Molecular biology and structure of a novel penaeid shrimp densovirus elucidate convergent parvoviral host capsid evolution.

The giant tiger prawn (Penaeus monodon) is a decapod crustacean widely reared for human consumption. Currently, viruses of two distinct lineages of parvoviruses (PVs, family Parvoviridae; subfamily Hamaparvovirinae) infect penaeid shrimp. Here, a PV was isolated and cloned from Vietnamese P. monodon specimens, designated Penaeus monodon metallodensovirus (PmMDV). This is the first member of a third divergent lineage shown to infect penaeid decapods. PmMDV has a transcription strategy unique among invertebrate PVs, using extensive alternative splicing and incorporating transcription elements characteristic of vertebrate-infecting PVs. The PmMDV proteins have no significant sequence similarity with other PVs, except for an SF3 helicase domain in its nonstructural protein. Its capsid structure, determined by cryoelectron microscopy to 3-Å resolution, has a similar surface morphology to Penaeus stylirostris densovirus, despite the lack of significant capsid viral protein (VP) sequence similarity. Unlike other PVs, PmMDV folds its VP without incorporating a ßA strand and displayed unique multimer interactions, including the incorporation of a Ca2+ cation, attaching the N termini under the icosahedral fivefold symmetry axis, and forming a basket-like pentamer helix bundle. While the PmMDV VP sequence lacks a canonical phospholipase A2 domain, the structure of an EDTA-treated capsid, determined to 2.8-Å resolution, suggests an alternative membrane-penetrating cation-dependent mechanism in its N-terminal region. PmMDV is an observed example of convergent evolution among invertebrate PVs with respect to host-driven capsid structure and unique as a PV showing a cation-sensitive/dependent basket structure for an alternative endosomal egress.

Progressive emergence of an S153F plus R263K combination of integrase mutations in the proviral DNA of one individual successfully treated with dolutegravir.

OBJECTIVES: The development of HIV drug resistance against the integrase strand transfer inhibitor dolutegravir is rare. We report here the transient detection, by near full-genome ultradeep sequencing, of minority HIV-1 subtype B variants bearing the S153F and R263K integrase substitutions in the proviral DNA from blood cells of one patient who successfully initiated dolutegravir-based ART, over 24 weeks. Our objective was to study the effects of these substitutions. METHODS: Strand transfer and DNA-binding activities of recombinant integrase proteins were measured in cell-free assays. Cell-based resistance, infectivity and replicative capacities were measured using molecular clones. Structural modelling was performed to understand experimental results. RESULTS: R263K emerged first, followed by the addition of S153F at Week 12. By Week 24, both mutations remained present, but at lower prevalence. We confirmed the coexistence of S153F and R263K on single viral genomes. Combining S153F or S153Y with R263K decreased integration and viral replicative capacity and conferred high levels of drug resistance against all integrase inhibitors. Alone, S153Y and S153F did little to infectivity or dolutegravir resistance. We identified altered DNA binding as a mechanism of resistance. The patient remained with undetectable viral loads at all timepoints. CONCLUSIONS: Drug-resistant minority variants have often been reported under suppressive ART. Our study adds to these observations by unravelling a progression towards higher levels of resistance through a novel pathway despite continuous undetectable viral loads. Poorly replicative HIV drug-resistant minority proviral variants did not compromise viral suppression in one individual treated with dolutegravir.

Antimalarial drugs and their metabolites are potent Zika virus inhibitors.

Studies aimed at repurposing existing drugs revealed that some antimalarial compounds possess anti-Zika virus (anti-ZIKV) activity. Here, we further tested 14 additional antimalarial drugs and their metabolites or analogs for anti-ZIKV activity using a phenotypic screening approach. We identified four compounds with varying anti-ZIKV activity, including a metabolite of amodiaquine termed desethylamodiaquine (DAQ) and N-desethylchloroquine (DECQ), a metabolite of chloroquine, which both exhibited low micromolar effective concentrations against three different ZIKV strains. Two other compounds termed dihydroartemisinin (DHA) and quinidine (QD) exhibited only partial inhibition of ZIKV replication. Characterization of the inhibitory mechanisms of DAQ and DECQ showed that both drugs target the entry step as well as postentry events of the viral replication cycle. These hits represent attractive starting points for future optimization of new anti-ZIKV drug candidates derived from antimalarial drugs and their analogs.

HIV-1 Resistance Dynamics in Patients With Virologic Failure to Dolutegravir Maintenance Monotherapy.

