RNA Titers Research Articles

Retrospective study evaluating association of colorectal tumors and hepatitis C virus.

Chronic hepatitis C virus (HCV) has been associated with hepatic and extrahepatic malignancies. Limited studies have shown an association between colorectal adenomas and HCV populations. To study the prevalence of colorectal adenomas in patients with HCV compared to the general population and to evaluate if it is an independent risk factor for colorectal adenomas. Patients were divided into HCV and non-HCV based on their HCV RNA titers. Patients with alcoholic liver disease, hepatitis B infection, and inflammatory bowel disease were excluded. Continuous variables were analyzed using the Mann-Whitney U test, and categorical variables using χ 2 with P < 0.05 were considered statistically significant. The significant covariates (independent variables) were matched in both groups by propensity score matching, followed by multivariate regression analysis. Of the 415 patients screened, 109 HCV patients and 97 non-HCV patients with colonoscopy results were included in the study. HCV patients were older, had a smoking history, had less frequent aspirin use, and had a lower body mass index (BMI) (P < 0.05). The HCV cohort had a significantly increased number of patients with adenomas (adenoma detection rate of 53.2% vs 34%. P = 0.006). We performed a propensity-matched multivariate analysis where HCV infection was significantly associated with colorectal adenoma (OR: 2.070, P = 0.019). Our study shows a significantly higher rate of adenomas in HCV patients compared to the general population. Prospective studies would help determine if the increase in adenoma detection lowers the risk for colorectal cancer.

Larval Competition between Aedes and Culex Mosquitoes Carries over to Higher Arboviral Infection during Their Adult Stage.

The common house mosquito (Culex pipiens) is a native vector for West Nile virus (WNV). Invasive species like the tiger mosquito (Aedes albopictus) and Asian bush mosquito (Aedes japonicus) are rapidly spreading through Europe, posing a major threat as vectors for dengue, chikungunya (CHIKV), and Japanese encephalitis virus (JEV). These mosquitoes share a similar ecological niche as larvae, but the carry-over effects of aquatic larval interactions to the terrestrial adult stage remain largely unknown and their medical relevance requires further investigation. This study examines the context dependency of larval interactions among Aedes albopictus, Aedes japonicus, and Culex pipiens. The survival, development time, growth, and energetic storage were measured in different European populations within density-response (intraspecific) experiments and replacement (interspecific) experiments at 20 °C and 26 °C. Overall, Ae. japonicus was the weakest competitor, while competition between Ae. albopictus and Cx. pipiens varied with temperature. Adults emerging from this larval competition were infected as follows: Culex pipiens with WNV, Ae. albopictus with CHIKV, and Ae. japonicus with JEV. While no JEV infection was observed, mosquitoes experiencing interspecific interactions during their larval stages exhibited higher infection rates and viral RNA titers for CHIKV and WNV. This increased susceptibility to viral infection after larval competition suggests a higher risk of arbovirus transmission in co-occurring populations.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Development of a SARS-CoV-2 viral dynamic model for patients with COVID-19 based on the amount of viral RNA and viral titer.

The target-cell limited model, which is one of the mathematical modeling approaches providing a quantitative understanding of viral dynamics, has been applied to describe viral RNA profiles of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in previous studies. However, these models have been developed mainly using patient data from the early phase of the pandemic. Furthermore, no reports focused on the profiles of the viral titer. In this study, the dynamics of both viral RNA and viral titer were characterized using data reflecting the current clinical situation in which the Omicron variant has become epidemic and vaccines for SARS-CoV-2 have become available. Consecutive data for 5212 viral RNA levels and 5216 viral titers were obtained from 720 patients with coronavirus disease 2019 (COVID-19) in a phase II/III study for ensitrelvir. Our model assumed that productively infected cells would produce only infectious viruses, which could be transformed into non-infectious viruses, and has been used to describe the dynamics of both viral RNA levels and viral titer. The time from infection to symptom onset (tinf) of unvaccinated patients was estimated to be 3.0 days, which was shorter than that of the vaccinated patients. The immune-related parameter as a power function for the vaccinated patients was 1.1 times stronger than that for the unvaccinated patients. Our model allows the prediction of the viral dynamics in patients with COVID-19 from the time of infection to symptom onset. Vaccination status was identified as a factor influencing tinf and the immune function.

