Introduction Chikungunya virus (CHIKV) is an alphavirus in the family Togaviridae. Since 2005 the virus has caused a major epidemic of disease in humans, ranging from Central Africa, South-East Asia, Caribbean and more recently the Americas. The virus is spread by mosquitoes, most notably Aedes aegypti and Ae. albopictus. CHIKV causes an acute disease in humans, which is characterised by a rapid onset of high fever, rash, myalgia and arthralgia. The symptoms typically resolve within a week. Remarkably, up to a third of patients who recover from acute chikungunya develop chronic arthritis/arthralgia, which may last for months or years and has a large negative impact on the quality of life. The mechanism by which this occurs is not yet fully understood. CHIKV can infect human monocytes, and macrophages positive for CHIKV antigen have been observed in joint tissue from patients recovered from acute CHIKV infection but with chronic arthritis. Furthermore, it has been demonstrated that macrophages can be infected with CHIKV in vitro by a mechanism involving apoptotic debris from CHIKV-infected cells. Hypothesis and aims Infection of monocytes and macrophages with CHIKV contributes to clinical disease and virus persistence in vivo. The aim of this project was to investigate the mechanism by which alphaviruses infect macrophages in vitro, and to generate a CHIKV which is unable to replicate in monocytes and macrophages in vitro, and to study its pathogenicity in vivo. Materials and methods HeLa cells were infected with Semliki Forest virus (SFV), an alphavirus closely related to CHIKV, or SFV replicon particles (SFV VRP). Following cell death, whole cell supernatant or clarified cell supernatant from SFV- and SFV VRP-infected cells was passaged onto human monocyte-derived macrophages (MDMs). These cells were observed microscopically for expression of the fluorescent marker encoded by the SFV. Virus and VRP-infected apoptotic debris were inspected for the presence of alphavirus replication complexes by electron microscopy. Subsequently, a recognition element (RE) for a haematopoietic-specific miRNA (miR-142-3P) was incorporated into the genome of SFV (proof-of-concept) and CHIKV to investigate if blocking virus replication in cells of the mononuclear phagocyte system altered virus kinetics in vitro. The replication of the modified viruses was investigated in macrophage/monocyte cell lines Thp-1 and IC-21, and in HEK 293 cells modified to express miR-142-3P under the control of an inducible tetracycline promoter. Modified viruses were tested in animal models of disease (mouse for SFV and non-human primate for CHIKV) to investigate the pathogenicity of these viruses in vivo. Results The presence of apoptotic debris from SFV-infected cells was required to infect MDMs with SFV. The presence or absence of infectious virus particles in the apoptotic debris did not affect the infection rate. Intact alphavirus replication complexes were found within the apoptotic debris. MiR-142-3P RE was successfully incorporated into the genome of both SFV and CHIKV. RE-virus replication in all cells expressing miR-142-3P was reduced by 90-99% when compared to control viruses. RE-virus replication was not affected in cells which did not express miR- 142-3P. In interferon-α/β receptor knockout mice, RE-SFV generated viraemia comparable to the control virus, but could not infect efficiently the population of macrophages resident in the marginal zone of the spleen. RE-CHIKV was found to be genetically stable in vitro following multiple passages on BHK-21 cells in the absence of a selective pressure from miR-142-3P. RE-CHIKV was inoculated into two cynomolgus macaques. The data from this experiment are not yet available. Conclusion SFV was shown to infect MDM via apoptotic debris containing intact alphavirus replication complexes, which were the most likely infectious agent. SFV and CHIKV unable to replicate in haematopoietic cells in vitro were successfully engineered. The pathogenicity of modified SFV and CHIKV was investigated in vivo.

