire reports among people living with HIV belonging to a nationwide, multicentre observational, prospective cohort study. Results: Among this study population of 3479 participants, the majority (87%) indicated that being men who have sex with men (MSM) was their likely HIV transmission route; 51% were recruited from Berlin. HBV prevalence for acute/chronic and resolved infections decreased from 4.1% and 45% in 1996–1999 to 1.3% and 16% in 2019, respectively. Simultaneously, participants with a serological status indicating HBV vaccination increased from 25% in 1996–1999 to 69% in 2019. Among vaccinated participants with relevant information (n = 1135), 38% received their first HBV vaccination after HIV infection. The HBV incidence rate in 565 eligible participants decreased from 6.9/100 person‐years in 2004–2007 to 0.45/100 person‐years in 2015. Conclusion: Increasing vaccination coverage because of a general HBV vaccination recommendation and catch‐up vaccination efforts among risk groups decreased HBV infection prevalence over time among this study population of people living with HIV, primarily MSM and from Berlin. Despite this success, the prevalence and incidence of HBV remains higher than in the general population in Germany. This emphasizes the need for continued HBV prevention by promoting HBV vaccination and HBV screening at regular intervals based on the individual risk behaviour. [ABSTRACT FROM AUTHOR]

A large-scale amoeba-based screening procedure developed for the detection of genes required for the full virulence of Legionella pneumophila against Acanthamoeba castellanii is described. The procedure identified many known and new colonization factors of L. pneumophila, from which bdhA-patD operon is found to be critical for intracellular replication in amoebae and macrophages, for poly-3-hydroxy-butyrate (PHB) usage, and for phospholipase A (PLA) and lysophospholipase A (LPLA) activities.

The article discusses the findings of the molecular surveillance of newly diagnosed HIV cases that serves as the Robert Koch Institute's instrument for nationwide surveillance of primary HIV-1 drug resistance in Germany. The results showed resistance to tenofovir or emtricitabine, which have been approved for pre-exposure prophylaxis, or both agents in 80 of the newly diagnosed HIV cases during the period of 2017 to 2020. An HIV treatment regimen using newer agents is recommended.

Abstract Background The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. Conclusions Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.

Abstract Background The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.

Abstract Background XMRV, a xenotropic murine leukemia virus (MuLV)-related virus, was recently identified by PCR testing in 67% of persons with chronic fatigue syndrome (CFS) and in 3.7% of healthy persons from the United States. To investigate the association of XMRV with CFS we tested blood specimens from 51 persons with CFS and 56 healthy persons from the US for evidence of XMRV infection by using serologic and molecular assays. Blinded PCR and serologic testing were performed at the US Centers for Disease Control and Prevention (CDC) and at two additional laboratories. Results Archived blood specimens were tested from persons with CFS defined by the 1994 international research case definition and matched healthy controls from Wichita, Kansas and metropolitan, urban, and rural Georgia populations. Serologic testing at CDC utilized a Western blot (WB) assay that showed excellent sensitivity to MuLV and XMRV polyclonal or monoclonal antibodies, and no reactivity on sera from 121 US blood donors or 26 HTLV-and HIV-infected sera. Plasma from 51 CFS cases and plasma from 53 controls were all WB negative. Additional blinded screening of the 51 cases and 53 controls at the Robert Koch Institute using an ELISA employing recombinant Gag and Env XMRV proteins identified weak seroreactivity in one CFS case and a healthy control, which was not confirmed by immunofluorescence. PCR testing at CDC employed a gag and a pol nested PCR assay with a detection threshold of 10 copies in 1 ug of human DNA. DNA specimens from 50 CFS patients and 56 controls and 41 US blood donors were all PCR-negative. Blinded testing by a second nested gag PCR assay at the Blood Systems Research Institute was also negative for DNA specimens from the 50 CFS cases and 56 controls. Conclusions We did not find any evidence of infection with XMRV in our U.S. study population of CFS patients or healthy controls by using multiple molecular and serologic assays. These data do not support an association of XMRV with CFS.

Abstract Background A novel gammaretrovirus named xenotropic murine leukemia virus-related virus (XMRV) has been recently identified and found to have a prevalence of 40% in prostate tumor samples from American patients carrying a homozygous R462Q mutation in the RNaseL gene. This mutation impairs the function of the innate antiviral type I interferon pathway and is a known susceptibility factor for prostate cancer. Here, we attempt to measure the prevalence of XMRV in prostate cancer cases in Germany and determine whether an analogous association with the R462Q polymorphism exists. Results 589 prostate tumor samples were genotyped by real-time PCR with regard to the RNaseL mutation. DNA and RNA samples from these patients were screened for the presence of XMRV-specific gag sequences using a highly sensitive nested PCR and RT-PCR approach. Furthermore, 146 sera samples from prostate tumor patients were tested for XMRV Gag and Env antibodies using a newly developed ELISA assay. In agreement with earlier data, 12.9% (76 samples) were shown to be of the QQ genotype. However, XMRV specific sequences were detected at neither the DNA nor the RNA level. Consistent with this result, none of the sera analyzed from prostate cancer patients contained XMRV-specific antibodies. Conclusion Our results indicate a much lower prevalence (or even complete absence) of XMRV in prostate tumor patients in Germany. One possible reason for this could be a geographically restricted incidence of XMRV infections.

