The roles of XJ13 and XJ44-specific mutations within the Candid #1 GPC in Junin virus attenuation.

Junin virus (JUNV) is a member of the Arenaviridae family of viruses and is the pathogen responsible for causing Argentine hemorrhagic fever, a potentially lethal disease endemic to Argentina. A live attenuated vaccine for human use, called Candid#1, is approved only in Argentina. Candid#1 vaccine strain of Junin virus was obtained through serial passage in mouse brain tissues followed by passage in Fetal Rhesus macaque lung fibroblast (FRhL) cells. Previously, the mutations responsible for attenuation of this virus in Guinea pigs were mapped in the gene encoding for glycoprotein precursor (GPC) protein. The resulting Candid#1 glycoprotein complex has been shown to cause endoplasmic reticulum (ER) stress in vitro resulting in the degradation of the GPC. To evaluate the attenuating properties of specific mutations within GPC, we created recombinant viruses expressing GPC mutations specific to key Candid#1 passages and evaluated their pathogenicity in our outbred Hartley guinea pig model of Argentine hemorrhagic fever. Here, we provide evidence that early mutations in GPC obtained through serial passaging attenuate the visceral disease and increase immunogenicity in guinea pigs. Specific mutations acquired prior to the 13th mouse brain passage (XJ13) are responsible for attenuation of the visceral disease while having no impact on the neurovirulence of Junin virus. Additionally, our findings demonstrate that the mutation within an N-linked glycosylation motif, acquired prior to the 44th mouse brain passage (XJ44), is unstable but necessary for complete attenuation and enhanced immunogenicity of Candid#1 vaccine strain. The highly conserved N-linked glycosylation profiles of arenavirus glycoproteins could therefore be viable targets for designing attenuating viruses for vaccine development against other arenavirus-associated illnesses.

Depletion of CD4 and CD8 T Cells Reduces Acute Disease and Is Not Associated with Hearing Loss in ML29-Infected STAT1-/- Mice.

Lassa virus (LASV) is a zoonotic virus endemic to western Africa that can cause a potentially lethal and hemorrhagic disease, Lassa fever (LF). Survivors suffer a myriad of sequelae, most notably sudden onset sensorineural hearing loss (SNHL), the mechanism of which remains unclear. Unfortunately, studies aiming to identify the mechanism of these sequelae are limited due to the biosafety level 4 (BSL4) requirements of LASV itself. ML29, a reassortant virus proposed as an experimental vaccine candidate against LASV, is potentially an ideal surrogate model of LF in STAT1-/- mice due to similar phenotype in these animals. We intended to better characterize ML29 pathogenesis and potential sequelae in this animal model. Our results indicate that while both CD4 and CD8 T cells are responsible for acute disease in ML29 infection, ML29 induces significant hearing loss in a mechanism independent of either CD4 or CD8 T cells. We believe that this model could provide valuable information for viral-associated hearing loss in general.

Machupo Virus with Mutations in the Transmembrane Domain and Glycosylation Sites of the Glycoprotein Is Attenuated and Immunogenic in Animal Models of Bolivian Hemorrhagic Fever.

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuates MACV in an interferon alpha/beta and gamma (IFN-α/ß and -γ) receptor knockout (R-/-) mouse model. However, some capability to induce neurological pathology still remained. The highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. An F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in the GPC TMD. The MACV mutant is fully attenuated in IFN-α/ß and -γ R-/- mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV completely protected guinea pigs from wild-type MACV lethal challenge. Last, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which provide insight into the rational design of potential vaccine candidates for highly pathogenic arenaviruses. IMPORTANCE For arenaviruses, the only vaccine available is the live attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.

Luminore CopperTouch Surface Coating Effectively Inactivates SARS-CoV-2, Ebola Virus, and Marburg Virus In Vitro.

We investigated the ability of Luminore CopperTouch copper and copper-nickel surfaces to inactivate filoviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The copper and copper-nickel surfaces inactivated 99.9% of Ebola and Marburg viruses after 30 min, and the copper surfaces inactivated 99% of SARS-CoV-2 in 2 h. These data reveal that Ebola virus, Marburg virus, and SARS-CoV-2 are inactivated by exposure to copper ions, validating Luminore CopperTouch as an efficacious tool for infection control.

Luminore CopperTouch™ surface coating effectively inactivates SARS-CoV-2, Ebola and Marburg viruses in vitro.

We investigated the ability of Luminore CopperTouch™ copper and copper-nickel surfaces to inactivate filoviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For this purpose, we compared viral titers in Vero cells from viral droplets exposed to copper surfaces for 30 min. The copper and copper-nickel surfaces inactivated 99.9% of the viral titer of both Ebola and Marburg viruses. The copper surfaces also inactivated 99% of SARS-CoV-2 titers in 2 hours to close to the limit of detection. These data add Ebolavirus, Marburgvirus, and SARS-CoV-2 (COVID-19) to the list of pathogens that can be inactivated by exposure to copper ions, validating Luminore CopperTouch™ technology (currently the only Environmental Protection Agency [EPA]-registered cold spray antimicrobial surface technology) as an efficacious, cost-friendly tool to improve infection control in hospitals, long-term care facilities, schools, hotels, buses, trains, airports, and other highly trafficked areas.

Vaccination has minimal impact on the intrahost diversity of H3N2 influenza viruses.

While influenza virus diversity and antigenic drift have been well characterized on a global scale, the factors that influence the virus' rapid evolution within and between human hosts are less clear. Given the modest effectiveness of seasonal vaccination, vaccine-induced antibody responses could serve as a potent selective pressure for novel influenza variants at the individual or community level. We used next generation sequencing of patient-derived viruses from a randomized, placebo-controlled trial of vaccine efficacy to characterize the diversity of influenza A virus and to define the impact of vaccine-induced immunity on within-host populations. Importantly, this study design allowed us to isolate the impact of vaccination while still studying natural infection. We used pre-season hemagglutination inhibition and neuraminidase inhibition titers to quantify vaccine-induced immunity directly and to assess its impact on intrahost populations. We identified 166 cases of H3N2 influenza over 3 seasons and 5119 person-years. We obtained whole genome sequence data for 119 samples and used a stringent and empirically validated analysis pipeline to identify intrahost single nucleotide variants at ≥1% frequency. Phylogenetic analysis of consensus hemagglutinin and neuraminidase sequences showed no stratification by pre-season HAI and NAI titer, respectively. In our study population, we found that the vast majority of intrahost single nucleotide variants were rare and that very few were found in more than one individual. Most samples had fewer than 15 single nucleotide variants across the entire genome, and the level of diversity did not significantly vary with day of sampling, vaccination status, or pre-season antibody titer. Contrary to what has been suggested in experimental systems, our data indicate that seasonal influenza vaccination has little impact on intrahost diversity in natural infection and that vaccine-induced immunity may be only a minor contributor to antigenic drift at local scales.