Background: A high genetic barrier to resistance to the integrase strand transfer inhibitor (INSTI) dolutegravir has been reported in vitro and in vivo. We describe the dynamics of INSTI resistance-associated mutations (INSTI-RAMs) and mutations in the 3'-polypurine tract (3'-PPT) in relation to virologic failure (VF) observed in the randomized Dolutegravir as Maintenance Monotherapy for HIV-1 study (DOMONO, NCT02401828). Methods: From 10 patients with VF, plasma samples were collected before the start of cART and during VF, and were used to generate Sanger sequences of integrase, the 5' terminal bases of the 3' long terminal repeat (LTR), and the 3'-PPT. Results: Median human immunodeficiency virus RNA load at VF was 3490 copies/mL (interquartile range 1440-4990 copies/mL). INSTI-RAMs (S230R, R263K, N155H, and E92Q+N155H) were detected in 4 patients, no INSTI-RAMs were detected in 4 patients, and sequencing of the integrase gene was unsuccessful in 2 patients. The time to VF ranged from 4 weeks to 72 weeks. In 1 patient, mutations developed in the highly conserved 3'-PPT. No changes in the terminal bases of the 3'-LTR were observed. Conclusions: The genetic barrier to resistance is too low to justify dolutegravir maintenance monotherapy because single INSTI-RAMs are sufficient to cause VF. The large variation in time to VF suggests that stochastic reactivation of a preexisting provirus containing a single INSTI-RAM is the mechanism for failure. Changes in the 3'-PPT point to a new dolutegravir resistance mechanism in vivo. Clinical Trials Registration: NCT02401828.

The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors.

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

Reprint of: Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy.

Effect on HIV-1 viral replication capacity of DTG-resistance mutations in NRTI/NNRTI resistant viruses.

BACKGROUND: Recommended regimens for HIV-positive individuals include the co-administration of dolutegravir (DTG) with two reverse transcriptase inhibitors (RTIs). Although rare, emerging resistance against DTG is often associated with the R263K substitution in integrase. In-vitro-selected R263K was associated with impaired viral replication capacity, DNA integration, and integrase strand-transfer activity, especially when accompanied by the secondary mutation H51Y. Given the reduced fitness of RTI-resistant viruses, we investigated potential impacts on viral replication of combining R263K and H51Y/R263K with major RTI-resistance substitutions including K65R, L74V, K103N, E138K, and M184I/V. RESULTS: We combined the R263K or H51Y/R263K with RTI-resistance mutations into the proviral plasmid pNL4.3 and measured the resulting viral infectiousness, replication capacity, and ability to integrate viral DNA into host cells. Infectiousness was determined by luciferase assay in TZM-bl cells. Replicative capacity was monitored over 7 days and viral DNA integration was studied by real-time Alu-qPCR in PM1 cells. We found that viral infectiousness, replication capacities and integration levels were greatly reduced in triple mutants, i.e. H51Y/R263K plus a RT mutation, and moderately reduced in double mutants, i.e. R263K plus a RT mutation, compared to wild-type and single RT-mutant viruses. CONCLUSIONS: Our findings help to explain the absence of RTI mutations in individuals who experienced DTG-treatment failure.

Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy.

Novel parvoviruses in reptiles and genome sequence of a lizard parvovirus shed light on Dependoparvovirus genus evolution.

Here, we report the detection and partial genome characterization of two novel reptilian parvoviruses derived from a short-tailed pygmy chameleon (Rampholeon brevicaudatus) and a corn snake (Pantherophis guttatus) along with the complete genome analysis of the first lizard parvovirus, obtained from four bearded dragons (Pogona vitticeps). Both homology searches and phylogenetic tree reconstructions demonstrated that all are members of the genus Dependoparvovirus. Even though most dependoparvoviruses replicate efficiently only in co-infections with large DNA viruses, no such agents could be detected in one of the bearded dragon samples, hence the possibility of autonomous replication was explored. The alternative ORF encoding the full assembly activating protein (AAP), typical for the genus, could be obtained from reptilian parvoviruses for the first time, with a structure that appears to be more ancient than that of avian and mammalian parvoviruses. All three viruses were found to harbour short introns as previously observed for snake adeno-associated virus, shorter than that of any non-reptilian dependoparvovirus. According to the phylogenetic calculations based on full non-structural protein (Rep) and AAP sequences, the monophyletic cluster of reptilian parvoviruses seems to be the most basal out of all lineages of genus Dependoparvovirus. The suspected ability for autonomous replication, results of phylogenetic tree reconstruction, intron lengths and the structure of the AAP suggested that a single Squamata origin instead of the earlier assumed diapsid (common avian-reptilian) origin is more likely for the genus Dependoparvovirus of the family Parvoviridae.