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

Development and preclinical evaluation of equine-derived hyperimmune serum against SARS-CoV-2 infection in K-18 hACE2 transgenic (Tg) mice.

This study aimed to develop an equine-derived hyperimmune serum against SARS-CoV-2 and evaluate its efficacy as a potential immunotherapy tool for the treatment of known and potential variants of COVID-19 in preclinical trials. The novelty of this study is the whole virus and ALUM gel adjuvant formula. The horses were immunized using a whole inactivated SARS-CoV-2 antigen, and the final purified hyperimmune serum showed high plaque reduction neutralization (PRNT 50) neutralizing titers. The efficacy of the hyperimmune serum was evaluated histopathologically and biochemically in the lungs, hearts, and serum of K18 hACE2 transgenic mice (n=45), which is an accepted model organism for SARS-CoV-2 studies and was challenged with live SARS-CoV-2. Serum treatment improved the general condition, resulting in lower levels of proinflammatory cytokines in the blood plasma, as well as reduced viral RNA titers in the lungs and hearts. Additionally, it reduced oxidative stress significantly and lessened the severity of interstitial pneumonia in the lungs when compared to infected positive controls. The study concluded that equine-derived anti-SARS-CoV-2 antibodies could be used for COVID-19 prevention and treatment, especially in the early stages of the disease and in combination with antiviral drugs and vaccines. This treatment will benefit special patient populations such as immunocompromised individuals, as specific antibodies against SARS-CoV-2 can neutralize the virus before it enters host cells. The rapid and cost-effective production of the serum allows for its availability during the acute phase of the disease, making it a critical intervention in preventing the spread of the disease and saving lives in new variants where a vaccine is not yet developed.

Heat inactivation of aqueous viable norovirus and MS2 bacteriophage.

This study aimed to compare the heat inactivation kinetics of viable human norovirus with the surrogate, MS2 bacteriophage as well as assess the decay of the RNA signal. Human intestinal enteroids were used to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage, which was cultured using a plaque assay. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50°C, 60°C, and 70°C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50°C, 60°C, and 70°C, respectively. Norovirus had significantly higher decay rates than MS2 bacteriophage at all tested temperatures (P=.002-.007). No decrease of RNA titers as measured by reverse transcription-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation and treatment efficiency is underestimated. Overall, our data demonstrate that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with the persistence of viable norovirus under heat inactivation.

Airborne transmission efficiency of SARS-CoV-2 in Syrian hamsters is not influenced by environmental conditions

Several human respiratory viruses display a clear seasonal pattern with a higher incidence in the winter season in temperate regions. We previously determined that SARS-CoV-2 is more stable at low-temperature and low-humidity conditions compared to warmer temperature and higher-humidity. To determine if this translates into differential airborne transmission rates in vivo, we performed airborne transmission experiments in the Syrian hamster model under three different environmental conditions (10 °C, 45% relative humidity (RH), 22 °C, 45% RH, and 27 °C, 65% RH). We compared the ancestral SARS-CoV-2 Lineage A with the more transmissible Delta Variant of Concern (VOC). Airborne transmission was evaluated using SARS-CoV-2 infected donor animals at 24 h post inoculation. Sentinels were placed at a 90 cm distance in a transmission set-up and exposed for 1-h to infected donor animals. While environmental conditions moderately impacted lung RNA titers, the shedding kinetics of the donors were not affected by the environmental conditions and did not differ significantly between variants on day 1. Overall, the highest transmission efficiency was observed at 22 °C, 40%RH for Delta (62.5%, based on seroconversion), and ranged between 37.5 and 50% for all other conditions. However, these differences were not significant. To elucidate this further, we performed aerosol stability comparisons and found that infectious virus remained stable during a 1-h time window across all conditions. Our data suggest that even when environmental conditions affect the stability of SARS-CoV-2, this may not directly be translatable to measurable impacts on transmission in an experimental setting when exposure time is restricted.

