Venezuelan Equine Encephalitis Virus Induces Apoptosis through the Unfolded Protein Response Activation of EGR1.

UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing (RNA-Seq) of poly(A) and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response (UPR) was observed. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the UPR was activated, as the expression of both activating transcription factor 4 (ATF4) and CHOP (DDIT3), critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 (EGR1) was induced in a PERK-dependent manner. EGR1(-/-) mouse embryonic fibroblasts (MEFs) demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE: Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 (EGR1) contributed to VEEV-induced cell death.

Detection of human virulence signatures in H5N1.

A method for detecting the emergence of potential pandemic-causing influenza strains has been developed. The system first uses real-time RT-PCR to detect H5, the highly pathogenic avian influenza subtype most likely to cause a pandemic. Pyrosequencing is then employed to scan for codon changes encoding amino acids known to define human influenza versus avian influenza signatures. The pyrosequencing assays were developed to screen at the nucleotide level for 52 amino acid changes defined as avian- or human-specific. A library has been built to screen the sequence data generated and properly identify the strain in question as a potential threat. This method can be used to screen samples for influenza and to determine if the detected virus contains mutations that may make the virus more infective or virulent to humans, potentially thwarting a pandemic outbreak.

Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry.

Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.