Expression strategy of Aedes albopictus densovirus.

The transcription map of the Aedes albopictus densovirus (AalDNV) brevidensovirus was identified by Northern blotting, rapid amplification of cDNA ends (RACE) analysis, and RNase protection assays. AalDNV produced mRNAs of 3,359 (NS1), 3,345 (NS2), and 1,246 (VP) nucleotides. The two overlapping P7/7.4 NS promoters employed closely located alternate transcription initiation sites, positioned at either side of the NS1 initiation codon. All NS mRNAs coterminated with VP mRNA. All promoters, explored using luciferase assays, were functional in insect and human cell lines.

Pseudoplusia includens densovirus genome organization and expression strategy.

The genome of a densovirus of a major phytophagous pest, Pseudoplusia includens, was analyzed. It contained 5,990 nucleotides (nt) and included inverted terminal repeats of 540 nt with terminal Y-shaped hairpins of 120 nt. Its DNA sequence and ambisense organization with 4 typical open reading frames demonstrated that it belonged to the genus Densovirus in the subfamily Densovirinae of the family Parvoviridae.

Bipartite genome and structural organization of the parvovirus Acheta domesticus segmented densovirus.

Parvoviruses (family Parvoviridae) are currently defined by a linear monopartite ssDNA genome, T = 1 icosahedral capsids, and distinct structural (VP) and non-structural (NS) protein expression cassettes within their genome. We report the discovery of a parvovirus with a bipartite genome, Acheta domesticus segmented densovirus (AdSDV), isolated from house crickets (Acheta domesticus), in which it is pathogenic. We found that the AdSDV harbors its NS and VP cassettes on two separate genome segments. Its vp segment acquired a phospholipase A2-encoding gene, vpORF3, via inter-subfamily recombination, coding for a non-structural protein. We showed that the AdSDV evolved a highly complex transcription profile in response to its multipartite replication strategy compared to its monopartite ancestors. Our structural and molecular examinations revealed that the AdSDV packages one genome segment per particle. The cryo-EM structures of two empty- and one full-capsid population (3.3, 3.1 and 2.3 Å resolution) reveal a genome packaging mechanism, which involves an elongated C-terminal tail of the VP, "pinning" the ssDNA genome to the capsid interior at the twofold symmetry axis. This mechanism fundamentally differs from the capsid-DNA interactions previously seen in parvoviruses. This study provides new insights on the mechanism behind ssDNA genome segmentation and on the plasticity of parvovirus biology.

Impact of a switch to immediate release on the patient viewing of diagnostic test results in an online portal at an academic medical center.

Patient portals allow patients to access their personal health information. The 21st Century Cures Act in the United States sought to eliminate 'information blocking', requiring timely release upon request of electronic health information including diagnostic test results. Some health systems, including the one in the present study, chose a systematic switch to immediate release of all or nearly all diagnostic test results to patient portals as part of compliance with the Cures Act. Our primary objective was to study changes in the time to view test results by patients before and after implementation of Cures Act-related changes. This retrospective pre-post study included data from two 10-month time periods before and after implementation of Cures Act-related changes at an academic medical center. The study included all patients (adult and pediatric) with diagnostic testing (laboratory and imaging) performed in the outpatient, inpatient, or emergency department settings. Between February 9, 2020 and December 9, 2021, there was a total of 3 809 397 diagnostic tests from 204 605 unique patients (3 320 423 tests for adult patients; 488 974 for pediatric patients). Overall, 56.5% (115 627) of patients were female, 84.1% (172 048) white, and 96.5% (197 517) preferred English as primary language. The odds of viewing test results within 1 and 30 days after portal release increased monthly throughout both time periods before and after the Cures Act for all patients. The rate of increase was significantly higher after implementation only in the subgroup of tests belonging to adult patients with active MyChart accounts. Immediate release shifted a higher proportion of result/report release to weekends (3.2% pre-Cures vs 15.3% post-Cures), although patient viewing patterns by day of week and time of day were similar before and after immediate release changes. The switch to immediate release of diagnostic test results to the patient portal resulted in a higher fraction of results viewed within 1 day across outpatient, inpatient, and emergency department settings.

