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Infectious route in competent vectors
Among the many species of mosquitoes, only a small number can acquire arbovirus infection and transmit it to an uninfected vertebrate. It seems that there is a specificity in the vector-arbovirus interaction, combining vector intrinsic factors such as mosquito physiology, genetic or antiviral cellular responses and consequently viral replication and propagation.
In comparison to the vertebrate host, arboviruses do not seem to cause significant pathology in the mosquito vector. From the oral acquisition of a viremic bloodmeal to the transmission to a new uninfected vertebrate host, the arbovirus replicates in the cells of the arthropod and must cope with antiviral pathways such as cell death and innate immune responses and several tissue barriers from the midgut until the saliva. Indeed, among the many species of mosquito, some are permissive (competent) and other are resistant to the infection and part of this competence is linked to the several tissue barriers that can be crossed or not during viral dissemination. Figure 7 shows a schematic presentation of the typical route in a permissive mosquito and the four principal tissue barriers (Midgut Infection Barrier MIB; Midgut Escape Barrier MEB; Salivary Gland Infection Barrier SGIB and Salivary Gland Escape Barrier SGEB).
In this part we describe the typical route of alphavirus infection in competent and permissive mosquitos, defined by 1) the acquisition of the virus; 2) passage through different barriers, and 3) replication and dissemination to salivary glands to successful transmission to another vertebrate host. The role of vector immune and cell death responses in determining vector competence (VC) in overcoming tissue barriers will be discussed in the following part.
The initial site of infection: the midgut
The digestive tube of the mosquito is divided into three major regions: the foregut, the midgut, and the hindgut. The foregut is a region extending from the mouth to the midgut and the foregut extending to the anus. The midgut epithelium is surrounded by an extracellular matrix, called basal lamina (BL) on the internal side of the mosquito. After a viremic bloodmeal, viruses enter the midgut lumen and are close to the single layer of epithelial cells (Figure 7). It has been shown that a small number of cells can be infected during this initial infection. For example, VEEV infects an average of 28 midgut cells in the Aedes mosquito and WNV less than 15 midgut cells in Culex (Scholle and Mason, 2004; Smith and Weaver, 2008). Moreover, specific regions of the midgut (posterior, and/or anterior regions) are preferentially infected. From this step, the virus is confronted with the midgut tissue barrier composed of an epithelial layer separating the lumen of the midgut from the hemocoel (blood-containing body cavity and function as part of the circulatory system). Once the virus enters a cell, viral replication starts, and the place of viral maturation and accumulation can also vary, according to the virus-mosquito combination. The endoplasmic reticulum (ER) has been shown to be involved in EEEV synthesis in Ae. triseriatus but, in contrast, it seems that CHIKV does not enter the cisternae of the ER in Ae. aegypti cells (Franz et al. 2015). These differences could be linked to a different temporal analysis of infection or a virus-vector specific interaction. New virions are accumulated at the plasma membrane facing the basal surface and, after budding, viruses are in contact with the basal lamina of the epithelium and need to pass through it to enter the hemocoel and disseminate. The basal lamina predominantly consists of laminin and collagen IV and presents small pores with a size exclusion of 9-12 nm. The BL acts as a very strong mechanical barrier for viruses and the pores are too small for alphaviruses to pass through (60-80 nm).
However, during bloodmeal digestion, it has been shown that the basal lamina is degraded by matrix metalloproteinases (MMPs) and becomes permissive. This change in basal lamina integrity was observed in Culex pipiens infected by WNV. However, in Aedes aegypti infected by CHIKV it has been shown that, curiously, overexpression of the Ae.aegypti tissue inhibitor of metalloproteinases (AeTIMP) induces the efficient dissemination of CHIKV (Dong, Balaraman, et al. 2017). As basal lamina degradation/remodeling involves activities of metalloproteinases and inhibitors, they hypothesized that the expression of inhibitors could be accompanied by the transient restructuring of basal lamina allowing the passage of CHIKV.
Following dissemination from the midgut, the virus disseminates up to the salivary glands and saliva. To disseminate to secondary organs , the hemocytes (insect blood cells) have been shown to be an important vehicle in the case of SINV infection of Ae.aegypti (Parikh, Oliver, and Bartholomay 2009). Moreover, it has been proposed that the infection of hemocytes leads to a high rate of infection which is required before the infection of salivary glands. Indeed, since only a few epithelial cells of the midgut are infected, the quantity of viral particles after the passage through basal lamina is small. It has been proposed that hemocytes are a vehicle for the virus to the salivary glands and allow the replication of the virus to produce enough particles for efficient infection of the mosquito’s head.
