Virology and Bioinformatics from Virology.ca

The spike (S) protein of the recently emerged human coronavirus (MERS-CoV) mediates infection by binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). Here we mapped the receptor-binding domain in the S protein to a 231-amino acid fragment (residues 358-588) by evaluating the interaction of spike truncation variants with receptor expressing cells and soluble DPP4. Antibodies to this domain - much less so to the preceding N-terminal region - efficiently neutralize MERS-CoV infection.

Influenza A viruses possess RNA genomes that mutate frequently in response to immune pressures. The mutations in the hemagglutinin genes are particularly significant, as the hemagglutinin proteins mediate attachment and fusion to host cells, thereby influencing viral pathogenicity and species specificity. Large-scale influenza A genome sequencing efforts have been ongoing to understand past epidemics and pandemics and anticipate future outbreaks. Sequencing efforts thus far have generated nearly 9,000 distinct hemagglutinin amino acid sequences.

Description: Comparative models for all publicly available influenza A hemagglutinin protein sequences (8,769 to date) were generated using the Rosetta modeling suite. The C-alpha root mean square deviations between a randomly chosen test set of models and their crystallographic templates were less than 2 A, suggesting that the modeling protocols yielded high-quality results. The models were compiled into an online resource, the Hemagglutinin Structure Prediction (HASP) server. The HASP server was designed as a scientific tool for researchers to visualize hemagglutinin protein sequences of interest in a three-dimensional context. With a built-in molecular viewer, hemagglutinin models can be compared side-by-side and navigated by a corresponding sequence alignment. The models and alignments can be downloaded for offline use and further analysis.

The modeling protocols used in the HASP server scale well for large amounts of sequences and will keep pace with expanded sequencing efforts. The conservative approach to modeling and the intuitive search and visualization interfaces allow researchers to quickly analyze hemagglutinin sequences of interest in the context of the most highly related experimental structures, and allow them to directly compare hemagglutinin sequences to each other simultaneously in their two- and three-dimensional contexts. The models and methodology have shown utility in current research efforts and the ongoing aim of the HASP server is to continue to accelerate influenza A research and have a positive impact on global public health.

IMPORTANCE Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology frequently remains unknown, which hampers development of therapeutic or preventive strategies. Hence, identification of novel pathogens is essential and is facilitated by current next-generation sequencing-based methods. Using such technology, we identified and characterized the full genome of a novel cyclovirus in cerebrospinal fluid (CSF) specimens from two Vietnamese patients with CNS infections of unknown etiology, which was subsequently detected in none of 122 CSF specimens from patients with noninfectious neurological disorders but 4% of 642 CSF specimens from Vietnamese patients with suspected or confirmed CNS infections. Similar detection rates in feces from healthy children suggested food-borne or orofecal transmission routes, while frequent detection in feces from Vietnamese pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further studies are needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza. Antibodies to N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (mAbs), which exhibit different reactivity spectra with H1N1 and H5N1 viruses, and used these mAbs to map N1 antigenic domains. We identified 12 amino acids essential for mAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341 and 343 are recognized by strain-specific group A mAbs, while residues 273, 338 and 339 are within conserved epitope(s), which allows cross-reactive group B mAbs to bind the NA of seasonal H1N1, the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B mAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1, and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another 3 N1 residues (at positions 396, 397 and 456) are essential for binding of cross-reactive group E mAbs, which differ from group B mAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses, and demonstrates the potential for developing broadly-protective NA-specific antibody treatments for influenza.

Through the use of intravital imaging of the liver, we demonstrate a collaborative role for platelets with Kupffer cells (KCs) in eradicating blood-borne bacterial infection. Under basal conditions, platelets, via the platelet-adhesion receptor GPIb, formed transient 'touch-and-go' interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria such as Bacillus cereus and methicillin-resistant Staphylococcus aureus (MRSA) were rapidly caught by KCs and triggered platelets to switch from 'touch-and-go' adhesion to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GPIbα-deficient mice had more endothelial and KC damage than did their wild-type counterparts, which led to more fluid leakage, substantial polycythemia and rapid mortality. Our study identifies a previously unknown surveillance mechanism by which platelets survey macrophages that rapidly converts to a critical host response to blood-borne bacteria.

Armed with new methods, researchers are interrogating the DNA of centuries-old pathogens extracted from the bones and teeth of victims.