Comprehensive mutational analysis reveals p6Gag phosphorylation to be dispensable for HIV-1 morphogenesis and replication.

The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.

Correction: Comparison of Four Active SARS-CoV-2 Surveillance Strategies in Representative Population Sample Points: Two-Factor Factorial Randomized Controlled Trial.

[This corrects the article DOI: 10.2196/44204.].

Limitations introduced by a low participation rate of SARS-CoV-2 seroprevalence data.

BACKGROUND: There has been a large influx of COVID-19 seroprevalence studies, but comparability between the seroprevalence estimates has been an issue because of heterogeneities in testing platforms and study methodology. One potential source of heterogeneity is the response or participation rate. METHODS: We conducted a review of participation rates (PR) in SARS-CoV-2 seroprevalence studies collected by SeroTracker and examined their effect on the validity of study conclusions. PR was calculated as the count of participants for whom the investigators had collected a valid sample, divided by the number of people invited to participate in the study. A multivariable beta generalized linear model with logit link was fitted to determine if the PR of international household and community-based seroprevalence studies was associated with the factors of interest, from 1 December 2019 to 10 March 2021. RESULTS: We identified 90 papers based on screening and were able to calculate the PR for 35 out of 90 papers (39%), with a median PR of 70% and an interquartile range of 40.92; 61% of the studies did not report PR. CONCLUSIONS: Many SARS-CoV-2 seroprevalence studies do not report PR. It is unclear what the median PR rate would be had a larger portion not had limitations in reporting. Low participation rates indicate limited representativeness of results. Non-probabilistic sampling frames were associated with higher participation rates but may be less representative. Standardized definitions of participation rate and data reporting necessary for the PR calculations are essential for understanding the representativeness of seroprevalence estimates in the population of interest.

Comparison of Four Active SARS-CoV-2 Surveillance Strategies in Representative Population Sample Points: Two-Factor Factorial Randomized Controlled Trial.

BACKGROUND: The COVID-19 pandemic is characterized by rapid increases in infection burden owing to the emergence of new variants with higher transmissibility and immune escape. To date, monitoring the COVID-19 pandemic has mainly relied on passive surveillance, yielding biased epidemiological measures owing to the disproportionate number of undetected asymptomatic cases. Active surveillance could provide accurate estimates of the true prevalence to forecast the evolution of the pandemic, enabling evidence-based decision-making. OBJECTIVE: This study compared 4 different approaches of active SARS-CoV-2 surveillance focusing on feasibility and epidemiological outcomes. METHODS: A 2-factor factorial randomized controlled trial was conducted in 2020 in a German district with 700,000 inhabitants. The epidemiological outcome comprised SARS-CoV-2 prevalence and its precision. The 4 study arms combined 2 factors: individuals versus households and direct testing versus testing conditioned on symptom prescreening. Individuals aged ≥7 years were eligible. Altogether, 27,908 addresses from 51 municipalities were randomly allocated to the arms and 15 consecutive recruitment weekdays. Data collection and logistics were highly digitized, and a website in 5 languages enabled low-barrier registration and tracking of results. Gargle sample collection kits were sent by post. Participants collected a gargle sample at home and mailed it to the laboratory. Samples were analyzed with reverse transcription loop-mediated isothermal amplification (RT-LAMP); positive and weak results were confirmed with real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Recruitment was conducted between November 18 and December 11, 2020. The response rates in the 4 arms varied between 34.31% (2340/6821) and 41.17% (2043/4962). The prescreening classified 16.61% (1207/7266) of the patients as COVID-19 symptomatic. Altogether, 4232 persons without prescreening and 7623 participating in the prescreening provided 5351 gargle samples, of which 5319 (99.4%) could be analyzed. This yielded 17 confirmed SARS-CoV-2 infections and a combined prevalence of 0.36% (95% CI 0.14%-0.59%) in the arms without prescreening and 0.05% (95% CI 0.00%-0.108%) in the arms with prescreening (initial contacts only). Specifically, we found a prevalence of 0.31% (95% CI 0.06%-0.58%) for individuals and 0.35% (95% CI 0.09%-0.61%) for households, and lower estimates with prescreening (0.07%, 95% CI 0.0%-0.15% for individuals and 0.02%, 95% CI 0.0%-0.06% for households). Asymptomatic infections occurred in 27% (3/11) of the positive cases with symptom data. The 2 arms without prescreening performed the best regarding effectiveness and accuracy. CONCLUSIONS: This study showed that postal mailing of gargle sample kits and returning home-based self-collected liquid gargle samples followed by high-sensitivity RT-LAMP analysis is a feasible way to conduct active SARS-CoV-2 population surveillance without burdening routine diagnostic testing. Efforts to improve participation rates and integration into the public health system may increase the potential to monitor the course of the pandemic. TRIAL REGISTRATION: Deutsches Register Klinischer Studien (DRKS) DRKS00023271; https://tinyurl.com/3xenz68a. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s13063-021-05619-5.

Diagnostic performance of anti-Zika virus IgM, IgAM and IgG ELISAs during co-circulation of Zika, dengue, and chikungunya viruses in Brazil and Venezuela.

BACKGROUND: Serological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela. METHODOLOGY/PRINCIPAL FINDINGS: Acute (day of illness 1-5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%. CONCLUSIONS/SIGNIFICANCE: Our findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks.

The vaccinia virus F11L gene product facilitates cell detachment and promotes migration.