The salivary glands of female mosquitoes are paired organs located in the thorax and each gland is composed of three lobes (two lateral and one median). Each lobe presents a central internal duct containing a cavity for saliva storage and surrounded by a monolayer of epithelial and acinar cells that connect the lobes. The monolayer is bounded externally by a BL. Once again, the salivary glands consist of a tissue barrier that must be crossed by the virus to reach the saliva. The CHIKV, present in the hemocoel must penetrate the BL to join the acinar cells and replicate inside to then be disseminated into the saliva cavity and released during blood feeding. It seems that CHIKV can infect both the lateral and median lobes of Ae. aegypti (Janzen, Rhodes, and Doane 1970), whereas SINV can infect only the lateral lobe in Ae. albopictus (Bowers, Abell, and Brown 1995). In Ae. albopictus, CHIKV particles were observed in acinar cells and in the apical cavity where the virus can be stored in saliva. Interestingly, Vega-Rua and colleagues showed that the nucleocapsid could bud at the plasma membrane of acinar cells and at the membrane of vesicles located in the apical cavity (Vega-Rúa et al. 2015). Once in the cavities of acinar cells during new blood feeding, the virus is released with saliva into the vertebrate host.
The saliva of the mosquito contains several proteins such as degrading enzymes, antimicrobials, and anti-inflammatory peptides. The proteins in the salivary glands of Ae. aegypti infected by CHIKV were triggered at 3- and 6-days post-infection (dpi) and showed the regulation of about ten secreted proteins at both times. Proteins involved in the anti-inflammatory effect, blood feeding and some protease inhibitors were increased while anti-oxidant related proteins like protein disulfide isomerase (PDI) involved in re-establishment of redox homeostasis were downregulated (Tchankouo-Nguetcheu et al. 2012). The proteins found in the saliva of infected mosquitoes could participate in the primo infection in the skin of host vertebrates. Indeed, the presence of anti-inflammatory protein and protease inhibitors could lower the host anti-viral immune response and subsequently create an efficient micro-environment for viral infection. However, it has been shown that the host vertebrate immune response of a murine model can induce specific antibody IgG response against the salivary protein called 34k2 from Aedes albopictus and Aedes aegypti. Interestingly, as the proteins showed very a low level of immune cross-reactivity, they proposed to use this protein as species-specific markers of host exposure (Montero et al. 2019).
Human infection: clinical signs
CHIKV disease in humans is symptomatic in 72-95 % of cases with an incubation period of 2 to 4 days. The infection is characterized by an acute phase with febrile illness: fever in 90% of patients (>39°C) and frequently accompanied by muscle (myalgia) and joint (arthralgia) pains in about 85% of patients. Other symptoms include headache, cutaneous manifestations, and digestive troubles. The acute phase generally disappears after one or two weeks. However, for around 35% of patients, joint pain can persist for several weeks to months and present similarity with rheumatoid arthritis. A systematic review has shown that around 25% of CHIKV cases would develop post-CHIKV chronic inflammatory rheumatism for more than 15 months and 14% would present chronic arthritis (Rodríguez-Morales et al. 2016). In addition to typical symptoms, during the CHIKV outbreak on La Réunion, some neurological complications were described for the first time, such as encephalitis and encephalopathy, showing a new CHIKV tropism for the central nervous system (Matusali et al. 2019).
Acute and chronic phases
In this part we present the dissemination of CHIKV from the skin to secondary organs and cellular tropism. Moreover, knowledge of characteristic-associated alphavirus chronicity will be introduced. Acute CHIKV infection is generally associated with high fever, rashes, and severe muscle and joint pains for 7 to 10 days. It follows the chronic CHIKV disease typically defined by musculoskeletal disease for several months and sometimes for years, beyond the onset of acute disease. Chronic CHIKV disease has been reported principally after the outbreak in La Réunion in 2006-2007. While the majority of individuals fully recovered, a large proportion of individuals experienced chronic CHIKV disease and long-term impaired quality of life has been reported for some individuals (McCarthy, Davenport, and Morrison 2018).
Following CHIKV inoculation through mosquito bite, viral particles enter the subcutaneous capillaries, where they are in contact with susceptible cells in the skin (Figure 8). The main infected cells are fibroblasts, endothelial cells, and macrophages (Dupuis-Maguiraga et al. 2012). Local replication in the skin is associated with a very early Type-I interferon response. Indeed, IFNα/β was detected very early in the infection and its concentration is correlated with viral load in plasma. On the one hand, the production of IFNα for ten days was associated with viral clearance without detection of adaptative immunity through IFNγ in serum (Chow et al. 2011).
On the other hand, Hoarau et al. observed a high level of IFNγ and cytokines IL-12 in patients from La Réunion (Dupuis-Maguiraga et al. 2012) and Wauquier et al. showed an overproduction of IFNα and IFNγ during the acute phase in infected individuals in Gabon (Wauquier et al. 2011) (Figure 9). The skin represents the site of local viral replication following a mosquito bite, and the new particles produced are probably transported to secondary lymphoid organs not far from the site of inoculation. Moreover, blood transports viral particles either in free virion form or in the form of infected monocytes, to the target organs like liver, muscle, joints, and the central nervous system (Schwartz and Albert 2010). While the evidence of production of viral particles from monocytes has not been demonstrated, Her et al. have detected CHIKV antigens in monocytes exposed to MOI 10 to 50 and this result was supported by the detection of antigen-positive monocytes from acutely infected patients (Her et al. 2010).
