Potential Role of Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP2 in Apoptosis Inhibition

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the pork industry, and its pathogenesis needs further investigations. To study the role of two structural proteins of PRRSV in virus-host cells interactions, two stable cell lines (MARC-2a and MARC-N) expressing GP2 and N proteins, respectively, were established. We induced apoptosis in these cells by treating them with staurosporine and found a significant reduction in the number of apoptotic cells in MARC-2a as compared to MARC-N and MARC-145 cells. In addition, we found significantly higher activities of transcriptional factors (NF-B and AP-1) in both cell lines as compared to MARC-145 (parent cells). Overall, our data suggest that, although both stable cell lines activate NF-B and AP-1, GP2 triggers the antiapoptotic process through an intermediate step that needs to be further investigated

Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species

TNF-α promoter polymorphism: a factor contributing to the different immunological and clinical phenotypes in Japanese encephalitis

<p>Abstract</p> <p>Background</p> <p>More than three billion populations are living under the threat of Japanese encephalitis in South East Asian (SEA) countries including India. The pathogenesis of this disease is not clearly understood and is probably attributed to genomic variations in viral strains as well as the host genetic makeup. The present study is to determine the role of polymorphism of TNF-alpha promoter regions at positions -238G/A, -308G/A, -857C/T and -863C/A in the severity of Japanese encephalitis patients.</p> <p>Methods</p> <p>Total of 142 patients including 66 encephalitis case (IgM/RT-PCR positive), 16 fever cases (IgM positive) without encephalitis and 60 apparently healthy individuals (IgG positive) were included in the study. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) using site specific restriction enzymes were implemented for polymorphism study of TNF alpha promoter.</p> <p>Results</p> <p>Following the analysis of the digestion patterns of four polymorphic sites of the TNF- alpha promoter region, a significant association was observed between the allele -308A and -863C with the patients of Japanese encephalitis.</p> <p>Conclusions</p> <p>TNF- alpha 308 G/A has been shown to be associated with elevated TNF- alpha transcriptional activity. On the other hand, polymorphism at position -863C/A in the promoter region has been reported to be associated with reduced TNF- alpha promoter activity and lower plasma TNF levels. As per the literature search, this is the first study to identify the role of TNF- alpha promoter in JE infection. Our results show that subjects with - 308A and -863C alleles are more vulnerable to the severe form of JE infection.</p

Recent Advances in Arboviral Vaccines: Emerging Platforms and Promising Innovations

Arboviruses are a group of viruses that are transmitted by arthropods, such as mosquitoes, and cause significant morbidity and mortality worldwide. Currently, there are only a few options, with restricted use, for effective vaccines against these viruses. However, recent advances in arboviral vaccine development have shown promising innovations that have potential in preclinical and clinical studies. Insect-specific viruses have been explored as a novel vaccine platform that can induce cross-protective immunity against related arboviruses. Nanoparticle-based vaccines have also been developed to enhance the immunogenicity and stability of viral antigens. Additionally, vaccines against mosquito salivary proteins that can modulate the host immune response and interfere with arboviral transmission are being explored. Synonymous recoding, such as random codon shuffling, codon deoptimization, and codon-pair deoptimization, is being investigated as a strategy to attenuate the replication of arboviruses in vertebrate cells, reducing the risk of reverting to wild-type virulence. Finally, mRNA vaccines have been developed to rapidly generate and express viral antigens in the host cells, eliciting robust and durable immune responses. The challenges and opportunities for arboviral vaccine development are outlined, and future directions for research and innovation are discussed

Vector competence of selected North American Anopheles and Culex mosquitoes for Zika virus

Zika virus (ZIKV) is a vector-borne flavivirus that has caused recent outbreaks associated with serious disease in infants and newborns in the Americas. Aedes mosquitoes are the primary vectors for ZIKV, but little is known about the diversity of mosquitoes that can transmit ZIKV in North America. We chose three abundant North American mosquito species (Anopheles freeborni, Anopheles quadrimaculatus, and Culex tarsalis) and one known vector species (Aedes aegypti), fed them blood meals supplemented with a recent outbreak ZIKV strain, and tested bodies, legs, and saliva for infectious ZIKV. ZIKV was able to infect, disseminate, and be transmitted by Aedes aegypti. However, Anopheles freeborni, Anopheles quadrimaculatus, and Culex tarsalis were unable to be infected. We conclude that these species are unlikely to be involved in ZIKV transmission in North America. However, we should continue to examine the ability for other mosquito species to potentially act as ZIKV vectors in North America

Anopheles mosquitoes may drive invasion and transmission of Mayaro virus across geographically diverse regions.

