Limited Effects of Type I Interferons on Kyasanur Forest Disease Virus in Cell Culture.

BACKGROUND:The tick-borne flavivirus, Kyasanur Forest disease virus (KFDV) causes seasonal infections and periodic outbreaks in south-west India. The current vaccine offers poor protection with reported issues of coverage and immunogenicity. Since there are no approved prophylactic therapeutics for KFDV, type I IFN-α/β subtypes were assessed for antiviral potency against KFDV in cell culture. METHODOLOGY/PRINCIPAL FINDINGS:The continued passage of KFDV-infected cells with re-administered IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production whereas the IFN-sensitive, green fluorescent protein-expressing vesicular stomatitis virus (VSV-GFP) infection was controlled. Further evaluation of the other IFN-α/β subtypes versus KFDV infection indicated that single treatments of either IFN-αWA and IFN-αΚ appeared to be more effective than IFN-α2a at reducing KFDV titres. Concentration-dependent analysis of these IFN-α/β subtypes revealed that regardless of subtype, low concentrations of IFN were able to limit cytopathic effects (CPE), while significantly higher concentrations were needed for inhibition of virion release. Furthermore, expression of the KFDV NS5 in cell culture before IFN addition enabled VSV-GFP to overcome the effects of IFN-α/β signalling, producing a robust infection. CONCLUSIONS/SIGNIFICANCE:Treatment of cell culture with IFN does not appear to be suitable for KFDV eradication and the assay used for such studies should be carefully considered. Further, it appears that the NS5 protein is sufficient to permit KFDV to bypass the antiviral properties of IFN. We suggest that other prophylactic therapeutics should be evaluated in place of IFN for treatment of individuals with KFDV disease

Evaluating Environmental Persistence and Disinfection of the Ebola Virus Makona Variant

Background: The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unprecedented morbidity and lethality given its geographic reach and sustained transmission. Sodium hypochlorite and ethanol are well-accepted decontamination agents, however little published evidence supports the selection of appropriate concentrations and contact times. The present study addresses the environmental robustness of EBOV/Mak and evaluates the effectiveness of sodium hypochlorite and ethanol as disinfectants. Methods: EBOV/Mak was suspended in a simulated organic soil load and dried onto surfaces. Viability was measured at 1 hour, 24 hours, 72 hours, and 192 hours. For the evaluation of disinfectants, EBOV/Mak in a simulated organic soil was dried onto stainless steel carriers and disinfected with 0.01% (v/v), 0.1% (v/v), 0.5% (v/v) and 1% (v/v) sodium hypochlorite solutions or 67% (v/v) ethanol at contact times of 1, 5 or 10 minutes. Results: EBOV/Mak persisted longer on steel and plastic surfaces (192 hours) than cotton (<24 hours). Dilute sodium hypochlorite (0.01% and 0.1%) showed little antiviral action, whereas 0.5% and 1% sodium hypochlorite solutions demonstrated recoverable virus at one minute but sterilized surfaces in five minutes. Disinfection with 67% ethanol did not fully clear infectious virions from 3/9 carriers at 1 minute but sterilized all carriers at 5 and 10 minutes. Conclusions: Sodium hypochlorite and ethanol effectively decontaminate EBOV/Mak suspended in a simulated organic load; however, selection of concentration and contact time proves critical

Nasopharyngeal angiotensin converting enzyme 2 (ACE2) expression as a risk-factor for SARS-CoV-2 transmission in concurrent hospital associated outbreaks

Abstract Background Widespread human-to-human transmission of the severe acute respiratory syndrome coronavirus two (SARS-CoV-2) stems from a strong affinity for the cellular receptor angiotensin converting enzyme two (ACE2). We investigate the relationship between a patient’s nasopharyngeal ACE2 transcription and secondary transmission within a series of concurrent hospital associated SARS-CoV-2 outbreaks in British Columbia, Canada. Methods Epidemiological case data from the outbreak investigations was merged with public health laboratory records and viral lineage calls, from whole genome sequencing, to reconstruct the concurrent outbreaks using infection tracing transmission network analysis. ACE2 transcription and RNA viral load were measured by quantitative real-time polymerase chain reaction. The transmission network was resolved to calculate the number of potential secondary cases. Bivariate and multivariable analyses using Poisson and Negative Binomial regression models was performed to estimate the association between ACE2 transcription the number of SARS-CoV-2 secondary cases. Results The infection tracing transmission network provided n = 76 potential transmission events across n = 103 cases. Bivariate comparisons found that on average ACE2 transcription did not differ between patients and healthcare workers (P = 0.86). High ACE2 transcription was observed in 98.6% of transmission events, either the primary or secondary case had above average ACE2. Multivariable analysis found that the association between ACE2 transcription (log2 fold-change) and the number of secondary transmission events differs between patients and healthcare workers. In health care workers Negative Binomial regression estimated that a one-unit change in ACE2 transcription decreases the number of secondary cases (β = -0.132 (95%CI: -0.255 to -0.0181) adjusting for RNA viral load. Conversely, in patients a one-unit change in ACE2 transcription increases the number of secondary cases (β = 0.187 (95% CI: 0.0101 to 0.370) adjusting for RNA viral load. Sensitivity analysis found no significant relationship between ACE2 and secondary transmission in health care workers and confirmed the positive association among patients. Conclusion Our study suggests that ACE2 transcription has a positive association with SARS-CoV-2 secondary transmission in admitted inpatients, but not health care workers in concurrent hospital associated outbreaks, and it should be further investigated as a risk-factor for viral transmission

Screening interferon-α/β subtypes against KFDV.