Florida's mild climate supports year round enzootic transmission of arthropod-borne (arbo) viruses, such as St. Louis Encephalitis virus (SLEV), West Nile virus (WNV), and Eastern Equine Encephalomyelitis virus (EEEV). First isolated in 1960 from two Florida blue jays, Highlands J virus (HJV) is endemic to the state and vectored by the same mosquitoes as EEEV (Henderson et al, 1962). EEEV and HJV are both alphaviruses, but HJV is not pathogenic to humans, occasionally causes encephalitis in horses, and is a recognized pathogen in some bird species (turkeys, emus, etc) (Cilnis et al, 1996). The Florida Sentinel Chicken Arboviral Surveillance Program, established in 1978, utilizes sentinel chickens to detect arboviral activity throughout the state. Current serologic antibody detection methods include the hemagglutination inhibition (HAI), IgM antibody capture enzyme-linked immunosorbent (MAC-ELISA), and serum neutralization plaque reduction (PRNT) assays (Blackmore et al, 2003). In 2003, sentinel chickens detected significantly greater alphavirus activity than seen in the previous 15 years (Stark & Kazanis, 2003). This increase raised concerns that bridging into the human population had become a serious threat as well as an important issue for veterinary health. The objective of this study was to determine if cross-reactions with Highlands J virus were impacting the serologic diagnostic tests routinely performed for identification of EEEV. For 2003, the HAI test detected 476 alphavirus positive sentinels. We tested 316 of these chickens in the PRNT, which identified 176 EEEV positive sentinels and 75 HJV positive sentinels. Our results indicate that Highlands J virus is extensively cross-reactive in the HAI test and that the MAC-ELISA is more specific for the detection of antibodies solely to EEEV. We demonstrated that EEEV antibody titers in the HAI test were positively correlated to antibody titers in the PRNT assay. Analysis of alphaviral activity by county indicates widespread transmission of HJV across the northern and panhandle regions of the state; however EEEV activity was greater than HJV activity in all but four counties. Consequently, distinguishing between the two agents can reduce the expenditure of resources on unnecessary vector control and medical alerts to protect the public health from Highlands J virus.

<p dir="ltr">Sindbis virus (SINV) is a mosquito-borne alphavirus with a positive-sense single-stranded RNA genome. It can cause febrile illness in humans, marked by rash, arthritis, and muscle pain. Due to its genetic and structural similarities with other medically significant alphaviruses, SINV serves as a model for studying alphavirus biology. One key SINV protein is a small 6 kDa hydrophobic protein called ‘6K’ that functions as a viroporin—a viral ion channel. Despite evidence suggesting that 6K forms ion channels and is involved in virus budding, its precise mechanisms are not well understood. Understanding the function and inhibition potential of 6K can unveil insights into alphavirus biology and open avenues for therapeutic intervention. There are currently no antiviral therapies targeting alphavirus infections.</p><p dir="ltr">In our study, we focus on 6K's ion channel function and its importance in viral growth, glycoprotein trafficking, and viral entry. We show that deleting 6K severely impairs viral growth, glycoprotein trafficking and viral budding. Chimeras in which the 6K transmembrane domain is replaced with ion channels from HIV-1 Vpu, IAV M2, or HCV p7, display partially restored function, demonstrating the conserved functions of viroporins from unrelated viruses.</p><p dir="ltr">In addition, we developed a novel chimeric SINV system to study the SARS-CoV-2 Envelope (E) protein’s ion channel activity under biosafety level 2 (BSL-2) conditions. E protein is highly conserved across SARS-CoV-2 variants of concern, making it an attractive target for antiviral drug development. Our system demonstrates that the E protein transmembrane domain (ETM) can partially replace the function of the SINV 6K ion channel, allowing us to screen inhibitors of the E protein. As validation, we show that known ion channel inhibitors such as amantadine and amilorides exhibit similar inhibition profiles in this chimeric system as reported in authentic BSL3 SARS-CoV-2 systems.</p><p dir="ltr">Overall, these studies advance our understanding of viroporins in viral infections and support the development of broad-spectrum antivirals against alphaviruses and SARS-CoV-2.</p>

Expanding urbanization, climate change, and population growth contribute to increased transmission and spread of arthropod-borne viruses (arboviruses), many of which cause severe disease in humans. Pathogenic arboviruses include dengue, Zika, tick-borne encephalitis, and sindbis viruses, which together threaten more than half the global population. Thus, there is a constant need for safe, specific, and sensitive molecular tests to identify early-stage infections for accurate diagnosis and molecular epidemiological data for disease prevention and control. The study tested the biosafety of using FTA™ cards when working with pathogenic arboviruses by conducting an infectivity assay using sindbis virus. Conditions for RNA extraction and storage of arboviruses on FTA were analyzed by measuring viral RNA (vRNA) stability using a SYBR-Green, Pan-Flavi RT-qPCR method composed of degenerate primers able to detect a variety of flaviviruses. Data from a Pan-Flavi RT-qPCR study comprising of 222 clinical blood and serum samples collected from a 2018 dengue virus outbreak in Hanoi (Vietnam) was analyzed to establish applicability of FTA for molecular epidemiology and diagnosis. Results showed that sindbis virus infectivity was inhibited by FTA-adsorption. FTA-adsorbed arboviruses were extracted with the highest yield using Trizol extraction and were preserved at storage at 4-20ºC for up to 30 days. The results showed that clinical blood samples acquired higher yields of vRNA for molecular testing than serum samples and that it may be possible to perform sequencing for genomic analysis. The study suggests that FTA cards may facilitate the storage and transportation of adsorbed arboviruses for downstream molecular epidemiological and diagnostic tests.