Virucidal efficacy of guanidine-free inactivants and rapid test buffers against SARS-CoV-2.

A pathogen inactivation step during collection or processing of clinical samples has the potential to reduce infectious risks associated with diagnostic procedures. It is essential that these inactivation methods are demonstrated to be effective, particularly for non-traditional inactivation reagents or for commercial products where the chemical composition is undisclosed. This study assessed inactivation effectiveness of twenty-four next-generation (guanidine-free) nucleic acid extraction lysis buffers and twelve rapid antigen test buffers against SARS-CoV-2, the causative agent of COVID-19. These data have significant safety implications for SARS-CoV-2 diagnostic testing and support the design and evidence-based risk assessment of these procedures.

Effective in vitro inactivation of SARS-CoV-2 by commercially available mouthwashes.

Infectious SARS-CoV-2 can be recovered from the oral cavities and saliva of COVID-19 patients with potential implications for disease transmission. Reducing viral load in patient saliva using antiviral mouthwashes may therefore have a role as a control measure in limiting virus spread, particularly in dental settings. Here, the efficacy of SARS-CoV-2 inactivation by seven commercially available mouthwashes with a range of active ingredients were evaluated in vitro. We demonstrate ≥4.1 to ≥5.5 log10 reduction in SARS-CoV-2 titre following a 1 min treatment with commercially available mouthwashes containing 0.01-0.02 % stabilised hypochlorous acid or 0.58 % povidone iodine, and non-specialist mouthwashes with both alcohol-based and alcohol-free formulations designed for home use. In contrast, products containing 1.5 % hydrogen peroxide or 0.2 % chlorhexidine gluconate were ineffective against SARS-CoV-2 in these tests. This study contributes to the growing body of evidence surrounding virucidal efficacy of mouthwashes/oral rinses against SARS-CoV-2, and has important applications in reducing risk associated with aerosol generating procedures in dentistry and potentially for infection control more widely.

Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives.

The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.

Utilisation of Chimeric Lyssaviruses to Assess Vaccine Protection against Highly Divergent Lyssaviruses.

Lyssaviruses constitute a diverse range of viruses with the ability to cause fatal encephalitis known as rabies. Existing human rabies vaccines and post exposure prophylaxes (PEP) are based on inactivated preparations of, and neutralising antibody preparations directed against, classical rabies viruses, respectively. Whilst these prophylaxes are highly efficient at neutralising and preventing a productive infection with rabies virus, their ability to neutralise other lyssaviruses is thought to be limited. The remaining 15 virus species within the lyssavirus genus have been divided into at least three phylogroups that generally predict vaccine protection. Existing rabies vaccines afford protection against phylogroup I viruses but offer little to no protection against phylogroup II and III viruses. As such, work involving sharps with phylogroup II and III must be considered of high risk as no PEP is thought to have any effect on the prevention of a productive infection with these lyssaviruses. Whilst rabies virus itself has been characterised in a number of different animal models, data on the remaining lyssaviruses are scarce. As the lyssavirus glycoprotein is considered to be the sole target of neutralising antibodies we generated a vaccine strain of rabies using reverse genetics expressing highly divergent glycoproteins of West Caucasian Bat lyssavirus and Ikoma lyssavirus. Using these recombinants, we propose that recombinant vaccine strain derived lyssaviruses containing heterologous glycoproteins may be a suitable surrogate for wildtype viruses when assessing vaccine protection for the lyssaviruses.

Rescue of a vaccine strain of peste des petits ruminants virus: In vivo evaluation and comparison with standard vaccine.

Across the developing world peste des petits ruminants virus places a huge disease burden on agriculture, primarily affecting the production of small ruminant. The disease is most effectively controlled by vaccinating sheep and goats with live attenuated vaccines that provide lifelong immunity. However, the current vaccines and serological tests are unable to enable Differentiation between naturally Infected and Vaccinated Animals (DIVA). This factor precludes meaningful assessment of vaccine coverage and epidemiological surveillance based on serology, in turn reducing the efficiency of control programmes. The availability of a recombinant PPRV vaccine with a proven functionality is a prerequisite for the development of novel vaccines that may enable the development of DIVA tools for PPRV diagnostics. In this study, we have established an efficient reverse genetics system for PPRV Nigeria 75/1 vaccine strain and, further rescued a version of PPRV Nigeria 75/1 vaccine strain that expresses eGFP as a novel transcription cassette and a version of PPRV Nigeria 75/1 vaccine strain with mutations in the haemagglutinin (H) gene to enable DIVA through disruption of binding to H by the C77 monoclonal antibody used in the competitive (c) H-ELISA. All three rescued viruses showed similar growth characteristics in vitro in comparison to parent vaccine strain and, following in vivo assessment the H mutant provided full protection in goats. Although the C77 monoclonal antibody used in the cH-ELISA was unable to bind to the mutated form of H in vitro, the mutation was not sufficient to enable DIVA in vivo.