Monitoring specific antibodies derived from whole-cell immunization through cell-based ELISA methods poses challenges due to humoral responses against various cell proteins. In this report, we outline a technique involving pre-adsorption on cells to remove undesirable antibodies from immune serum. This step provides the subsequent monitoring of antibodies specific to the targeted antigen using a tANCHOR-based ELISA. Notably, this approach accelerates result acquisition, eliminating the necessity to purify the expressed antigen or obtain a customized peptide for coating assay plates., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Conventional neutralizing enzyme-linked immunosorbent assay (ELISA) systems for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mimic the protein-protein interaction between angiotensin-converting enzyme 2 (ACE2) and the receptor-binding domain (RBD). However, an easy and rapidly adaptative ELISA-based system for testing neutralizing antibodies against upcoming SARS-CoV-2 variants is urgently needed. In this study, we closed this gap by developing a tANCHOR-cell-based RBD neutralization assay that avoids time-consuming protein expression and purification followed by coating on ELISA plates. This cell-based assay can be rapidly adopted to monitor neutralizing antibodies (NAbs) against upcoming SARS-CoV-2 variants. We show that the results obtained with the tANCHOR-cell-based assay system strongly correlate with commercially available surrogate assays for testing NAbs. Moreover, this technique can directly measure binding between cell-surface-exposed RBDs and soluble ACE2. With this technique, the degree of antibody escape elicited by emerging SARS-CoV-2 variants in current vaccination regimens can be determined rapidly and reliably., Competing Interests: The non-profit organization Peter und Traudl Engelhorn Foundation holds a patent application for the tANCHOR system where D.I. is listed as an inventor. J.M. is employed at the company ATG:biosynthetics GmbH (founder H.B.)., (© 2024 The Author(s).)

Enzyme-linked immunosorbent assay (ELISA) systems use plates coated with peptides or expressed and purified proteins to monitor immunoglobulins derived from patient serum. However, there is currently no easy, flexible, and fast adaptive ELISA-based system for testing antibodies directed against new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In this study, we utilized the tANCHOR protein display system that provides a cell surface decorated with the receptor-binding domain (RBD) to monitor specific antibodies derived from SARS-CoV-2 convalescent and vaccinated individuals directed against it. To test sera from vaccinees or convalescent individuals, only the RBD coding sequence needs to be cloned in the tANCHOR vector system and transfected into HeLa cells. Time-consuming protein expression, isolation, and purification followed by coating assay plates are not necessary. With this technique, the immune evasion of new SARS-CoV-2 variants from current vaccination regimes can be examined quickly and reliably., Competing Interests: The non-profit Peter und Traudl Engelhorn Foundation holds a patent application for the tANCHOR system, where D.I. is listed as an inventor. J.M. is employed at the company ATG: biosynthetics GmbH (founded by H.B.)., (© The Author(s) 2024. Published by Oxford University Press.)

Viruses that carry a positive-sense, single-stranded (+ssRNA) RNA translate their genomes soon after entering the host cell to produce viral proteins, with the exception of retroviruses. A distinguishing feature of retroviruses is reverse transcription, where the +ssRNA genome serves as a template to synthesize a double-stranded DNA copy that subsequently integrates into the host genome. As retroviral RNAs are produced by the host cell transcriptional machinery and are largely indistinguishable from cellular mRNAs, we investigated the potential of incoming retroviral genomes to directly express proteins. Here we show through multiple, complementary methods that retroviral genomes are translated after entry. Our findings challenge the notion that retroviruses require reverse transcription to produce viral proteins. Synthesis of retroviral proteins in the absence of productive infection has significant implications for basic retrovirology, immune responses and gene therapy applications., (© 2024. The Author(s).)

Syncytin-1 and -2 are glycoproteins encoded by human endogenous retrovirus (hERV) that, through their fusogenic properties, are needed for the formation of the placental syncytiotrophoblast. Previous studies suggested that these proteins, in addition to the EnvP(b) envelope protein, are also involved in other cell fusion events. Since galectin-1 is a β-galactoside-binding protein associated with cytotrophoblast fusion during placental development, we previously tested its effect on Syncytin-mediated cell fusion and showed that this protein differently modulates the fusogenic potential of Syncytin-1 and -2. Herein, we were interested in comparing the impact of galectin-1 on hERV envelope proteins in different cellular contexts. Using a syncytium assay, we first demonstrated that galectin-1 increased the fusion of Syncytin-2- and EnvP(b)-expressing cells. We then tested the infectivity of Syncytin-1 and -2 vs. VSV-G-pseudotyped viruses toward Cos-7 and various human cell lines. In the presence of galectin-1, infection of Syncytin-2-pseudotyped viruses augmented for all cell lines. In contrast, the impact of galectin-1 on the infectivity of Syncytin-1-pseudotyped viruses varied, being cell- and dose-dependent. In this study, we report the functional associations between three hERV envelope proteins and galectin-1, which should provide information on the fusogenic activity of these proteins in the placenta and other biological and pathological processes.

