Determinants of time to viral clearance among SARS-CoV-2 infected individuals at Millennium COVID-19 care center in Ethiopia: A prospective observational study

Background: Understanding the COVID-19 disease course in terms of viral shedding is important to assist in providing a tailored isolation and treatment practice. Therefore, the current study aimed to estimate time to viral clearance and identify determinants among SARS-CoV-2 infected individuals admitted to Millennium COVID-19 Care Center in Ethiopia. Methods: A Prospective observational study was conducted among 360 randomly selected SARS-CoV-2 infected individuals who were on follow up from 2nd June to 5th July 2020. Kaplan Meier plots, median survival times, and Log-rank test were used to describe the data and compare survival distribution between groups. Association between time to viral clearance and determinants was assessed using the Cox proportional hazard survival model, where hazard ratio, P-value, and 95% CI for hazard ratio were used for testing significance Results: The Median time to viral clearance was 16 days. The log-rank test shows that having moderate and severe disease, one or more symptoms at presentation, and presenting with respiratory and constitutional symptoms seems to extend the time needed to achieve viral clearance. The Final Cox regression result shows that the rate of achieving viral clearance among symptomatic patients was 44% lower than patients who were asymptomatic (AHR=0.560, 95% CI=0.322-0.975, p-value=0.040). Conclusions: Presence of symptoms was found to be associated with delayed viral clearance implying that symptomatic patients are more likely to be infectious and therefore, attention should be paid to the practices regarding isolation and treatment of COVID-19 patients.

IL-17A involved in respiratory syncytial virus-associated pathogenesis by promoting IFN-γand inhib-iting viral clearance in mice / 中华微生物学和免疫学杂志

Objective To identify the role of IL-17A during respiratory syncytial virus (RSV) in-fection in a mouse model. Methods Female wild-type C57BL/6 mice and IL-17A knockout ( IL-17A-/-) mice at the age of 6 to 8 weeks were both randomly divided into two groups:control and RSV groups. Mice in the control groups were given the supernatant of Hep-2 cell culture, while those in the RSV groups were treated with RSV A2 through intranasal administration. Leukocytes in bronchoalveolar lavage fluid ( BALF) samples were counted. Left lung tissues were stained with hematoxylin and eosin ( HE ) to evaluate his-topathological scores. Airway hyperresponsiveness ( AHR) was measured by whole-body plethysmography. The concentrations of IFN-γ were determined with ELISA. RSV titers were measured by plaque assay. To assess the effects of IL-17A on IFN-γproduction and its role in RSV infection, IL-17A-/- mice were treated with exogenous recombinant murine IFN-γ or IL-17A, while wild-type mice were given IFN-γ neutralizing antibody intervention. Results The counts of inflammatory cells and neutrophils in BALF, lung tissue his-topathological scores, AHR, IFN-γlevels and virus titers of the wild-type group were higher than those of the IL-17A-/-group after RSV infection. IFN-γlevels, inflammatory cell counts in BALF, AHR and lung tissue histopathological scores were significantly increased in RSV-infected IL-17A-/- mice after the intervention of recombinant murine IL-17A or IFN-γ. RSV titers were much higher in the recombinant murine IL-17A-trea-ted group, but not affected by the recombinant murine IFN-γ intervention. Inflammatory cell counts in BALF, AHR and lung tissue histopathological scores were significantly decreased in RSV-infected wild-type mice following IFN-γ neutralizing antibody treatment, but no significant changes were found in RSV titers. Conclusions IL-17A might be involved in the pathogenesis of pulmonary diseases during RSV infection through promoting IFN-γ production and inhibiting viral clearance in mice.

Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant.