Cellular nuclear-localized U2AF2 protein is hijacked by the flavivirus 3’UTR for viral replication complex formation and RNA synthesis

Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3’ untranslated region (3’UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3’UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3’UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3’ UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.

A Composite Score for Predicting Vertical Transmission of Hepatitis C: A Multicenter Study.

Prevention of the vertical transmission of the hepatitis C virus (HCV) presents an obstetric challenge. There are no approved antiviral medications for the treatment or prevention of HCV for pregnant patients. We aimed to create a composite score to accurately identify a population of pregnant patients with HCV who have high potential for vertical transmission. In a retrospective, multicenter cohort study, we identified pregnant patients with hepatitis C with linked data to their infants who have had HCV RNA or HCV antibody testing. Demographic data, including age and race/ethnicity, as well as clinical and laboratory data, including tobacco/alcohol use, infections, liver function tests, the HCV RNA titer, HCV antibody, HCV genotype, absolute lymphocyte count, and platelet count, were collected. Data were analyzed using logistic regression and receiver operating characteristics (ROCs) and internally validated using the forward selection bootstrap method. We identified 157 pregnant patients and 163 corresponding infants. The median maternal delivery age was 29 (IQR: 25-33) years, and the majority (141, or 89.8%) were White. A high HCV RNA titer, high absolute lymphocyte count, and high platelet count were associated with vertical transmission. A high HCV RNA titer had an AUROC of 0.815 with sensitivity, specificity, a positive predictive value, and a negative predictive value of 100.0%, 59.1%, 17.6%, and 100.0%, respectively. A composite score combining the three risk factors had an AUROC of 0.902 (95% CI = 0.840-0.964) but with a risk of overfitting. An HCV RNA titer alone or a composite score combining the risk factors for HCV vertical transmission can potentially identify a population of pregnant patients where the rate of vertical transmission is high, allowing for potential interventions during antepartum care.

Antiviral effect of alkaloids-free Ephedra Herb extract on respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children. Despite decades of efforts, no effective therapies are available. We recently reported that extracts of Ephedra Herb and Cinnamon Bark interacted with the G attachment protein of RSV to inhibit infectivity. The present in vitro study aimed to investigate the antiviral effect of ephedrine alkaloids-free Ephedra Herb extract (EFE), which is characterized by free of harmful effects of ephedrine alkaloids in Ephedra Herb, on experimental RSV infection. Infection of RSV into A549 cells simultaneously with EFE resulted the significant reduction of RSV RNA, viral protein, and viral titers after the incubation of the cells. We found that RSV attachment to the cell surface was inhibited both in the presence of EFE and when RSV particles were pre-treated with EFE. We also found that EFE specifically interacted with the central conserved domain of RSV G protein by surface plasmon resonance, demonstrating that specific binding of G protein to the cellular receptor was inhibited by EFE. Another mechanism was found in which a higher concentration of EFE inhibited the viral load immediately after the viral entry into host cells, suggesting the inhibition of viral RNA replication. These results demonstrate that EFE worked against RSV infection through multiple antiviral mechanisms, a unique feature of this crude drug extract.

