Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Novel linear DNA vaccines induce protective immune responses against lethal infection with influenza virus type A/H5N1.

Vaccine development for possible influenza pandemics has been challenging. Conventional vaccines such as inactivated and live attenuated virus preparations are limited in terms of production speed and capacity. DNA vaccination has emerged as a potential alternative to conventional vaccines against influenza pandemics. In this study, we use a novel, cell-free DNA manufacturing process (synDNA) to produce prototype linear DNA vaccines against the influenza virus type A/H5N1. This synDNA process does not require bacterial fermentation, so it avoids the use of antibiotic resistance genes and other nucleic acid sequences unrelated to the antigen gene expression in the actual therapeutic DNA construct. The efficacy of various vaccines expressing the hemagglutinin and neuraminidase proteins (H5N1 synDNA), hemagglutinin alone (H5 synDNA) or neuraminidase alone (N1 synDNA) was evaluated in mice. Two of the constructs (H5 synDNA and H5N1 synDNA) induced a robust protective immune response with up to 93% of treated mice surviving a lethal challenge of a virulent influenza A/Vietnam/1203/04 H5N1 isolate. In combination with a potent biological activity and simplified production footprint, these characteristics make DNA vaccines prepared with our synDNA process highly suitable as alternatives to other vaccine preparations.

Recombinant alphaviruses are safe and useful serological diagnostic tools.

Serological assays for diagnosis of Venezuelan equine encephalitis virus (VEEV) currently require bio-safety level 3 facilities and select agent certification to produce antigens, reference sera, or viral stocks. Rapid identification of VEEV infection is required to respond to human and equine outbreaks of encephalitis caused by that virus and can be useful for epidemiologic surveillance. Alphavirus (Sindbis)-based recombinant viruses that express VEEV structural proteins are attenuated in animal models, thus representing an alternative to the handling of virulent infectious virus. Virus and viral antigens from recombinant Sindbis/VEE constructs engineered to express structural proteins from multiple VEEV subtypes were evaluated as diagnostic reagents in VEEV-specific serological assays, e.g., plaque reduction neutralization test (PRNT), hemagglutination inhibition (HI) assay, and complement fixation (CF) test. Chimeric viruses were produced efficiently in cell culture and were as effective as the parental virus for identifying infection of humans, horses, and rodents in these serological assays.

Production of IL-8, IL-17, IFN-gamma and IP-10 in human astrocytes correlates with alphavirus attenuation.

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995 indicate that VEEV still poses a serious public health threat. Astrocytes may be target cells in human and mouse infection and they play an important role in repair through gliosis. In this study, we report that virulent VEEV efficiently infects cultured normal human astrocytes, three different murine astrocyte cell lines and astrocytes in the mouse brain. The attenuation of virus replication positively correlates with the increased levels of production of IL-8, IL-17, IFN-gamma and IP-10. In addition, VEEV infection induces release of basic fibroblast growth factor and production of potent chemokines such as RANTES and MIP-1-beta from cultured human astrocytes. This growth factor and cytokine profile modeled by astrocytes in vitro may contribute to both neuroprotection and repair and may play a role in leukocyte recruitment in vivo.

Encephalitic alphaviruses.

This review will cover zoonotic, encephalitic alphaviruses in the family Togaviridae. Encephalitic alphaviruses, i.e. Western- (WEEV), Eastern- (EEEV), Venezuelan equine encephalitis virus (VEEV) and, more rarely, Ross River virus, Chikungunya virus and Highlands J virus (HJV), are neuroinvasive and may cause neurological symptoms ranging from mild (e.g., febrile illness) to severe (e.g., encephalitis) in humans and equines. Among the naturally occurring alphaviruses, WEEV, EEEV and VEEV have widespread distributions in North, Central and South America. WEEV has found spanning the U.S. from the mid-West (Michigan and Illinois) to the West coast and extending to Canada with human cases reported in 21 states. EEEV is found along the Gulf (Texas to Florida) and Atlantic Coast (Georgia to New Hampshire), as well as in the mid-West (Wisconsin, Illinois and Michigan) and in Canada, with human cases reported in 19 states. In contrast, transmission of VEEV occurs predominantly in Central and South America. As with their geographical distribution, equine encephalitis viruses differ in their main mosquito vector species and their zoonotic potential.

CD4+ T cells provide protection against acute lethal encephalitis caused by Venezuelan equine encephalitis virus.

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Superior efficacy of a recombinant flagellin:H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin.

Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model.

Inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers.

The genus Alphavirus contains members that threaten human health, both as natural pathogens and as potential biological weapons. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) enter cells readily and can inhibit viral replication through sequence-specific steric blockade of viral RNA. Sindbis virus (SINV) has low pathogenicity in humans and is regularly utilized as a model alphavirus. PPMO targeting the 5'-terminal and AUG translation start site regions of the SINV genome blocked the production of infectious SINV in tissue culture. PPMO designed against corresponding regions in Venezuelan equine encephalitis virus (VEEV) were likewise found to be effective in vitro against several strains of VEEV. Mice treated with PPMO before and after VEEV infection were completely protected from lethal outcome while mice receiving only post-infection PPMO treatment were partially protected. Levels of virus in tissue samples correlated with animal survival. Uninfected mice suffered no apparent ill-effects from PPMO treatment. Thus, PPMO appear promising as candidates for therapeutic development against alphaviruses.

Pathogenesis of XJ and Romero strains of Junin virus in two strains of guinea pigs.

