Presence and Persistence of Andes Virus RNA in Human Semen.

When infecting humans, Andes orthohantavirus (ANDV) may cause a severe disease called hantavirus cardiopulmonary syndrome (HCPS). Following non-specific symptoms, the infection may progress to a syndrome of hemorrhagic fever combined with hyper-acute cardiopulmonary failure. The case fatality rate ranges between 25-40%, depending on the outbreak. In this study, we present the follow-up of a male patient who recovered from HCPS six years ago. We demonstrate that the ANDV genome persists within the reproductive tract for at least 71 months. Genome sequence analysis early and late after infection reveals a low number of mutations (two single nucleotide variants and one deletion), suggesting limited replication activity. We can exclude the integration of the viral genome into the host genome, since the treatment of the specimen with RNAse led to a loss of signal. We demonstrate a long-lasting, strong neutralizing antibody response using pseudovirions expressing the ANDV glycoprotein. Taken together, our results show that ANDV has the potential for sexual transmission.

Virucidal activity of three standard chemical disinfectants against Ebola virus suspended in tripartite soil and whole blood.

Proper disinfection and inactivation of highly pathogenic viruses is an essential component of public health and prevention. Depending on environment, surfaces, and type of contaminant, various methods of disinfection must be both efficient and available. To test both established and novel chemical disinfectants against risk group 4 viruses in our maximum containment facility, we developed a standardized protocol and assessed the chemical inactivation of the two Ebola virus variants Mayinga and Makona suspended in two different biological soil loads. Standard chemical disinfectants ethanol and sodium hypochlorite completely inactivate both Ebola variants after 30 s in suspension at 70% and 0.5% v/v, respectively, concentrations recommended for disinfection by the World Health Organization. Additionally, peracetic acid is also inactivating at 0.2% v/v under the same conditions. Continued vigilance and optimization of current disinfection protocols is extremely important due to the continuous presence of Ebola virus on the African continent and increased zoonotic spillover of novel viral pathogens. Furthermore, to facilitate general pandemic preparedness, the establishment and sharing of standardized protocols is very important as it allows for rapid testing and evaluation of novel pathogens and chemical disinfectants.

Author Correction: In vitro virucidal activity of Echinaforce®, an Echinacea purpurea preparation, against coronaviruses, including common cold coronavirus 229E and SARS-CoV-2.

The original version of the acknowledgement unfortunately contained a mistake.

In vitro virucidal activity of Echinaforce®, an Echinacea purpurea preparation, against coronaviruses, including common cold coronavirus 229E and SARS-CoV-2.

BACKGROUND: Coronaviruses (CoVs) were long thought to only cause mild respiratory and gastrointestinal symptoms in humans but outbreaks of Middle East Respiratory Syndrome (MERS)-CoV, Severe Acute Respiratory Syndrome (SARS)-CoV-1, and the recently identified SARS-CoV-2 have cemented their zoonotic potential and their capacity to cause serious morbidity and mortality, with case fatality rates ranging from 4 to 35%. Currently, no specific prophylaxis or treatment is available for CoV infections. Therefore we investigated the virucidal and antiviral potential of Echinacea purpurea (Echinaforce®) against human coronavirus (HCoV) 229E, highly pathogenic MERS- and SARS-CoVs, as well as the newly identified SARS-CoV-2, in vitro. METHODS: To evaluate the antiviral potential of the extract, we pre-treated virus particles and cells and evaluated remaining infectivity by limited dilution. Furthermore, we exposed cells to the extract after infection to further evaluate its potential as a prophylaxis and treatment against coronaviruses. We also determined the protective effect of Echinaforce® in re-constituted nasal epithelium. RESULTS: In the current study, we found that HCoV-229E was irreversibly inactivated when exposed to Echinaforce® at 3.2 µg/ml IC50. Pre-treatment of cell lines, however, did not inhibit infection with HCoV-229E and post-infection treatment had only a marginal effect on virus propagation at 50 µg/ml. However, we did observe a protective effect in an organotypic respiratory cell culture system by exposing pre-treated respiratory epithelium to droplets of HCoV-229E, imitating a natural infection. The observed virucidal activity of Echinaforce® was not restricted to common cold coronaviruses, as both SARS-CoV-1 and MERS-CoVs were inactivated at comparable concentrations. Finally, the causative agent of COVID-19, SARS-CoV-2 was also inactivated upon treatment with 50µg/ml Echinaforce®. CONCLUSIONS: These results show that Echinaforce® is virucidal against HCoV-229E, upon direct contact and in an organotypic cell culture model. Furthermore, MERS-CoV and both SARS-CoV-1 and SARS-CoV-2 were inactivated at similar concentrations of the extract. Therefore we hypothesize that Echinacea purpurea preparations, such as Echinaforce®, could be effective as prophylactic treatment for all CoVs due to their structural similarities.