Morbillivirus vaccines: recent successes and future hopes.

The impact of morbilliviruses on both human and animal populations is well documented in the history of mankind. Indeed, prior to the development of vaccines for these diseases, morbilliviruses plagued both humans and their livestock that were heavily relied upon for food and motor power within communities. Measles virus (MeV) was responsible for the death of millions of people annually across the world and those fortunate enough to escape the disease often faced starvation where their livestock had died following infection with rinderpest virus (RPV) or peste des petits ruminants virus (PPRV). Canine distemper virus has affected dog populations for centuries and in the past few decades appears to have jumped species, now causing disease in a number of non-canid species, some of which are been pushed to the brink of extinction by the virus. During the age of vaccination, the introduction and successful application of vaccines against rinderpest and measles has led to the eradication of the former and the greater control of the latter. Vaccines against PPR and canine distemper have also been generated; however, the diseases still pose a threat to susceptible species. Here we review the currently available vaccines against these four morbilliviruses and discuss the prospects for the development of new generation vaccines.

A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion.

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.

A novel approach to generating morbillivirus vaccines: negatively marking the rinderpest vaccine.

The eradication of rinderpest virus (RPV) from the globe was possible through the availability of a safe and effective live attenuated vaccine and a suitable companion diagnostic test. However, the inability to serologically 'Differentiate between naturally Infected and Vaccinated Animals' (DIVA) meant that both the time taken to complete the eradication programme and the economic burden on countries involved was significantly greater than if a vaccine and companion diagnostic test that fulfilled the DIVA concept had been available. During the RPV eradication campaign serosurveillance for RPV was primarily based on a competitive ELISA using a RPV specific (C1) monoclonal antibody (mAb) directed against the viral haemagglutinin (H) protein but this test was not able to meet DIVA requirements. To provide proof of concept for the generation of novel morbillivirus DIVA vaccines we have identified, by phage display, and mutated residues critical for C1 mAb binding and assessed the functionality of mutants in an in vitro fusion assay. Finally we have incorporated mutated epitopes into a full length clone and rescued recombinant RPV using reverse genetics techniques. Here we describe a novel mechanism of marking morbillivirus vaccines, using RPV as a proof of concept, and discuss the applicability of this method to the development of marked vaccines for peste des petits ruminants virus (PPRV).

A real time RT-PCR assay for the specific detection of Peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) causes a devastating disease of small ruminants present across much of Africa and Asia. Recent surveillance activities and phylogenetic analyses have suggested that the virus is an emerging problem as it is now being detected in areas previously free of the disease. As such, the virus not only is threatening small ruminant production and agricultural stability in the developing world, but also poses an economic threat to livestock in the European Union (EU) through introduction from European Turkey and North Africa. This report describes the development of a high throughput, rapid, real time RT-PCR method for the sensitive and specific detection of PPRV using robotic RNA extraction. This assay targets the nucleocapsid (N) gene of PPRV and has been shown to detect all four genetic lineages of PPRV in tissues, ocular and nasal swabs and blood samples collected in the field. The lowest detection limit achieved was approximately 10 genome copies/reaction, making this assay an ideal tool for the sensitive and rapid detection of PPRV in diagnostic laboratories.

Epidemiology, pathology, and genetic analysis of a canine distemper epidemic in Namibia.

Severe population declines have resulted from the spillover of canine distemper virus (CDV) into susceptible wildlife, with both domestic and wild canids being involved in the maintenance and transmission of the virus. This study (March 2001 to October 2003) collated case data, serologic, pathologic, and molecular data to describe the spillover of CDV from domestic dogs (Canis familiaris) to black-backed jackals (Canis mesomelas) during an epidemic on the Namibian coast. Antibody prevalence in jackals peaked at 74.1%, and the clinical signs and histopathologic observations closely resembled those observed in domestic dog cases. Viral RNA was isolated from the brain of a domestic dog from the outbreak area. Sequence data from the phosphoprotein (P) gene and the hemagglutinin (H) genes were used for phylogenetic analyses. The P gene sequence from the domestic dog shared 98% identity with the sequence data available for other CDV isolates of African carnivores. For the H gene, the two sequences available from the outbreak that decimated the lion population in Tanzania in 1994 were the closest match with the Namibian sample, being 94% identical across 1,122 base pairs (bp). Phylogenetic analyses based on this region clustered the Namibian sample with the CDV that is within the morbilliviruses. This is the first description of an epidemic involving black-backed jackals in Namibia, demonstrating that this species has the capacity for rapid and large-scale dissemination of CDV. This work highlights the threat posed to endangered wildlife in Namibia by the spillover of CDV from domestic dog populations. Very few sequence data are currently available for CDV isolates from African carnivores, and this work provides the first sequence data from a Namibian CDV isolate.