Argentine hemorrhagic fever (AHF), a systemic infectious disease caused by infection with Junin virus, affects several organs, and patients can show hematologic, cardiovascular, renal, or neurologic symptoms. We compared the virulence of two Junin virus strains in inbred and outbred guinea pigs with the aim of characterizing this animal model better for future vaccine/antiviral efficacy studies. Our data indicate that this passage of the XJ strain is attenuated in guinea pigs. In contrast, the Romero strain is highly virulent in Strain 13 as well as in Hartley guinea pigs, resulting in systemic infection, thrombocytopenia, elevated aspartate aminotransferase levels, and ultimately, uniformly lethal disease. We detected viral antigen in formalin-fixed, paraffin-embedded tissues. Thus, both guinea pig strains are useful animal models for lethal Junin virus (Romero strain) infection and potentially can be used for preclinical trials in vaccine or antiviral drug development.

Injectable peramivir mitigates disease and promotes survival in ferrets and mice infected with the highly virulent influenza virus, A/Vietnam/1203/04 (H5N1).

The post-exposure therapeutic efficacy of injectable peramivir against highly pathogenic avian influenza type A H5N1 was evaluated in mice and in ferrets. Seventy to eighty percent of the H5N1-infected peramivir-treated mice, and 70% in the oseltamivir treated mice survived the 15-day study period, as compared to 36% in control (vehicle) group. Ferrets were infected intranasally with H5N1 followed by treatment with multiple doses of peramivir. In two of three trials, a statistically significant increase in survival over a 16-18 day period resulted from peramivir treatment, with improved survival of 40-64% in comparison to mock-treated or untreated animals. Injected peramivir mitigates virus-induced disease, reduces infectious virus titers in the lungs and brains and promotes survival in ferrets infected intranasally with this highly neurovirulent isolate. A single intramuscular peramivir injection protected mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets.

Impairment of cell division of Trypanosoma cruzi epimastigotes.

The mechanisms that facilitate the adaptation of Trypanosoma cruzi to two distinct hosts, insect and vertebrate, are poorly understood, in part due to the limited ability to perform gene disruption studies by homologous recombination. This report describes a developmentally-defective phenotype that resulted from integration of a drug marker adjacent to the GAPDH gene in T. cruzi.

Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection.

We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (alphabeta) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta (gammadelta) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (microMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3(+) T cells are required for protection.

Recent developments in the molecular, biochemical and functional characterization of GPI8 and the GPI-anchoring mechanism [review].

Glycoconjugates are utilized by eukaryotic organisms ranging from yeast to humans for the cell surface expression of a wide variety of proteins and lipids. These glycoconjugates are expressed as enzymes or receptors and serve a diversity of functions, including cell signaling and cell survival. In parasitic protozoans, glycoconjugates play roles in infectivity, survival, virulence and immune evasion. Among the alternate glycoconjugate structures that have been identified, glycosylphosphatidylinositols (GPIs) represent a universal structure for the anchorage of proteins, lipids, and phosphosaccharides to cellular membranes. Biosynthesis of the GPI is a multi-step process that culminates in the attachment of the assembled GPI to a precursor protein. This final step in the transfer of the GPI to a protein is catalyzed by GPI8 of the putative transamidase complex (TAM). GPI8 functions dually to perform the proteolytic cleavage of the C-terminal signal sequence of the precursor protein, followed by the formation of an amide bond between the protein and the ethanolamine phosphate of the GPI. This review summarizes the current aggregate of biochemical, gene-disruption and active site mutagenesis studies, which provide evidence that GPI8 is responsible for the protein-GPI anchoring reaction. We describe recently published studies that have identified other potential components of the TAM complex and that have elucidated their likely role in protein-GPI attachment. Further, we discuss the biochemical, molecular and functional differences between protozoan and mammalian GPI8 and the protein-GPI anchoring machinery. Finally, we will present the implications of these studies for the development of anti-parasite drug therapies.

An overview of chagasic cardiomyopathy: pathogenic importance of oxidative stress.

There is growing evidence to suggest that chagasic myocardia are exposed to sustained oxidative stress-induced injuries that may contribute to disease progression. Pathogen invasion- and replication-mediated cellular injuries and immune-mediated cytotoxic reactions are the common source of reactive oxygen species (ROS) in infectious etiologies. However, our understanding of the source and role of oxidative stress in chagasic cardiomyopathy (CCM) remains incomplete. In this review, we discuss the evidence for increased oxidative stress in chagasic disease, with emphasis on mitochondrial abnormalities, electron transport chain dysfunction and its role in sustaining oxidative stress in myocardium. We discuss the literature reporting the consequences of sustained oxidative stress in CCM pathogenesis.

Impairment of cell division of Trypanosoma cruzi epimastigotes

The mechanisms that facilitate the adaptation of Trypanosoma cruzi to two distinct hosts, insect and vertebrate, are poorly understood, in part due to the limited ability to perform gene disruption studies by homologous recombination. This report describes a developmentally-defective phenotype that resulted from integration of a drug marker adjacent to the GAPDH gene in T. cruzi.

An overview of chagasic cardiomyopathy: pathogenic importance of oxidative stress

Há evidências que sugerem que as miocardites chagásicas são devidas aos danos induzidos pelo estresse oxidativo, podendo contribuir para a evolução da doença de Chagas. Em doenças infecciosas, a formação de espécies reativas do oxigênio (ROS) é, principalmente, derivada de danos celulares mediados pela invasão e replicação do patógeno e por reações citotóxicas mediadas pelo sistema imune. No entanto, como as ROS são formadas e sua função no estresse oxidativo na cardiomiopatia chagásica (CCM) não estão completamente elucidadas. Nesta revisão, nós discutimos as evidências para o aumento do estresse oxidativo na doença de Chagas, com ênfase nas anormalidades mitocondriais, na disfunção da cadeia de transporte de elétrons e seu papel na manutenção do estresse oxidativo no miocárdio. Discutimos ainda, os resultados da literatura que relatam as conseqüências da manutenção do estresse oxidativo na patogênese da CCM.