Standardized focus assay protocol for biosafety level four viruses.

Working in accordance with biosafety level four practices is highly complex and time-consuming. Therefore, the respective laboratory protocols should be as uniform as possible, simple to perform and straightforward in readout. Here we describe the successful application of a standardized 24-well plate focus assay protocol for the titration of Zaire ebolavirus (two isolates), Marburg virus (three isolates), Lassa virus (two isolates), Crimean Congo hemorrhagic fever virus (one isolate), and tick-borne encephalitis virus (two isolates). Viral titers are determined based on a simple visual readout. The protocol exhibits high precision, with coefficients of variation for interassay variability ranging between 0.05 and 0.21 and those for intraassay variability between 0.08 and 0.23. All reagents required for the test, including primary and secondary antibodies, are commercially available, facilitating the establishment of the protocol in other laboratories.

Limited Correlation of Shotgun Metagenomics Following Host Depletion and Routine Diagnostics for Viruses and Bacteria in Low Concentrated Surrogate and Clinical Samples.

The etiologic cause of encephalitis, meningitis or meningo-encephalitis is unknown in up to 70% of cases. Clinical shotgun metagenomics combined with host depletion is a promising technique to identify infectious etiologies of central nervous system (CNS) infections. We developed a straightforward eukaryotic host nucleic acid depletion method that preserves intact viruses and bacteria for subsequent shotgun metagenomics screening of clinical samples, focusing on cerebrospinal fluid (CSF). A surrogate CSF sample for a CNS infection paradigm was used to evaluate the proposed depletion method consisting of selective host cell lysis, followed by enzymatic degradation of the liberated genomic DNA for final depletion with paramagnetic beads. Extractives were subjected to reverse transcription, followed by whole genome amplification and next generation sequencing. The effectiveness of the host depletion method was demonstrated in surrogate CSF samples spiked with three 1:100 dilutions of Influenza A H3N2 virus (qPCR Ct-values 20.7, 28.8, >42/negative). Compared to the native samples, host depletion increased the amount of the virus subtype reads by factor 7127 and 132, respectively, while in the qPCR negative sample zero vs. 31 (1.4E-4 %) virus subtype reads were detected (native vs. depleted). The workflow was applied to thirteen CSF samples of patients with meningo-/encephalitis (two bacterial, eleven viral etiologies), a serum of an Andes virus infection and a nose swab of a common cold patient. Unlike surrogate samples, host depletion of the thirteen human CSF samples and the nose swab did not result in more reads indicating presence of damaged pathogens due to, e.g., host immune response. Nevertheless, previously diagnosed pathogens in the human CSF samples (six viruses, two bacteria), the serum, and the nose swab (Human rhinovirus A31) were detected in the depleted and/or the native samples. Unbiased evaluation of the taxonomic profiles supported the diagnosed pathogen in two native CSF samples and the native and depleted serum and nose swab, while detecting various contaminations that interfered with pathogen identification at low concentration levels. In summary, damaged pathogens and contaminations complicated analysis and interpretation of clinical shotgun metagenomics data. Still, proper consideration of these issues may enable future application of metagenomics for clinical diagnostics.

Evaluation of antivirals against tick-borne encephalitis virus in organotypic brain slices of rat cerebellum.

Neurotropic tick borne encephalitis virus (TBEV) causes life-threatening disease, and accounts for most cases of tick-transmitted viral infections in Central and Eastern Europe and Russia. No specific treatment for TBEV infections exists, and vaccination is recommended for people at risk. So far, various nucleoside analogues have been investigated in vitro as potential candidates for treatment of TBEV infections. However, in vitro experiments with more complex cell culture systems, such as organotypic culture slices which model the sophisticated architecture of the target tissue are lacking. Using TBEV as a model, we investigated the suitability of rat organotypic cerebellum slices (OCS) to study the effectiveness of nucleoside analogues with a well-known anti-TBEV activity. In these OCS, 50 µM of the nucleoside analogues 2'-C-methyladenosine (2'-CMA) and especially 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) exhibited strong inhibitory effects on TBEV replication, reducing viral titers to an average of 103-fold and TBEV RNA content 60-90-fold. In contrast, the influence of 2'-C-methylcytidine (2'-CMC) on TBEV replication was very weak, reducing virus titers by 10-fold and TBEV RNA content by 3-fold. In agreement with other studies, there was no noticeable difference in TBEV titers between OCS treated with 50 µM of Ribavirin and the DMSO treated controls. All tested nucleoside analogues exhibited excellent cytotoxicity profiles at concentrations of 50 µM. Our findings in OCS were highly comparable to data obtained in cell line culture systems. Therefore, OCS represent an ideal in vitro approach to study antivirals against TBEV and possibly other neurotropic viruses.

