Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems

Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1–7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting

Identification of a pegivirus (GBV-like virus) that infects horses

The recent identification of nonprimate hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features and phylogenetic analyses confirmed that the tentatively named equine pegivirus (EPgV) represents a novel species within the Pegivirus genus. We also determined that EPgV causes persistent viremia whereas its clinical significance is undetermined

Ultrasound measurement of joint cartilage thickness in large and small joints in healthy children: a clinical pilot study assessing observer variability

<p>Abstract</p> <p>Background</p> <p>Loss of joint cartilage is a feature of destructive disease in JIA. The cartilage of most joints can be visualized with ultrasonography (US). Our present study focuses on discriminant validity of US in children. We studied reproducibility between and within a skilled and a non-skilled investigator of US assessment of cartilage thickness in small and large joints in healthy children.</p> <p>Methods and results</p> <p>In 11 healthy children (5 girls/6 boys), aged 9.6 years (9.3–10 years), 110 joints were examined. Cartilage thickness of the right and left hip, knee, ankle, 2^ndmetacarpophalangeal (MCP), and 2^ndproximal interphalangeal (PIP) joint independently. The joints were examined twice, two days apart by a skilled and a non-skilled investigator. Mean cartilage thickness in the five joints was: hip 2.59 ± 0.41, knee 3.67 ± 0.64, ankle 1.08 ± 0.31, MCP 1.52 ± 0.27 and PIP 0.73 ± 0.15 mm. We found the same mean differences in CTh of 0.6 mm in the inter-observer part with regard of the PIP joint. Within investigators (intra-observer), the smallest mean difference of CTh was found in the MCP joint with -0.004 (skilled) and 0.013 mm (non-skilled).</p> <p>Conclusion</p> <p>We found the level of agreement between observers within a 95% Confidence Interval in assessment of cartilage thickness in hip-, knee-, ankle-, MCP-, and PIP joints in healthy children. Observer variability seems not to relate to joint size but to the positioning of the joints and the transducer. These factors seem to be of major importance for reproducible US measurements. The smallest difference in measurement of cartilage thickness <it>between observers </it>was found in the PIP joint, and <it>within observers </it>in the MCP joint and it seems that using EULAR standard US guidelines is feasible for a pediatric setting. The use of US in children is promising. Studies on larger groups of children are needed to confirm the validation and variability of US in children as well as determining the smallest detectable difference of US measures.</p

SARS-COV-2 production in a single-use scalable high cell density bioreactor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the fast development of first-generation vaccines have demonstrated the value of applying a variety of vaccine technology platforms (1). Inactivated vaccines represent a well-known approach, and their manufacturing depends on high-yield virus production in appropriate biosafety level facilities. This study (2) aimed to establish efficient Vero (WHO) cell-based and animal component-free SARS-CoV-2 production in the CelCradle bioreactor (Esco Aster Pte Ltd.). The single-use culture vessels pre-packed with 0.1 L BioNOCII carriers are highly useful for small scale cultivation of adherent cell lines. Cultures were seeded with 1.5 × 10⁸ cells and total cell numbers peaked at 9 days post cell seeding (dpcs) with 2.7–2.8 × 10⁹ cells/vessel in non-infected cultures. To produce virus, cultures were infected at 7 dpcs at total cell numbers of 2.2–2.5 × 10⁹ cells/vessel at a multiplicity of infection of 0.006. Harvest of virus-containing supernatant twice instead of once per day improved the virus yield by 2–7 fold. Introducing a temperature shift from 37°C to 33°C upon the time of infection improved virus yield by 2–9 fold with a considerable decline of infectious titer only after 72 h post infection (hpi). Infectious titers peaked at 7.3 log₁₀ 50% tissue culture infectious dose (TCID₅₀)/mL at 72 hpi, and a total of 10.5 log₁₀ TCID₅₀ were produced in ~5 L (11 harvests). While trypsin has been reported to enhance SARS-CoV-2 spread in cell culture, addition of 0.5% recombinant trypsin from the time of infection did not affect virus yield. Overall, animal component-free production of SARS-CoV-2 in Vero (WHO) cells was successfully established in a single-use packed-bed bioreactor. β-propiolactone inactivated SARS-CoV-2 from this study was immunogenic and induced neutralizing antibodies in mice with mean 50% neutralization titers of 1/150 or 1/580 after three immunizations with doses containing 0.1 µg or 0.5 µg S1 protein, respectively. The CelCradle represents a scalable technology and is a small version of the TideXCell system applying single-use culture vessels with packed-bed volumes of up to 100 L. The potential rapid response to outbreaks with inactivated vaccines has been demonstrated in the SARS-CoV-2 pandemic (1). Vero cells are susceptible to a wide range of viral pathogens (3), and these scalable single-use bioreactors provide a high level of flexibility and potentially decreased response time for production of future emerging viruses for vaccine purposes. References: (1) Poland 2020 Lancet [PMID: 33065034], (2) Offersgaard 2021 Vaccines [PMID: 34209694], (3) Barret 2009 Expert Rev. Vaccines [PMID: 19397417

miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5’UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5’ end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity

Inter -and intraobserver variation of ultrasonographic cartilage thickness assessments in small and large joints in healthy children

<p>Abstract</p> <p>Background</p> <p>There is an increasing interest among pediatric rheumatologist for using ultrasonography (US) in the daily clinical examination of children with juvenile idiopathic arthritis (JIA). Loss of joint cartilage may be an early feature of destructive disease in JIA. However, US still needs validation before it can be used as a diagnostic bedside tool in a pediatric setting. This study aims to assess the inter- and intraobserver reliability of US measurements of cartilage thickness in the joints of healthy children.</p> <p>Methods</p> <p>740 joints of 74 healthy Caucasian children (27 girls/47 boys), aged 11.3 (7.11 – 16) years were examined with bilateral US in 5 preselected joints to assess the interobserver variability. In 17 of these children (6 girls/11 boys), aged 10.1(7.11–11.1) years, 170 joints was examined in an intraobserver sub study, with a 2 week interval between the first and second examination.</p> <p>Results</p> <p>In this study we found a good inter- and intraobserver agreement expressed as a coefficient of variation (CV) less than 10% in the knee (CV = 9.5%_{interobserver}and 5.9%_{intraobservserI}, 9.3%_{intraobserverII}respectively for the two intraobserver measurements) and fairly good for the MCP joints (CV = 11.9%_{interobserver}, 12.9%_{intraobserverI}and 11.9%_{intraobsevrerII}). In the ankle and PIP joints the inter- and intraobserver agreement was within an acceptable limit (CV<20%) but not for the wrist joint (CV>26%). We found no difference in cartilage thickness between the left and right extremity in the investigated joints.</p> <p>Conclusion</p> <p>We found a good inter -and intraobserver agreement when measuring cartilage thickness with US. The inter- and intraobserver variation seemed not to be related to joint size. These findings suggest that positioning of the joint and the transducer is of major importance for reproducible US measurements. We found no difference in joint cartilage thickness between the left and right extremity in any of the examined joint of the healthy children. This is an important finding giving the opportunity of using the non-affected extremity as a reference when assessing articular joint cartilage damage in JIA.</p

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M&gt;70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0&lt;e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM