Comparison of Nanopore and Synthesis-Based Next-Generation Sequencing Platforms for SARS-CoV-2 Variant Monitoring in Wastewater.

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION's versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic.

Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing.

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.

Detection of Nonenveloped Hepatitis E Virus in Plasma of Infected Blood Donors.

BACKGROUND: Transfusion-transmitted hepatitis E virus (HEV) infections have raised many concerns regarding the safety of blood products. To date, enveloped HEV particles have been described in circulating blood, whereas nonenveloped HEV virions have only been found in feces; however, no exhaustive studies have been performed to fully characterize HEV particles in blood. METHODS: Using isopycnic ultracentrifugation, we determined the types of HEV particles in plasma of HEV-infected blood donors. RESULTS: Nonenveloped HEV was detected in 8 of 23 plasma samples, whereas enveloped HEV was found in all of them. No association was observed between the presence of nonenveloped HEV and viral load, gender, or age at infection. However, samples with HEV-positive serology and/or increased levels of liver injury markers contained a higher proportion of nonenveloped HEV than samples with HEV-negative serology and normal levels of liver enzymes. These results were further confirmed by analyzing paired donation and follow-up samples of 10 HEV-infected donors who were HEV seronegative at donation but had anti-HEV antibodies and/or increased levels of liver enzymes at follow up. CONCLUSIONS: The HEV-contaminated blood products may contain nonenveloped HEV, which may pose an additional risk to blood safety by behaving differently to pathogen inactivation treatments or increasing infectivity.

Partial restoration of immune response in Hepatitis C patients after viral clearance by direct-acting antiviral therapy.

BACKGROUND & AIMS: HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. METHODS: We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Ɣ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Ɣ) and CFSE-based proliferation assays. RESULTS: We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Ɣ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. CONCLUSIONS: Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA's therapies.

Pathogenicity and virulence of hepatitis A virus.

Hepatitis A is an acute infection of the liver, which is mostly asymptomatic in children and increases the severity with age. Although in most patients the infection resolves completely, in a few of them it may follow a prolonged or relapsed course or even a fulminant form. The reason for these different outcomes is unknown, but it is generally accepted that host factors such as the immunological status, age and the occurrence of underlaying hepatic diseases are the main determinants of the severity. However, it cannot be ruled out that some virus traits may also contribute to the severe clinical outcomes. In this review, we will analyze which genetic determinants of the virus may determine virulence, in the context of a paradigmatic virus in terms of its genomic, molecular, replicative, and evolutionary features.

Exosome mimicry by a HAVCR1-NPC1 pathway of endosomal fusion mediates hepatitis A virus infection.

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis, binding and uptake of exosomes are well characterized4,5, delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome9, are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway, which Ebola virus exploits to infect cells9, mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.

HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection.

The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV.IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the mechanism by which the subsequent necroinflammatory process clears the infection remain a puzzle that most likely involves the HAV-HAVCR1 interaction. Based on negative data, a recent paper from the S. M. Lemon and W. Maury laboratories (A. Das, A. Hirai-Yuki, O. Gonzalez-Lopez, B. Rhein, S. Moller-Tank, R. Brouillette, L. Hensley, I. Misumi, W. Lovell, J. M. Cullen, J. K. Whitmire, W. Maury, and S. M. Lemon, mBio 8:e00969-17, 2017, https://doi.org/10.1128/mBio.00969-17) suggested that HAVCR1 is not a functional HAV receptor, nor it is it required for HAV infection. However, our data, based on regain of the HAV receptor function in HAVCR1 knockout cells transfected with HAVCR1 cDNA, disagree with their findings. Our positive data show conclusively that HAVCR1 is indeed a functional HAV receptor and lays the ground for the identification of alternative HAV receptors and how they interact with HAVCR1 in cell entry and the pathogenesis of HAV.

Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81.IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid-binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis.

Improving virus production through quasispecies genomic selection and molecular breeding.

Virus production still is a challenging issue in antigen manufacture, particularly with slow-growing viruses. Deep-sequencing of genomic regions indicative of efficient replication may be used to identify high-fitness minority individuals suppressed by the ensemble of mutants in a virus quasispecies. Molecular breeding of quasispecies containing colonizer individuals, under regimes allowing more than one replicative cycle, is a strategy to select the fittest competitors among the colonizers. A slow-growing cell culture-adapted hepatitis A virus strain was employed as a model for this strategy. Using genomic selection in two regions predictive of efficient translation, the internal ribosome entry site and the VP1-coding region, high-fitness minority colonizer individuals were identified in a population adapted to conditions of artificially-induced cellular transcription shut-off. Molecular breeding of this population with a second one, also adapted to transcription shut-off and showing an overall colonizer phenotype, allowed the selection of a fast-growing population of great biotechnological potential.