The Togavirus (Alphavirus) Mayaro virus (MAYV) was initially described in 1954 from Mayaro County (Trinidad) and has been responsible for outbreaks in South America and the Caribbean. Imported MAYV cases are on the rise, leading to invasion concerns similar to Chikungunya and Zika viruses. Little is known about the range of mosquito species that are competent MAYV vectors. We tested vector competence of 2 MAYV genotypes in laboratory strains of six mosquito species (Aedes aegypti, Anopheles freeborni, An. gambiae, An. quadrimaculatus, An. stephensi, Culex quinquefasciatus). Ae. aegypti and Cx. quinquefasciatus were poor MAYV vectors, and had either poor or null infection and transmission rates at the tested viral challenge titers. In contrast, all Anopheles species were able to transmit MAYV, and 3 of the 4 species transmitted both genotypes. The Anopheles species tested are divergent and native to widely separated geographic regions (Africa, Asia, North America), suggesting that Anopheles may be important in the invasion and spread of MAYV across diverse regions of the world

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

International audienceMosquito bites transmit a number of pathogens via salivary droplets deposited during bloodfeeding, resulting in potentially fatal diseases. Little is known about the genomic content of these nanodroplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform enabling high-throughput molecular interrogation of individual mosquito bites. We introduce an ultra-thin PDMS membrane which acts as a biting interface to arrays of micro-wells. Freely-behaving mosquitoes deposit saliva droplets by biting into these micro-wells. By modulating membrane thickness, we observe species-dependent differences in mosquito biting capacity, utilizable for selective sample collection. We demonstrate RT-PCR and focus-forming assays on-chip to detect mosquito DNA, Zika virus RNA, as well as quantify infectious Mayaro virus particles transmitted from single mosquito bites. The Vectorchip presents a promising approach for singlebite-resolution laboratory and field characterization of vector-pathogen communities, and could serve as a powerful early warning sentinel for mosquito-borne diseases

Heat shock protein 70 (Hsp70) mediates Zika virus entry, replication, and egress from host cells

ABSTRACTZika virus (ZIKV) is a historically neglected mosquito-borne flavivirus that has caused recent epidemics in the western hemisphere. ZIKV has been associated with severe symptoms including infant microcephaly and Guillain-Barré syndrome, stimulating interest in understanding factors governing ZIKV infection. Heat shock protein 70 (Hsp70) has been shown to be an infection factor for multiple viruses, leading us to investigate the role of Hsp70 in the ZIKV infection process. ZIKV infection induced Hsp70 expression in host cells 48-h post-infection. Inducing Hsp70 expression in mammalian cells increased ZIKV production, whereas inhibiting Hsp70 activity reduced ZIKV viral RNA production and virion release from the cell. Hsp70 was localized both on the cell surface where it could interact with ZIKV during the initial stages of the infection process, and intracellularly where it localized with viral RNA. Blocking cell surface-localized Hsp70 using antibodies decreased ZIKV cell infection rates and production of infectious virus particles, as did competition with recombinant Hsp70 protein. Overall, Hsp70 was found to play a functional role in both the pre- and post-ZIKV infection processes affecting viral entry, replication, and egress. Understanding the interactions between Hsp70 and ZIKV may lead to novel therapeutics for ZIKV infection

Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing

Delivery of Cas9 ribonucleoprotein complexes can be done via microinjection into eggs, though this is a technically challenging procedure. Here the authors demonstrate ReMOT Control, delivery of RNPs to the mosquito germline by tagging with peptide derived from yolk protein 1