Successful induction of antibodies in model organisms like mice depends strongly on antigen design and delivery. New antigen designs for immunization are helpful for developing future therapeutic monoclonal antibodies (mAbs). One of the gold standards to induce antibodies in mice is to express and purify the antigen for vaccination. This is especially time-consuming when mAbs are needed rapidly. We closed this gap and used the display technology tetraspanin anchor to develop a reliable immunization technique without the need to purify the antigen. This technique is able to speed up the immunization step enormously and we have demonstrated that we were able to induce antibodies against different proteins with a focus on the receptor-binding domain of SARS-CoV-2 and the extracellular loop of canine cluster of differentiation 20 displayed on the surface of human cells., Competing Interests: The non-profit organization Peter und Traudl Engelhorn Foundation holds a patent application for the tANCHOR system where D.I. is listed as an inventor. J.M. is employed at the company ATG:biosynthetics GmbH (founder H.B.). A.S. and K.H. are associated with the company new/era/mabs GmbH, which provides a proprietary technology platform to generate customized antibodies., (© The Author(s) 2023. Published by Oxford University Press.)

Background: The transmission of resistant HIV variants jeopardizes the effective use of antiretrovirals for therapy and prophylaxis. Molecular surveillance of new HIV diagnoses with a focus on prevalence and type of resistance associated mutations and the subtype of circulating viruses is mandatory., Method: From 2017 to 2020, 11,527 new HIV diagnoses were reported in Germany to the Robert Koch Institute (RKI). Protease (PR) and reverse-transcriptase (RT) sequences were obtained from 4559 (39.6%) cases, and PR, RT and integrase (IN) sequences were obtained from 3097 (26.9%) cases. The sequences were analyzed with data from the national HIV reports., Results: Among all cases in the analysis, the proportion of primary resistance was 4.3% for nucleoside reverse-transcriptase inhibitors (NRTIs), 9.2% for non-NRTI (NNRTIs), 3.3% for protease inhibitors (PIs) and 1.4% for integrase inhibitors (INIs). Dual-class resistance was highest for NRTIs/NNRTIs with 1.2%. There was no trend in the proportion of viruses resistant to drug classes. Most individual key mutations associated with relevant resistance had a prevalence below 1% including K65R (0.1%) and M184V (0.6%). A notable exception was K103NS, with a prevalence of 2.9% and a significant increase (p Trend =0.024) during 2017-2020. In this period, diagnoses of infections with HIV-1 subtype B were the most common at 58.7%, but its prevalence was declining (p Trend =0.049) while the frequency of minority subtypes (each < 1%) increased (p Trend =0.007). Subtype B was highest (75.6%) in men who have sex with men (MSM) and lowest in reported heterosexual transmissions (HETs, 22.6%)., Conclusion: The percentage of primary resistance was high but at a stable level. A genotypic determination of resistance is therefore still required before the start of therapy. The subtype diversity of circulating HIV-1 is increasing., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: We investigated the impact of HIV pre-exposure prophylaxis (PrEP) as a new service of the statutory health insurance (SHI) on the incidence of HIV and other sexually transmitted infections (STIs) in Germany. In addition, PrEP needs and access barriers were analyzed., Methods: The following data were evaluated as part of the evaluation project: HIV and syphilis notification data and extended surveillance by the Robert Koch Institute (RKI), pharmacy prescription data, SHI routine data, PrEP use in HIV-specialty care centers, Checkpoint, the BRAHMS and PrApp studies, as well as a community board., Results: The majority of PrEP users were male (98-99%), primarily aged between 25-45 years, and predominantly of German nationality or origin (67-82%). The majority were men who have sex with men (99%). With regard to HIV infections, PrEP proved to be highly effective. There were only isolated cases of HIV infections (HIV incidence rate 0.08/100 person years); in most cases the suspected reason was low adherence. The incidences of chlamydia, gonorrhea, and syphilis did not increase but remained almost the same or even decreased. A need for information on PrEP for people in trans*/non-binary communities, sex workers, migrants, and drug users emerged. Needs-based services for target groups at increased risk of HIV are necessary., Discussion: PrEP proved to be a very effective HIV prevention method. The partly feared indirect negative influences on STI rates were not confirmed in this study. Due to the temporal overlap with the containment measures during the COVID-19 pandemic, a longer observation period would be desirable for a conclusive assessment., (© 2023. The Author(s).)