How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles

Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct "replication factories" inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology

Cytomegalovirus and polyomavirus BK posttransplant

Virus replication and progression to disease in transplant patients is determined by patient-, graft- and virus-specific factors. This complex interaction is modulated by the net state of immunosuppression and its impact on virus-specific cellular immunity. Due to the increasing potency of immunosuppressive regimens, graft rejections have decreased, but susceptibility to infections has increased. Therefore, cytomegalovirus (CMV) remains the most important viral pathogen posttransplant despite availability of effective antiviral drugs and validated strategies for prophylactic, preemptive and therapeutic intervention. CMV replication can affect almost every organ system, with frequent recurrences and increasing rates of antiviral resistance. Together with indirect long-term effects, CMV significantly reduces graft and patient survival after solid organ and hematopoietic stem cell transplantation. The human polyomavirus called BK virus (BKV), on the other hand, only recently surfaced as pathogen with organ tropism largely limited to the reno-urinary tract, manifesting as polyomavirus-associated nephropathy in kidney transplant and hemorrhagic cystitis in hematopoetic stem cell transplant patients. No licensed anti-polyoma viral drugs are available, and treatment relies mainly on improving immune functions to regain control over BKV replication. In this review, we discuss diagnostic and therapeutic aspects of CMV and BKV replication and disease posttransplantatio

Prevalence of Polyomavirus BK and JC Infection and Replication in 400 Healthy Blood Donors

BackgroundThe replication of BK virus (BKV) and JC virus (JCV) is linked to polyomavirus-associated nephropathy, hemorrhagic cystitis, and multifocal leukoencephalopathy in immunodeficient patients, but the behavior of these viruses in immunocompetent individuals has hardly been characterized MethodsWe used EIA to study samples obtained from 400 healthy blood donors aged 20-59 years for BKV- and JCV-specific antibodies against virus-like particles. We also studied BKV and JCV loads in plasma and urine among these individuals by use of real-time polymerase chain reaction ResultsIgG seroprevalence was 82% (328 of 400 donors) for BKV and 58% (231 of 400) for JCV. As age increased (age groups were divided by decade), the seroprevalence of BKV decreased from 87% (87 of 100) in the youngest group (aged 20-29 years) to 71% (71 of 100) in the oldest group (aged 50-59 years) (P=.006), whereas the seroprevalence of JCV increased from 50% (50 of 100) in the youngest group to 68% (68 of 100) in the oldest group (P=.06). Asymptomatic urinary shedding of BKV and JCV was observed in 28 (7%) and 75 (19%) of 400 subjects, respectively, with median viral loads of 3.51 and 4.64 log copies/mL, respectively (P<.001). Unlike urinary BKV loads, urinary JCV loads were positively correlated with IgG levels. The shedding of JCV was more commonly observed among individuals who were seropositive only for JCV, compared with individuals who were seropositive for both BKV and JCV, suggesting limited cross-protection from BKV immunity. Noncoding control regions were of archetype architecture in all cases, except for 1 rearranged JCV variant. Neither BKV nor JCV DNA was detected in plasma ConclusionsOur study provides important data about polyomavirus infection and replication in healthy, immunocompetent individuals. These data indicate significant differences between BKV and JCV with respect to virus-host interaction and epidemiolog

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5'-cap-structures of higher eukaryote mRNAs are ribose 2'-O-methylated. Likewise, a number of viruses replicating in the cytoplasm of eukayotes have evolved 2'-O-methyltransferases to modify autonomously their mRNAs. However, a defined biological role of mRNA 2'-O-methylation remains elusive. Here we show that viral mRNA 2'-O-methylation is critically involved in subversion of type-I-interferon (IFN-I) induction. We demonstrate that human and murine coronavirus 2'-O-methyltransferase mutants induce increased IFN-I expression, and are highly IFN-I sensitive. Importantly, IFN-I induction by 2'-O-methyltransferase-deficient viruses is dependent on the cytoplasmic RNA sensor melanoma differentiation-associated gene 5 (MDA5). This link between MDA5-mediated sensing of viral RNA and mRNA 2'-O-methylation suggests that RNA modifications, such as 2'-O-methylation, provide a molecular signature for the discrimination of self and non-self mRNA

Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles

Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6. IMPORTANCE Although the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well

Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity

Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts

Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology

Immunosuppression is required for BK viremia and polyomavirus BK–associated nephropathy (PVAN) in kidney transplants (KTs), but the role of viral determinants is unclear. We examined BKV noncoding control regions (NCCR), which coordinate viral gene expression and replication. In 286 day–matched plasma and urine samples from 129 KT patients with BKV viremia, including 70 with PVAN, the majority of viruses contained archetypal (ww-) NCCRs. However, rearranged (rr-) NCCRs were more frequent in plasma than in urine samples (22 vs. 4%; P < 0.001), and were associated with 20-fold higher plasma BKV loads (2.0 × 104/ml vs. 4.4 × 105/ml; P < 0.001). Emergence of rr-NCCR in plasma correlated with duration and peak BKV load (R2 = 0.64; P < 0.001). This was confirmed in a prospective cohort of 733 plasma samples from 227 patients. For 39 PVAN patients with available biopsies, rr-NCCRs were associated with more extensive viral replication and inflammation. Cloning of 10 rr-NCCRs revealed diverse duplications or deletions in different NCCR subregions, but all were sufficient to increase early gene expression, replication capacity, and cytopathology of recombinant BKV in vitro. Thus, rr-NCCR BKV emergence in plasma is linked to increased replication capacity and disease in KTs

New insights on the role of paired membrane structures in coronavirus replication

The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication

Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results

Background: Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTMCMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014). Objectives: To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT. Study design: The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction. Results: Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by > 90% indicating the presence of unprotected CMV genomic DNA. Conclusions: Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons.Peer reviewe

Usefulness of the GenMark ePlex RPP assay for the detection of respiratory viruses compared to the FTD21 multiplex RT-PCR

Cartridge-based multiplex panels covering numerous pathogens offer an advantage of minimal hands-on-time and short time to result to commercial RT-PCR assays. In this study, we evaluated the performance of the ePlex respiratory pathogen panel (RPP) compared to the Fast Track Diagnostics Respiratory pathogens 21 multiplex RT-PCR assay (FTD21) using 400 clinical respiratory samples. Discrepant results were further analysed by a reference nucleic acid amplification testing (NAT) and a composite reference approach was used for final interpretation. Discordant results were observed in 56 targets corresponding to 54 samples. Sensitivities and specificities were 85.5% and 99.9% for the ePlex RPP and 95.8% and 99.7% for the FTD21 system, respectively. Altogether, the ePlex RPP is a valuable tool for the rapid detection of a number of different respiratory viruses with the exception of the coronavirus family (low sensitivity ranging from 50-80%) and samples with a low pathogen load (Ct values >33)