<p>Cultures of A549 cells were pre-treated (grey bars) 24 hours prior to infection or post-treated (black bars) 1 hour after infection with 1, 000 U/mL of IFN-α (B2, C, D, F, G, H2, I, J1, K, WA, 2a, 2b or 4b), IFN-β (beta-1) and a recombinant IFN-α (Universal) subtypes and infected with KFDV at a MOI of 1. Supernatants were harvested for each treatment after 72 hours of incubation and quantified (expressed in log₁₀ scale TCID₅₀/mL) on BHK-21 (ATCC) when the mock-treated control cells displayed CPE near 100%. Pre-infection treatment experiments were assayed in two biological replicates and post-infection treatment experiments were assayed in three biological replicates; the resulting averages and standard deviations are presented. Mock, Mock-treated with IFN. UI, Un-infected control. IFN-α2b was excluded from 24-hour pre-infection treatment. * Significant compared to mock-treated samples (P < 0.1). *** Significant compared to mock-treated samples (P < 0.01).</p

IFN-α2a does not clear KFDV infection in BHK-21 cells.

<p>BHK-21 cells were infected with a 11 TCID₅₀ units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID₅₀ assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log₁₀ scale TCID₅₀/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging Cell monolayers were photographed prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.</p

Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.

<p>Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.</p

KFDV NS5 impedes the cellular antiviral state.

<p><b>(A)</b> VeroE6 (ATCC) cells were transfected with plasmid encoding KFDV NS proteins and Ebola virus VP24 and treated with 1, 000 U/mL of Universal IFN, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, pictures were taken with fluorescent (top panel) and light (bottom panel) microscopy 24 hours later. <b>(B)</b> VeroE6 (ATCC) cells were transfected with KFDV NS5-pCAGGS and treated with 1, 000 U/mL of commercially available type I IFNs, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, 24 hours later, the virus-containing supernatants were harvested for virus quantification. Dark grey bars indicate experiments in which cells were un-transfected. Light grey bars indicate NS5-expressing cells. Mock, no IFN treatment of cells lacking NS5 expression (dark gray bar) and with NS5 expression (light gray bar); UI represents uninfected/un-treated cells. Universal IFN controls included VP24-pCAGGS as anti-IFN control. The graph represents the log₁₀ scale TCID₅₀/mL averages and standard deviations from three biological repetitions. ***, Significant difference of NS5-expressing cells compared to VP24-expressing cells (P < 0.01).</p

Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.

<p>Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.</p

IFN-α2a does not clear KFDV infection in A549 cells.

<p>A549 cells were infected with a 11 TCID₅₀ units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID₅₀ assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log₁₀ scale TCID₅₀/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.</p

Characterizing Longitudinal Antibody Responses in Recovered Individuals Following COVID-19 Infection and Single-Dose Vaccination: A Prospective Cohort Study

Background: Investigating antibody titers in individuals who have been both naturally infected with SARS-CoV-2 and vaccinated can provide insight into antibody dynamics and correlates of protection over time. Methods: Human coronavirus (HCoV) IgG antibodies were measured longitudinally in a prospective cohort of qPCR-confirmed, COVID-19 recovered individuals (k = 57) in British Columbia pre- and post-vaccination. SARS-CoV-2 and endemic HCoV antibodies were measured in serum collected between Nov. 2020 and Sept. 2021 (n = 341). Primary analysis used a linear mixed-effects model to understand the effect of single dose vaccination on antibody concentrations adjusting for biological sex, age, time from infection and vaccination. Secondary analysis investigated the cumulative incidence of high SARS-CoV-2 anti-spike IgG seroreactivity equal to or greater than 5.5 log10 AU/mL up to 105 days post-vaccination. No re-infections were detected in vaccinated participants, post-vaccination by qPCR performed on self-collected nasopharyngeal specimens. Results: Bivariate analysis (complete data for 42 participants, 270 samples over 472 days) found SARS-CoV-2 spike and RBD antibodies increased 14&ndash;56 days post-vaccination (p &lt; 0.001) and vaccination prevented waning (regression coefficient, B = 1.66 [95%CI: 1.45&ndash;3.46]); while decline of nucleocapsid antibodies over time was observed (regression coefficient, B = &minus;0.24 [95%CI: &minus;1.2-(&minus;0.12)]). A positive association was found between COVID-19 vaccination and endemic human &beta;-coronavirus IgG titer 14&ndash;56 days post vaccination (OC43, p = 0.02 &amp; HKU1, p = 0.02). On average, SARS-CoV-2 anti-spike IgG concentration increased in participants who received one vaccine dose by 2.06 log10 AU/mL (95%CI: 1.45&ndash;3.46) adjusting for age, biological sex, and time since infection. Cumulative incidence of high SARS-CoV-2 spike antibodies (&gt;5.5 log10 AU/mL) was 83% greater in vaccinated compared to unvaccinated individuals. Conclusions: Our study confirms that vaccination post-SARS-CoV-2 infection provides multiple benefits, such as increasing anti-spike IgG titers and preventing decay up to 85 days post-vaccination