Vaccine-associated respiratory pathology correlates with viral clearance and protective immunity after immunization with self-amplifying RNA expressing the spike (S) protein of SARS-CoV-2 in mouse models

In this study, we evaluated the immunogenicity and protective immunity of in vitro transcribed Venezuelan equine encephalitis virus (VEEV TC-83 strain) self-amplifying RNA (saRNA) encoding the SARS-CoV-2 spike (S) protein in wild type (S-WT) and stabilized pre-fusion conformations (S-PP). Immunization with S-WT and S-PP saRNA induced specific neutralizing antibody responses in both K18-Tg hACE2 (K18) and BALB/c mice, as assessed using SARS-CoV-2 pseudotyped viruses.Protective immunity was assessed in challenge experiments. Two immunizations with S-WT and S-PP induced protective immunity, evidenced by lower mortality, lower weight loss and more than one log10 lower subgenomic virus RNA titers in the upper and lower respiratory tracts in both K18 and BALB/c mice. Histopathologic examination of lungs post-challenge showed that immunization with S-WT and S-PP resulted in a higher degree of immune cell infiltration and inflammatory changes, compared with control mice, characterized by high levels of T- and B-cell infiltration. No substantial differences were found in the presence and localization of eosinophils, macrophages, neutrophils, and natural killer cells. CD4 and CD8 T-cell depletion post immunization resulted in reduced lung inflammation post challenge but also prolonged virus clearance. These data indicate that immunization with saRNA encoding the SARS-CoV-2 S protein induces immune responses that are protective following challenge, that virus clearance is associated with pulmonary changes caused by T-cell and B-cell infiltration in the lungs, but that this T and B-cell infiltration plays an important role in viral clearance.

Serum alpha-fetoprotein level (&gt; 10,000 U/mL) is a marker of hepatocellular carcinoma: A case study

Hepatocellular Carcinoma (HCC) development in young adults is strongly multifactorial. Important risk factors for the same are smoking, alcohol abuse and hepatitis C virus (HCV) infection at an early age. However, suspecting HCC is enigmatic at early age in which biochemical marker may aid in identifying such patients. In this article, we describe an unusual case of a 27-years-old male who presented with complaints of vague right upper abdominal fullness, non-bilious, non-projectile vomiting, loss of appetite, and significant weight loss for the past 2 months. On examination, there was firm, non-tender hepatomegaly. Patient had normal liver enzymes with very high alpha-fetoprotein (AFP) level (&gt; 10,000 U/mL). Ultrasoundabdomen suggested hetero-echoic lesion in liver along with chronic kidney disease (CKD) changes. The triple phase CT of the abdomen showed a liver mass with arterial enhancement and delayed washout suggestive of HCC. Chronic HCV infection was confirmed with high RNA titres (&gt; 50,000 IU/mL). Patient was diagnosed as HCC at an early stage, which allowed for timely initiation of treatment. This early age onset of HCC in a young adult may be due to multiple factors such as HCV infection, alcoholism, cirrhosis, and CKD. This single case study confirms that alcohol-induced liver injury increases the risk of developing early age HCC in persons infected with HCV and complicated with CKD. And AFP measurement helps inearly identification of HCC, especially at such high level &gt; 10,000 U/mL.

Assessment of Serum Interleukin-35 Levels in Iraqi Patients with Hepatitis C Virus Infection

Background: It is unclear how IL-35 contributes to chronic hepatitis C virus (HCV) infection. Objective: To evaluate the association of IL-35 with the viral load in HCV infections. Methods: Fifty patients were included, and their age and sex were matched with 50 healthy subjects. Seven ml of venous blood was obtained and tested for anti-HCV antibodies using the ELISA kit, quantitative measurement of HCV RNA by PCR, and estimation of serum levels of IL-35. Results: The HCV viral RNA titers ranged from 165 to 55595000 copies/mL and were positive for anti-HCV antibodies. The control group was negative. The serum level of IL-35 in the patients was significantly lower than in the controls. The correlation coefficient for HCV viral loads and IL-35 in patients is equal to 0.114. The serum IL-35 level was highest in the age group of 30–39 years. The HCV patients on treatment have a mean viral load equal to 406,3051.90 copies/ml and a mean IL-35 level equal to 151.98 ng/ml; those without treatment have a mean viral load equal to 765,525.94 copies/ml and an IL-35 level equal to 165.09 ng/ml. The gender distribution of HCV viral load revealed non-significant differences. Conclusion: Most of the HCV patients did not achieve rapid or sustained virologic responses after treatments. Serum IL-35 was significantly reduced and shows a weak positive correlation with HCV viral load. Older HCV patients usually had lower serum IL-35 levels.

Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR)

Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment.

Pre-existing, treatment-specific resistance-associated substitutions in hepatitis C virus genotype 1 and 3 and viral RNA titers during treatment with direct-acting antivirals.

The introduction of direct-acting antiviral (DAA) treatment of hepatitis C virus (HCV) infected patients has greatly increased treatment success rates. However, viral response kinetics to DAA treatment may depend on pre-existing resistance-associated substitutions (RASs) in HCV. The aim of this study was to describe how pre-existing RASs affect DAA treatment-induced reduction in HCV RNA titers in HCV genotypes 1- and 3-infected individuals. Patients with HCV genotype 1 infection (N = 31) treated with either sofosbuvir/ledipasvir/ribavirin or paritaprevir/ombitasvir/ritonavir/dasabuvir/ribavirin and HCV genotype 3-infected patients (N = 16) treated with either sofosbuvir/daclatasvir/ribavirin or sofosbuvir/ribavirin were analyzed. HCV RNA levels were determined at baseline and frequently during treatment, and RAS profiles were obtained by deep sequencing at baseline. In total, 33/47 (70.2%) of the patients had baseline RASs. However, treatment-specific RASs were detected at baseline only in 12.9% and 18.8% of HCV genotypes 1- and 3-infected patients, respectively. In genotype 1-infected individuals, reduction in HCV RNA titer during the first week of treatment was not affected by evidence of either treatment-specific RASs or cirrhosis or treatment regimen. In genotype 3-infected individuals receiving sofosbuvir/daclatasvir/ribavirin, the presence of daclatasvir-specific NS5A RASs at baseline correlated with a reduced decline of HCV RNA in the first treatment week. For both genotypes 1- and 3-infected individuals, cirrhosis but not treatment-specific RAS were associated with the time of clearance of HCV RNA. It is, however, important to note that this study involves DAA regimens that were used only during the original introduction of interferon-free DAA-based treatments.

Low Replication Efficiency of a Japanese Rabbit Hepatitis E Virus Strain in the Human Hepatocarcinoma Cell Line PLC/PRF/5.

A Japanese rabbit hepatitis E virus (HEV) strain, JP-59, has been identified in a feral rabbit. When this virus was transmitted to a Japanese white rabbit, it caused persistent HEV infection. The JP-59 strain shares an <87.5% nucleotide sequence identity with other rabbit HEV strains. Herein, to isolate JP-59 by cell culture, we used a 10% stool suspension recovered from a JP-59-infected Japanese white rabbit and contained 1.1 × 107 copies/mL of the viral RNA and using it to infect a human hepatocarcinoma cell line, PLC/PRF/5. No sign of virus replication was observed. Although long-term virus replication was observed in PLC/PRF/5 cells inoculated with the concentrated and purified JP-59 containing a high titer of viral RNA (5.1 × 108 copies/mL), the viral RNA of JP-59c that was recovered from the cell culture supernatants was <7.1 × 104 copies/mL during the experiment. The JP-59c strain did not infect PLC/PRF/5 cells, but its intravenous inoculation caused persistent infection in rabbits. The nucleotide sequence analyses of the virus genomes demonstrated that a total of 18 nucleotide changes accompanying three amino acid mutations occurred in the strain JP-59c compared to the original strain JP-59. These results indicate that a high viral RNA titer was required for JP-59 to infect PLC/PRF/5 cells, but its replication capability was extremely low. In addition, the ability of rabbit HEVs to multiply in PLC/PRF/5 cells varied depending on the rabbit HEV strains. The investigations of cell lines that are broadly susceptible to rabbit HEV and that allow the efficient propagation of the virus are thus needed.