We previously showed that infection with vaccinia virus (VV) induces cell motility, characterized by contractility and directed migration. Motility is temporally regulated because cells are motile immediately after infection, whereas late in infection motility ceases and cells resettle. Motility and its cessation are accompanied by temporal rearrangements of both the microtubule and the actin networks. Because the F11L gene has previously been implicated in VV-induced migration, we now explore the role of F11L in contractility, migration, the cessation of motility and the cytoskeletal rearrangements. By live cell imaging using a VV that lacks an intact F11L gene, we show that F11L facilitates cell detachment and is required for migration but not for contractility. By light microscopy, F11L expression induces a remodeling of the actin, but not the microtubule, network. The lack of migration correlates with smaller plaques, indicating that this process facilitates cell-to-cell spreading of VV. Late in infection, when motility ceases, cells re-establish cell-to-cell contacts in an F11L-independent manner. We finally show that VV-induced motility and its cessation correlate with a temporal regulation of the guanosine triphosphatase RhoA as well as the expression levels of F11L during the infectious cycle.

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Understanding the relation between Zika virus infection during pregnancy and adverse fetal, infant and child outcomes: a protocol for a systematic review and individual participant data meta-analysis of longitudinal studies of pregnant women and their infants and children.

INTRODUCTION: Zika virus (ZIKV) infection during pregnancy is a known cause of microcephaly and other congenital and developmental anomalies. In the absence of a ZIKV vaccine or prophylactics, principal investigators (PIs) and international leaders in ZIKV research have formed the ZIKV Individual Participant Data (IPD) Consortium to identify, collect and synthesise IPD from longitudinal studies of pregnant women that measure ZIKV infection during pregnancy and fetal, infant or child outcomes. METHODS AND ANALYSIS: We will identify eligible studies through the ZIKV IPD Consortium membership and a systematic review and invite study PIs to participate in the IPD meta-analysis (IPD-MA). We will use the combined dataset to estimate the relative and absolute risk of congenital Zika syndrome (CZS), including microcephaly and late symptomatic congenital infections; identify and explore sources of heterogeneity in those estimates and develop and validate a risk prediction model to identify the pregnancies at the highest risk of CZS or adverse developmental outcomes. The variable accuracy of diagnostic assays and differences in exposure and outcome definitions means that included studies will have a higher level of systematic variability, a component of measurement error, than an IPD-MA of studies of an established pathogen. We will use expert testimony, existing internal and external diagnostic accuracy validation studies and laboratory external quality assessments to inform the distribution of measurement error in our models. We will apply both Bayesian and frequentist methods to directly account for these and other sources of uncertainty. ETHICS AND DISSEMINATION: The IPD-MA was deemed exempt from ethical review. We will convene a group of patient advocates to evaluate the ethical implications and utility of the risk stratification tool. Findings from these analyses will be shared via national and international conferences and through publication in open access, peer-reviewed journals. TRIAL REGISTRATION NUMBER: PROSPERO International prospective register of systematic reviews (CRD42017068915).

Partial downregulation of MAD1 causes spindle checkpoint inactivation and aneuploidy, but does not confer resistance towards taxol.

The mitotic spindle assembly checkpoint ensures proper chromosome segregation during mitosis by inhibiting the onset of anaphase until all kinetochores are attached to the mitotic spindle and tension across the kinetochores is generated. Here, we report that the stable partial downregulation of the spindle checkpoint gene MAD1, which is observed in human cancer, leads to a functional inactivation of the spindle checkpoint resulting in gross aneuploidy. Interestingly, although Mad1 is thought to act as a kinetochore based activator of Mad2 during checkpoint activation, we show that normal levels of Mad2, but not of Mad1, are required for preventing premature sister chromatid separation and for maintaining the timing of an undisturbed mitosis, suggesting a Mad1 independent function of Mad2 that operates independent of its checkpoint function. Most significantly, a partial repression of either MAD1 or MAD2 confers resistance to nocodazole, a drug that inhibits microtubule attachment. In contrast, sensitivity to clinically relevant drugs like taxol or monastrol that inhibit the generation of tension across kinetochores is not modulated by partial downregulation of MAD1, suggesting a functional bifurcation of spindle checkpoint dependent apoptotic pathways.

Seasonal Distribution and Climatic Correlates of Dengue Disease in Dhaka, Bangladesh.

Dengue has been regularly reported in Dhaka, Bangladesh, since a large outbreak in 2000. However, to date, we have limited information on the seasonal distribution of dengue disease and how case distribution correlates with climate. Here, we analyzed dengue cases detected at a private diagnostic facility in Dhaka during 2010-2014. We calculated Pearson cross-correlation coefficients to examine the relationship between the timing of cases and both rainfall and temperature. There were 2,334 cases diagnosed during the study period with 76% over the age of 15 years. Cases were reported in every month of the study; however, 90% of cases occurred between June and November. Increases in rainfall were correlated with increases in cases 2 months later (correlation of 0.7). The large proportion of adult cases is consistent with substantial population susceptibility and suggests Dhaka remains at risk for outbreaks. Although cases occurred year-round, public health preparedness should be focused during peak months.

[Chest granuloma secondary to methyl methacrylate. Case report]. / Granuloma de tórax secundario a metilmetacrilato. Reporte de caso.

We present the case of a patient with a history of a massive left hemithorax crushing injury in 1985; the exact management of the lesion is unknown. Twenty years later he had a thoracic fistula with a culture that was reported as positive for Enteroccocus faecalis and Staphyloccocus epidermidis. The patient was referred by the chest surgery service with the diagnosis of rib osteomyelitis once complementary imaging tests were performed (plain X-rays, CAT scan and MRI). The patient underwent surgery at our service; a granulomatous reaction secondary to a foreign body (methyl methacrylate and Ethibon) was reported. Chest reconstruction for massive lesions is possible with methyl methacrylate. Imaging studies involve the well-known difficulty to identify this material, given that it may produce signals and densities that are difficult to interpret by specialized physicians.