Biology subjects

Gilliland, T.

Publications and source records attributed to Gilliland, T..

6 recordsLinked to original sources

bioRxiv2023

Structural and functional basis of VLDLR receptor usage by Eastern equine encephalitis virus

The very low-density lipoprotein receptor (VLDLR) is comprised of eight LDLR type A (LA) domains and supports entry of distantly related Eastern equine encephalitis (EEEV) and Semliki Forest (SFV) alphaviruses. Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage different LA domains simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection, highlighting complexity in domain usage. Whereas all EEEV strains show conservation of two VLDLR binding sites, the EEEV PE-6 strain and other EEE complex members feature a single amino acid substitution that mediates binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.

microbiology↗

bioRxiv2023

Seeding Activity of Skin Misfolded Tau as a Novel Biomarker for Tauopathies

Tauopathies are a group of age-related neurodegenerative diseases with a molecular hallmark of the prion-like propagation and accumulation of pathologically phosphorylated tau protein in the brain. They include Alzheimers disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Picks disease (PiD). Currently, in the peripheral tissues and body fluids there are no reliable diagnostic biomarkers available that are able to directly reflect the capability of propagation and spreading of the misfolded tau aggregates. Here, we revealed significantly increased amounts of phosphorylated tau in the skin of AD patients compared to those in other tauopathies and normal controls. Moreover, the seed-amplification assay (SAA) by the ultrasensitive real-time quaking-induced conversion (RT-QuIC) displayed that the prion-like seeding activity of pathological tau in the skin of cadavers with neuropathologically confirmed tauopathies including AD, PSP, CBD, PiD was dramatically higher than that in normal controls, yielding 75-80% sensitivity and 95-100% specificity, respectively, depending on different tau substrates used. The increased tau-seeding activity was also observed in biopsy skin samples from living AD and PSP patients. Moreover, analysis of the end products of skin-tau SAA confirmed that the increased seeding activity is accompanied with formation of tau aggregates that are of different physicochemical properties determined by the different tau-substrates used. Our study provides proof-of-concept that the skin tau-SAA can differentiate tauopathies from normal controls, suggesting that the seeding activity of the skin misfolded tau can serve as an accurate diagnostic biomarker of tauopathies.

neuroscience↗

bioRxiv2023

No evidence for enhanced disease with polyclonal SARS-CoV-2 antibody in the ferret model.

Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-enhanced disease (AED) as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for AED, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.

microbiology↗

bioRxiv2021

Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the CNS, were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, viral genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

immunology↗

bioRxiv2021

Protection of human ACE2 transgenic Syrian hamsters from SARS CoV-2 variants by human polyclonal IgG from hyper-immunized transchromosomic bovines

Pandemic SARS CoV-2 has been undergoing rapid evolution during spread throughout the world resulting in the emergence of many Spike protein variants, some of which appear to either evade antibody neutralization, transmit more efficiently, or potentially exhibit increased virulence. This raises significant concerns regarding the long-term efficacy of protection elicited after primary infection and/or from vaccines derived from single virus Spike (S) genotypes, as well as the efficacy of anti-S monoclonal antibody based therapeutics. Here, we used fully human polyclonal human IgG (SAB-185), derived from hyperimmunization of transchromosomic bovines with DNA plasmids encoding the SARS-CoV-2 Wa-1 strain S protein or purified ectodomain of S protein, to examine the neutralizing capacity of SAB-185 in vitro and the protective efficacy of passive SAB-185 antibody (Ab) transfer in vivo. The Ab preparation was tested for neutralization against five variant SARS-CoV-2 strains: Munich (Spike D614G), UK (B.1.1.7), Brazil (P.1) and SA (B.1.3.5) variants, and a variant isolated from a chronically infected immunocompromised patient (Spike {Delta}144-146). For the in vivo studies, we used a new human ACE2 (hACE2) transgenic Syrian hamster model that exhibits lethality after SARS-Cov-2 challenge and the Munich, UK, SA and {Delta}144-146 variants. SAB-185 neutralized each of the SARS-CoV-2 strains equivalently on Vero E6 cells, however, a control convalescent human serum sample was less effective at neutralizing the SA variant. In the hamster model, prophylactic SAB-185 treatment protected the hamsters from fatal disease and minimized clinical signs of infection. These results suggest that SAB-185 may be an effective treatment for patients infected with SARS CoV-2 variants.

microbiology↗

bioRxiv2020

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients

SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

microbiology↗