The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and ago-2 Expression.

Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus (Togaviridae) both in Aedes aegypti-derived cells and in vivo Antiviral activity of p400 was also demonstrated against chikungunya virus (Togaviridae) and Bunyamwera virus (Peribunyaviridae) but not Zika virus (Flaviviridae). p400 was found to be expressed across mosquito tissues and regulated ago-2 but not dcr-2 transcript levels in A. aegypti mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein's antiviral and RNA interference regulatory activities in Drosophila melanogasterIMPORTANCE Female Aedes aegypti mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito's antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that A. aegypti p400, whose ortholog Domino in Drosophila melanogaster is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls ago-2 expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in A. aegypti mosquitoes.

Aedes aegypti Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses.

The small interfering RNA (siRNA) pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here, we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5, and Piwi6) in an Aedes aegypti-derived cell line, Aag2. Analysis of small RNAs captured by Piwi4 revealed that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus-infected cells and, to a lesser extent, with viral piRNAs. By using a Dcr2 knockout cell line, we showed directly that Ago2 lost its antiviral activity, as it was no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathways in A. aegypti and identify Piwi4 as a noncanonical PIWI protein that interacts with members of the siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway. IMPORTANCE Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of their actions.

Fighting Arbovirus Transmission: Natural and Engineered Control of Vector Competence in Aedes Mosquitoes.

Control of aedine mosquito vectors, either by mosquito population reduction or replacement with refractory mosquitoes, may play an essential role in the fight against arboviral diseases. In this review, we will focus on the development and application of biological approaches, both natural or engineered, to limit mosquito vector competence for arboviruses. The study of mosquito antiviral immunity has led to the identification of a number of host response mechanisms and proteins that are required to control arbovirus replication in mosquitoes, though more factors influencing vector competence are likely to be discovered. We will discuss key aspects of these pathways as targets either for selection of naturally resistant mosquito populations or for mosquito genetic manipulation. Moreover, we will consider the use of endosymbiotic bacteria such as Wolbachia, which in some cases have proven to be remarkably efficient in disrupting arbovirus transmission by mosquitoes, but also the use of naturally occurring insect-specific viruses that may interfere with arboviruses in mosquito vectors. Finally, we will discuss the use of paratransgenesis as well as entomopathogenic fungi, which are also proposed strategies to control vector competence.

Development of an avidity assay for detection of recent HIV infections.

HIV avidity can measure the incidence of recent infections within the population. The aim of this study was to evaluate an HIV avidity assay, initially from a clinically defined group of patients and then apply the assay to a prospective study to determine the false recency rate and mean duration of recency for the assay. The assay is a commercial ELISA modified with 7 M urea. The validation of the assay used plasma from patients split into Group 1 (recently infected N=25) and group 2 (established infection N=301). The prospective study tested 178 newly diagnosed HIV patients for avidity. A total of 326 retrospective samples of known HIV status were collected and tested. The initial evaluation gave a sensitivity 100% (CI 86.16-100%) and specificity of 98.65% (95% CI 97.05-99.78%). The prospective study incorporating 178 newly diagnosed patients found 22 patients with low avidity. Follow-up samples obtained from low avidity patients determined the estimated mean duration of recency to be between 3 and 4 months with a false recency rate of 0.89% (CI: 0.24-2.3%). The assay described here compares well in sensitivity, specificity and false recency rate with that of other published avidity assays.

A hepatitis C avidity test for determining recent and past infections in both plasma and dried blood spots.

BACKGROUND: DBS testing has been used successfully to detect HCV antibody positive individuals. Determining how long someone has been infected is important for surveillance initiatives. Antibody avidity is a method that can be used to calculate recency of infection. OBJECTIVES: A HCV avidity assay was evaluated for both plasma and DBS. STUDY DESIGN: To measure antibody avidity a commercial HCV ELISA was modified using 7 M urea. The plasma samples were split into: group 1 (recently infected N = 19), group 2 (chronic carrier N = 300) and group 3 (resolved infection N = 82). Mock DBS made from group 1 (N = 12), group 2 (N = 50), group 3 (N = 25) and two seroconverter panels were evaluated. 133 DBS taken from patients known to have a resolved infection or be a chronic carrier were also tested. RESULTS: The avidity assay cut-off was set at AI≤30 for a recent infection. Using sequential samples the assay could detect a recent infection in the first 4-5 months from the point of infection. Most of the false positive results (AI < 30 among cases known not to have had recent infection) were detected among known resolved infections, in both the plasma and DBS; as a result, a testing algorithm has been designed incorporating both PCR and two dilution factors. The sensitivity and specificity of the assay on plasma was 100% and 99.3%, respectively, while DBS had 100% sensitivity and 98.3% specificity. CONCLUSION: The HCV avidity assay can be used to distinguish between chronic and recent infection using either plasma or DBS as the sample type.

CK2 protein kinase is stimulated and redistributed by functional herpes simplex virus ICP27 protein.

It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs.