The rapid emergence and spread of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants across the globe underscores the crucial need for continuous SARS-CoV-2 surveillance to ensure that potentially more pathogenic variants are detected early and contained. Whole genome sequencing (WGS) is currently the gold standard for COVID-19 surveillance; however, it remains cost-prohibitive and requires specialized technical skills. To increase surveillance capacity, especially in resource-scarce settings, supplementary methods that are cost- and time-effective are needed. Real-time multiplex PCR genotyping assays offer an economical and fast solution for screening circulating and emerging variants while simultaneously complementing existing WGS approaches. In this study we evaluated the AllplexTM SARS-CoV-2 Variants II multiplex real-time PCR genotyping assay, Seegene (South Korea), and implemented it in retrospectively characterizing circulating SARS-CoV-2 variants in a rural South African setting between April and October 2021, prior to the emergence of the Omicron variant in South Africa. The AllplexTM SARS-CoV-2 Variants II real-time PCR assay demonstrated perfect concordance with whole-genome sequencing in detecting Beta and Delta variants and exhibited high specificity, sensitivity and reproducibility. Implementation of the assay in characterization of SARS-CoV-2 variants between April and October 2021 in a rural South African setting revealed a rapid shift from the Beta to the Delta variant between April and June. All specimens successfully genotyped in April were Beta variants and the Delta variant was not detected until May. By June, 78% of samples genotyped were Delta variants and in July >95% of all genotyped samples were Delta variants. The Delta variant continued to predominate through to the end of our analysis in October 2021. Taken together, a commercial SARS-CoV-2 variant genotyping assay detected the rapid rate at which the Delta variant displaced the Beta variant in Limpopo, an under-monitored province in South Africa. Such assays provide a quick and cost-effective method of monitoring circulating variants and should be used to complement genomic sequencing for COVID-19 surveillance especially in resource-scarce settings.

Background Viral rebound during antiretroviral treatment (ART) is most often driven by suboptimal adherence in the absence of drug resistance. We assessed the diagnostic performance of point-of-care (POC) tenofovir (TFV) detection in urine for the prediction of viral rebound and drug resistance during ART. Methods We performed a nested case-control study within the ADVANCE randomized clinical trial (NCT03122262) in Johannesburg, South Africa. Adult outpatients living with HIV and newly initiating ART were randomized to receive either dolutegravir or efavirenz, tenofovir disoproxil fumarate or alafenamide, and emtricitabine. All participants with rebound ≥200 copies/mL between 24 and 96 weeks of follow-up were selected as cases and matched to controls with virological suppression <50 copies/mL. Rapid POC urine-TFV detection was performed retrospectively. Results We included 281 samples from 198 participants. Urine-TFV was detectable in 30.7% (70/228) of cases and in 100% (53/53) of controls. Undetectable urine-TFV predicted rebound with a sensitivity of 69% [95%CI 63-75] and specificity of 100% [93-100]. In cases with virological failure and sequencing data (n = 42), NRTI drug resistance was detected in 50% (10/20) of cases with detectable urine-TFV versus in 8.3% (2/24) of cases with undetectable urine-TFV. Detectable urine-TFV predicted NRTI resistance (OR 10.4 [1.8-114.4] p = 0.005) with a sensitivity of 83% [52-98] and specificity of 69% [50-84]. Conclusions Point-of-care objective adherence testing using a urine-TFV test predicted viral rebound with high specificity. In participants with rebound, urine-TFV testing predicted the selection of drug resistance. Objective adherence testing may be used to rapidly provide insight into adherence, suppression, and drug resistance during ART.

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HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions. IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.

BackgroundThe lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev.ResultsThe tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 Å, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding.ConclusionsOur results suggest that EIAV Rev binding to the RRE requires dimerization via a coiled-coil motif to juxtapose two RNA binding motifs, ARM-1 and ARM-2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0115-7) contains supplementary material, which is available to authorized users.

BackgroundRev-like proteins are post-transcriptional regulatory proteins found in several retrovirus genera, including lentiviruses, betaretroviruses, and deltaretroviruses. These essential proteins mediate the nuclear export of incompletely spliced viral RNA, and act by tethering viral pre-mRNA to the host CRM1 nuclear export machinery. Although all Rev-like proteins are functionally homologous, they share less than 30% sequence identity. In the present study, we computationally assessed the extent of structural homology among retroviral Rev-like proteins within a phylogenetic framework.ResultsWe undertook a comprehensive analysis of overall protein domain architecture and predicted secondary structural features for representative members of the Rev-like family of proteins. Similar patterns of α-helical domains were identified for Rev-like proteins within each genus, with the exception of deltaretroviruses, which were devoid of α-helices. Coiled-coil oligomerization motifs were also identified for most Rev-like proteins, with the notable exceptions of HIV-1, the deltaretroviruses, and some small ruminant lentiviruses. In Rev proteins of primate lentiviruses, the presence of predicted coiled-coil motifs segregated within specific primate lineages: HIV-1 descended from SIVs that lacked predicted coiled-coils in Rev whereas HIV-2 descended from SIVs that contained predicted coiled-coils in Rev. Phylogenetic ancestral reconstruction of coiled-coils for all Rev-like proteins predicted a single origin for the coiled-coil motif, followed by three losses of the predicted signal. The absence of a coiled-coil signal in HIV-1 was associated with replacement of canonical polar residues with non-canonical hydrophobic residues. However, hydrophobic residues were retained in the key ‘a’ and ‘d’ positions, and the α-helical region of HIV-1 Rev oligomerization domain could be modeled as a helical wheel with two predicted interaction interfaces. Moreover, the predicted interfaces mapped to the dimerization and oligomerization interfaces in HIV-1 Rev crystal structures. Helical wheel projections of other retroviral Rev-like proteins, including endogenous sequences, revealed similar interaction interfaces that could mediate oligomerization.ConclusionsSequence-based computational analyses of Rev-like proteins, together with helical wheel projections of oligomerization domains, reveal a conserved homogeneous structural basis for oligomerization by retroviral Rev-like proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-017-0366-1) contains supplementary material, which is available to authorized users.

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We describe a case of prolonged SARS-CoV-2 RNA shedding in an HIV-negative 21-year-old man recovering from abdominal and thoracic trauma. Nasopharyngeal (NP) swabs collected at 12 time points over a 95-day span all tested positive for SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR). Genotyping revealed canonical beta-variant E484K and N501Y mutations at earlier time points. Human rhinovirus, coronavirus NL63 and respiratory syncytial virus B were detected at different time points by RT-PCR. Full blood analysis at time point 9 (day 82) showed leukopenia with lymphocytosis. The patient’s NP swab tested negative for SARS-CoV-2 by RT-PCR 101 days after the first positive test. The prolonged duration of SARS-CoV-2 RNA shedding in the context of trauma presented here is unique and has important implications for COVID-19 diagnosis, management and policy guidelines