Aim: To investigate if any mutations in hepatitis C virus (HCV) internal ribosome entry site (IRES) can inhibit the translation of viral polyprotein. Materials and Methods: A 26-year-old male patient infected with HCV 10 years ago was followed up. After 9 years of chronic infection. The patient had managed to resolve the infection for a period of 9 months, after which the patient experienced a viral recurrence characterized by high viral load and diverse HCV quasispecies. The IRES structures of the viral strains that disappeared were comparable with those that are currently active using structural mutational analysis. Results: A novo mutational position 254 combined with a rarely observed mutation at position 253 in the stem of the IIId subdomain were observed and the new conformation had an octa-apical loop (AGUGUUGG) and a shift in the 3 ` GU from the loop to the stem. Conclusions: These mutations were found to be highly deleterious, and they affected the direct binding of the IIId loop to the 40S ribosomal subunit with a subsequent inhibition of translation of viral polyprotein and clearance of the virus.

Predictors of spontaneous viral clearance and outcomes of acute hepatitis C infection

BACKGROUND/AIMS: This study evaluated the predictors of spontaneous viral clearance (SVC), as defined by two consecutive undetectable hepatitis C virus (HCV) RNA tests performed > or =12 weeks apart, and the outcomes of acute hepatitis C (AHC) demonstrating SVC or treatment-induced viral clearance. METHODS: Thirty-two patients with AHC were followed for 12-16 weeks without administering antiviral therapy. RESULTS: HCV RNA was undetectable at least once in 14 of the 32 patients. SVC occurred in 12 patients (37.5%), among whom relapse occurred in 4. SVC was exhibited in 8 of the 11 patients exhibiting undetectable HCV RNA within 12 weeks. HCV RNA reappeared in three patients (including two patients with SVC) exhibiting undetectable HCV RNA after 12 weeks. SVC was more frequent in patients with low viremia than in those with high viremia (55.6% vs. 14.3%; P=0.02), and in patients with HCV genotype non-1b than in those with HCV genotype 1b (57.1% vs. 22.2%; P=0.04). SVC was more common in patients with a > or =2 log reduction of HCV RNA at 4 weeks than in those with a smaller reduction (90% vs. 9.1%, P or =2 log reduction of HCV RNA at 4 weeks was a follow-up predictor for SVC. Undetectable HCV RNA occurring after 12 weeks was not sustained. All patients receiving antiviral therapy achieved a sustained viral response. Antiviral therapy should be initiated in patients with detectable HCV RNA at 12 weeks after the diagnosis.

Study of the immunogenicity and virulence of replication-competent recombinant vaccinia virus in CD4+T cell-depleted mice / 中华微生物学和免疫学杂志

Objective To investigate the effects of CD4+T cell depletion in BALB/c mice on the immunogenicity and virulence of replication-competent recombinant vaccinia virus. Methods Twelve BALB/c mice were inoculated with recombinant Tiantan vaccinia ( rTV, n=8 ) or vaccinia virus Tiantan strain ( VTT, n=4) by tail scarification after the depletion of CD4+T cells with anti-CD4 monoclonal anti-body( McAb) injected intraperitoneally for three days before virus inoculation.A control group without anti-body treatment was set up accordingly.Several parameters including the body weight, pocks on tail, viral shedding and the percentage of CD4+T cells were monitored.In the fourth week after virus infection, ovaries were collected from mice and viral loads in the tissue were titrated by plaque forming assay on chick embryo fibroblast ( CEF) cells.The specific cellular immune responses against vaccinia virus and HIV induced by inoculation of VTT and rTV respectively were detected by intracellular cytokine staining ( ICS) assay.ELISA was used to detect antibodies against vaccinia virus and HIV-1 gp120.Results All mice with or without CD4 McAb treatment showed typical poxvirus pocks on tails after inoculation of vaccinia viruses, but none of them developed secondary or satellite lesions.It took a longer time for CD4+T cell-depleted mice to heal from lesions, to regain body weights and to release viruses than the mice in control group.No vaccinia virus was detected in the ovaries of CD4+T cell-depleted mice or mice in control group.The mean absorbance( A) values for the detection of HIV-specific and vaccinia virus-specific antibodies in CD4+T cell-depleted mice with ELISA were respectively 0.119 and 0.168, which were significantly lower than those in mice of control group.The titers of neutralizing antibodies against vaccinia virus in McAb/rTV treated mice (1 ∶321) were lower than those in rTV treated mice (1 ∶1286) (P<0.05).The percentages of CD4+T cells secreting IFN-γ(0.654%) in McAb/rTV treated mice were significantly lower than those in rTV treated mice in the fourth week after immunization (P <0.0004).No significant differences with the vaccinia virus-specificCD8+ T cell responses were observed among mice with or without CD4+T cells depletion.Conclusion Thereplication and dissemination of replication-competent recombinant vaccinia virus could be effectively controlledin the mice with CD4+ T cell-depletion.The depletion of CD4+ T cells significantly reduced the humoraland CD4+ T cell responses, but had no effect on CD8+ T cell responses.