CLEC18A Impairs Phagocytosis by Reducing FcγRIIA Expression and Arresting Autophagosome-Lysosome Fusion.

Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC (P < 0.005). We demonstrated that CLEC18A could inhibit FcγRIIA expression in a dose-dependent manner through the production of NOX-2-dependent reactive oxygen species to impair the uptake of ICs. Additionally, CLEC18A suppresses the Rab7 expression that is induced by starvation. Overexpressed CLEC18A does not affect autophagosome formation but does reduce the recruitment of Rab7 to autophagosomes, thereby retarding the maturation of autophagosomes and affecting autophagosome-lysosome fusion. We offer a novel molecular machinery with which to understand the association of HCV infection with autoimmunity and propose that CLEC18A may act as a candidate biomarker for HCV-associated MC. IMPORTANCE During infection, the host immune system produces cellular factors to protect against pathogen invasion. However, when the immune response overreacts and there is dysregulated cytokine homeostasis, autoimmunity occurs following an infection. We identified a cellular factor that is involved in HCV-related extrahepatic manifestation, namely, CLEC18A, which is expressed abundantly in hepatocytes and phagocytes. It inhibits HCV replication in hepatocytes by interacting with Rab5/7 and enhancing type I/III IFN expression. However, overexpressed CLEC18A inhibited FcγRIIA expression in phagocytes to impair phagocytosis. Furthermore, the interaction between CLEC18A and Rab5/7 may reduce the recruitment of Rab7 to autophagosomes and thereby retard autophagosome maturation and cause immune complex accumulation. A decreasing trend in CLEC18A levels that was accompanied by reduced HCV RNA titers and diminished cryoglobulin was observed in the sera of HCV-MC patients after direct-acting antiviral therapy. CLEC18A may be used for the evaluation of anti-HCV therapeutic drug effects and could be a potential predisposing factor for the development of MC syndrome.

Impact of SARS-CoV-2 RNA titer on the level of IgG antibodies in recovered patients with COVID-19 disease.

It is important to better understand the presence of neutralizing anti-SARS-CoV-2 antibodies in the population, as they potentially prevent (re)infection. To correlate the Cycle threshold (Ct value) of SARS-CoV-2 and its impact on specific Anti-SARS-CoV-2 IgG titer, to reveal the effect of age and disease severity on antibodies titer. A total of 153 infected participants laboratory-confirmed COVID-19 cases 4-11 months ago, aged 18-85 years old (mean = 43.58, SD ± 15.34) were enrolled in the study. They have not received any COVID-19 vaccine. A questionnaire was prepared including demographic data, age, gender, residence, and symptoms severity they suffered. Five mL of venous blood was taken from each participant to measure SARS-CoV-2 IgG antibodies against the receptor binding domain (RBD) by (VIDAS SARS-COV-2 IgG - Biomerieux kit). Ct values were measured by qRT-PCR (BIO-RAD-CFX96) kit which detected two virus genes, namely (RdRp-N genes). Lowest Ct values were detected significantly in age group 50-59 and 70-85 respectively. The highest mean of IgG was detected in age groups 70-85 and 50-59, and was found to be significantly correlated with disease severity. There is a direct relationship between Ct values and the titer of specific IgG, as increasing in viral load is associated with a higher level of antibodies. Antibodies were detected several months after infection with the highest mean after 10-11 months. Specific Anti-viral IgG are significantly associated with increasing age and disease severity, and the direct relation of IgG with viral load. Antibodies are detected several months post-infection but their protective efficacy is controversial.

Evaluation of a panel of therapeutic antibody clinical candidates for efficacy against SARS-CoV-2 in Syrian hamsters

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (−24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.