Prevalence of tick-borne pathogens in questing Ixodes ricinus ticks in urban and suburban areas of Switzerland.

BACKGROUND: Throughout Europe, Ixodes ricinus transmits numerous pathogens. Its widespread distribution is not limited to rural but also includes urbanized areas. To date, comprehensive data on pathogen carrier rates of I. ricinus ticks in urban areas of Switzerland is lacking. RESULTS: Ixodes ricinus ticks sampled at 18 (sub-) urban collection sites throughout Switzerland showed carrier rates of 0% for tick-borne encephalitis virus, 18.0% for Borrelia burgdorferi (sensu lato), 2.5% for Borrelia miyamotoi, 13.5% for Rickettsia spp., 1.4% for Anaplasma phagocytophilum, 6.2% for "Candidatus Neoehrlichia mikurensis", and 0.8% for Babesia venatorum (Babesia sp., EU1). Site-specific prevalence at collection sites with n > 45 ticks (n = 9) significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis", but were not related to the habitat type. Three hundred fifty eight out of 1078 I. ricinus ticks (33.2%) tested positive for at least one pathogen. Thereof, about 20% (71/358) were carrying two or three different potentially disease-causing agents. Using next generation sequencing, we could detect true pathogens, tick symbionts and organisms of environmental or human origin in ten selected samples. CONCLUSIONS: Our data document the presence of pathogens in the (sub-) urban I. ricinus tick population in Switzerland, with carrier rates as high as those in rural regions. Carriage of multiple pathogens was repeatedly observed, demonstrating the risk of acquiring multiple infections as a consequence of a tick bite.

Antiviral Innate Immune Activation in HIV-Infected Adults Negatively Affects H1/IC31-Induced Vaccine-Specific Memory CD4+ T Cells.

Tuberculosis (TB) remains a global health problem, with vaccination being a necessary strategy for disease containment and elimination. A TB vaccine should be safe and immunogenic as well as efficacious in all affected populations, including HIV-infected individuals. We investigated the induction and maintenance of vaccine-induced memory CD4(+) T cells following vaccination with the subunit vaccine H1/IC31. H1/IC31 was inoculated twice on study days 0 and 56 among HIV-infected adults with CD4(+) lymphocyte counts of >350 cells/mm(3). Whole venous blood stimulation was conducted with the H1 protein, and memory CD4(+) T cells were analyzed using intracellular cytokine staining and polychromatic flow cytometry. We identified high responders, intermediate responders, and nonresponders based on detection of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) expressing central (TCM) and effector memory CD4(+) T cells (TEM) 182 days after the first immunization. Amplicon-based transcript quantification using next-generation sequencing was performed to identify differentially expressed genes that correlated with vaccine-induced immune responses. Genes implicated in resolution of inflammation discriminated the responders from the nonresponders 3 days after the first inoculation. The volunteers with higher expression levels of genes involved in antiviral innate immunity at baseline showed impaired H1-specific TCM and TEM maintenance 6 months after vaccination. Our study showed that in HIV-infected volunteers, expression levels of genes involved in the antiviral innate immune response affected long-term maintenance of H1/IC31 vaccine-induced cellular immunity. (The clinical trial was registered in the Pan African Clinical Trials Registry [PACTR] with the identifier PACTR201105000289276.).

Safety and immunogenicity of H1/IC31®, an adjuvanted TB subunit vaccine, in HIV-infected adults with CD4+ lymphocyte counts greater than 350 cells/mm3: a phase II, multi-centre, double-blind, randomized, placebo-controlled trial.

BACKGROUND: Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1) formulated with the adjuvant IC31, was evaluated in HIV-infected adults. METHODS: HIV-infected adults with CD4+ T cell counts >350/mm3 and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5:1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay. RESULTS: 47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients (65.0% vs. 12.5%, p = 0.015). Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells. CONCLUSION: H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mm3. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response. TRIAL REGISTRATION: Pan African Clinical Trials Registry (PACTR) PACTR201105000289276.