Delayed Viral Clearance of Chronic Hepatitis C in Patients after Treatment Failure

Hepatitis C virus (HCV) infection usually progresses to chronic hepatitis, with rare cases of spontaneous viral eradication. We present herein four cases involving patients that were initially declared to have failed to respond to treatments, based on the presence of HCV RNA that was still detectable after completion of the standard treatment for chronic hepatitis C with genotype 2. However, the HCV RNA became undetectable, with a delayed response, after discontinuation of therapy. Two of the four patients were diagnosed as treatment failures after extended treatment, and the other two received no further treatment after the standard treatment. All four patients maintained a sustained virological response during the periodic follow-up after delayed viral clearance.

Evaluation of JEV and BVDV Clearance During the Purification of Recombinant HPV-16 L1 Virus-Like Particles

Insect cell-derived biotechnological products have a potential for viral contamination from cell line sources themselves or from adventitious introduction of virus during production. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs) using Japanese encephalitis virus (JEV) and bovine viral diarrhea virus (BVDV) as relevant viruses. The downstream process for the production of recombinant HPV-16 L1 VLPs was sequentially carried out employing detergent lysis (NP-40/PBS), sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. Recombinant HPV-16 L1 capsid protein (56 kD) expressed in Sf9 cell culture was clearly detected by SDS-PAGE and Western blotting analysis. Each purification step was evaluated to determine reduction factor for viral clearance by infectivity assay. In individual purification steps, detergent treatment (0.50% v/v, NP-40/PBS) and CsCl equilibrium density centrifugation were found to be effective in JEV and BVDV clearance. Overall cumulative reduction factors of JEV and BVDV infectivity titer for the purification procedure implemented in this study were 12.53 and 10.05 log TCID(50)/pool, respectively. The results suggest that the purification procedure employed in this study for the HPV-16 L1 VLPs produced from recombinant baculovirus-infected Sf9 cells will be effective over 10 log TCID(50)/pool reduction factor in the clearance of enveloped, adventitious viruses with a buoyant density lower than approximately 1.23 g/ml.

A Quantitative Assay of Japanese Encephalitis Virus as a Model Virus for Viral Clearance Validation in Insect Cell-Derived Biotechnology Products

Commercial interests towards insect cell cultures have greatly been increased recently, in part due to the widespread use of insect virus-based vectors for efficient expressions of foreign proteins. Insect cell-derived biotechnology products should be free of adventitious agents such as arboviruses and mycoplasmas. The objective of this study was to establish techniques for the viral safety evaluation of insect cell-derived biotechnology products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses which has been known to be infectious to insect cells such as Sf9 cells. Quantitative assays for viral infectivity, concentrations of an antigen or a genome are a prerequisite for the studies of viral clearance validation. Here, we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. Viral RNA extracted from stock suspension of JEV with known infectivity titer was made to 10-fold serial dilution, and each dilution was subjected to the assay for the generation of a standard curve. A JEV specific primer was selected from 3' untranslated region with the expected band size of 323 base pairs. The real-time RT-PCR assay resulted in a successful amplification within 5 log dilution ranges of JEV RNA samples, and the sensitivity of the assay was calculated to be approximately 15 TCID50 per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. The real-time RT-PCR assay for the quantification of JEV will be an efficient alternative tool for viral clearance validation studies of insect cell-derived biotechnology products.