The utility of renal medullary pyramidal thickness measurements on the first and second postnatal ultrasound in infants with congenital hydronephrosis.

INTRODUCTION: Prior studies with postnatal ultrasound measurements of the medullary pyramid thickness (PT) demonstrate a PT < 3 mm as a significant risk factor for the diagnosis of UPJ obstruction and pyeloplasty. These studies used the postnatal ultrasound demonstrating the largest degree of hydronephrosis. Since early identification of children at increased risk of obstruction and pyeloplasty would be clinically useful, we reviewed the PT on the first and second postnatal ultrasound in infants with congenital hydronephrosis. OBJECTIVE: The aims of the study were to determine the prognostic value of: 1) PT on the first and second postnatal ultrasound, 2) a change in PT between first and second ultrasounds, and 3) ratio of PT in the hydronephrotic kidney to the contralateral PT in the normal kidney in those with unilateral hydronephrosis. We hypothesized that a smaller PT on either the first or second ultrasound, as well as a decreasing PT between the first and second ultrasound, and a decreased ratio of hydronephrotic PT to the contralateral normal kidney, would each be early predictors of subsequent pyeloplasty. STUDY DESIGN: A retrospective chart and ultrasound review of children with a diagnosis of isolated high grade (SFU grade 3 or 4) hydronephrosis was performed. This study also analyzed the impact on predictive ability of the PT obtained on an ultrasound obtained before 3 days of life compared to those in which the first ultrasound was obtained after 3 days of life. 91 infants (77 boys and 14 girls) met eligibility criteria (105 kidneys). The median age (IQR) at first ultrasound was 1.5 (1.0-15.0) days and 54.0 (27.5-123.0) days at the second ultrasound. DISCUSSION AND CONCLUSION: For the group overall, a smaller PT on both the first and second ultrasound was associated with increased risk of pyeloplasty, however, a PT obtained on an ultrasound prior to 3 days of life was not demonstrated to be predictive. Of note, PT was predictive in this same group of patients on their second ultrasound. A PT of <3 mm on an ultrasound obtained beyond 3 days of life was associated with higher risk of pyeloplasty. The PT ratio of hydronephrotic to normal contralateral kidney of the children who had their first ultrasound after 3 days of life was also significant in predicting the odds of having surgery. In addition, a decreasing PT between the first and second ultrasound was also identified as a risk factor for pyeloplasty.

A Circo-Like Virus Isolated from Penaeus monodon Shrimps.

A virus with a circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) genome (PmCV-1) was isolated from Penaeus monodon shrimps in Vietnam. The gene structure of the 1,777-nucleotide (nt) genome was similar to that of circoviruses and cycloviruses, but the nucleic acid and protein sequence identities to these viruses were very low.

Acheta domesticus Volvovirus, a Novel Single-Stranded Circular DNA Virus of the House Cricket.

The genome of a novel virus of the house cricket consists of a 2,517-nucleotide (nt) circular single-stranded DNA (ssDNA) molecule with 4 open reading frames (ORFs). One ORF had a low identity to circovirus nucleotide sequences (NS). The unique properties of this volvovirus suggested that it belongs to a new virus family or genus.

New Volvovirus Isolates from Acheta domesticus (Japan) and Gryllus assimilis (United States).

A novel circular single-stranded DNA (ssDNA) virus, volvovirus, from the house cricket has been described recently. Here, we report the isolation of volvoviruses from Acheta domesticus in Japan and Gryllus assimilis in the United States. These Acheta domesticus volvovirus (AdVVV) isolates have genomes of 2,517 and 2,516 nucleotides (nt) and 4 large open reading frames (ORFs).

A Novel Ambisense Densovirus, Acheta domesticus Mini Ambidensovirus, from Crickets.

The genome structure of Acheta domesticus mini ambidensovirus, isolated from crickets, resembled that of ambisense densoviruses from Lepidoptera but was 20% smaller. It had the highest (<25%) protein sequence identity with the nonstructural protein 1 (NS1) of Iteravirus and VP of Densovirus members (both with 25% coverage) and smaller (0.2- versus 0.55-kb) Y-shaped inverted terminal repeats.

Junonia coenia Densovirus (JcDNV) Genome Structure.

The sequence of Junonia coenia densovirus was the first densovirus genome sequence published, but the first published sequence contained incomplete inverted terminal repeats and ambiguous nucleotides or indels leading to an incorrect map of the open reading frames. Our sequencing of clones of the complete genome demonstrated that this virus is closely related to other viruses in the Densovirus genus.