Figure 8: CHIKV dissemination and targets in the human. Transmission of CHIKV to humans occurs following a female Ae.aegypti or Ae.albopictus mosquito bite. The virus can replicate in the skin (fibroblasts and melanocytes cells) and disseminate to the liver (endothelial cells), lymphoid tissue, muscles (fibroblasts and satellite cells) and joints (fibroblasts) and the brain through the bloodstream. The persistent phase is characterized by persistent muscle and joint pains. The persistent phase seems to involve macrophage and monocyte inflammatory response (based on Matusali et al. 2019; Schwartz and Albert 2010).
The Chikungunya virus reaches muscles and joints where myalgia and/or arthralgia symptoms may persist for several weeks, months or even years. Following the outbreak at La Réunion, it was described in muscle biopsies from two patients that differentiated myotubes that were not permissive to CHIKV, whereas satellite cells (skeletal muscle progenitor cells) were positive for CHIKV (Ozden et al. 2007). The other characteristic disease symptom of CHIKV is arthralgia. Viral RNA has been detected in the synovial tissues and fluids during acute and chronic CHIKV infection, where synovial fibroblasts and macrophages were sensitive to CHIKV (Young et al. 2019). Chronic CHIKV disease characterized by persistent arthralgia seems to be an immune and cell death mediated response also associated with risk factors. Nevertheless, CHIKV antigens have been detected only during the persistent phase. The osteoblasts are infected, leading to the secretion of proinflammatory and pro-osteoclastic factors (IL-6, IL-1 β, CCL-2 and RANKL). In normal joints, the cytokine RANKL (receptor activator of the nuclear factor NF-κB ligand) can induce the differentiation of osteoblasts into osteoclasts and OPG (osteoprotegerin) can inhibit the action of RANKL. The RANKL:OPG ratio controls this differentiation. However, during CHIKV infection the RANKL:OPG ratio increases, favoring osteoclast differentiation and leading to bone loss and promoting inflammation (Chen et al. 2015). Viral persistence suggests that cells can be chronically infected and are perhaps able to control or block cell death. However, it has been observed that in chronic CHIKV individuals, the damaged synovial tissues present an intense immune response on the one hand and a strong programmed cell death characteristic on the other (Hoarau et al. 2010). Interestingly, it has been shown that the human monocyte MM6 cell line was infected with a very low rate of RRV replication and the infection led to a late apoptotic cell death feature without immune response control, suggesting that monocytes could be a viral reservoir during chronic RRV disease (Krejbich-Trotot et al. 2016). In parallel, the infection of macrophages in joints was associated with the production of cytokines and chemokines such as interleukins (IL)-6, IL-8, CCL-2/MCP-1 and, moreover, it has been proposed that the phagocytosis of apoptotic bodies from infected fibroblasts could also participate in viral persistence and the inflammation of musculoskeletal tissues in chronic CHIKV disease. Finally, Natural Killer NK cells and T cells together with monocytes were found activated and attracted to joint tissues (Dupuis-Maguiraga et al. 2012), (Figure 9). Nevertheless, the mechanisms involved in viral persistence are still poorly understood as are the beneficial and deleterious effect of local inflammation and cell death on viral persistence.
Vaccine, antiviral treatments, and strategies in humans and mosquitos
Two strategies against CHIKV and other arboviruses have emerged: the first is the development of a vaccine for humans and the second the inhibition of viral infection in mosquito.
Vaccine and antiviral treatments in humans
To date, no specific treatments or licensed vaccines are available. Up to now, patients have been treated with symptomatic treatments like anti-inflammatory drugs to treat chronic arthralgia and myalgia. Chloroquine, a known drug against malaria, initially showed promising results in patients, however, several years later it was documented that after 200 days of treatment, patients complained more about joint pain than control group (Brighton 1984; De Lamballerie et al. 2008). Another anti-inflammatory molecule, named bindarit, had been shown useful in the treatment of CHIKV-induced arthritis in a mouse model (Rulli et al. 2011).
Regarding vaccines, a recent publication reported the success of an experimental CHIKV vaccine (phase 2) which confers durable immune response and is safe for healthy volunteers aged 18 to 60 (Chen et al. 2020). The volunteers received two intramuscular doses of the vaccine and showed strong immune response to CHIKV after 72 weeks of the study (Chang et al. 2014; Chen et al. 2020). This vaccine approach is based on a virus-like particle (VLP) presenting structural proteins recognized by the immune system, suggesting a protective immune response to the CHIKV. Other vaccines are under development such as a chimeric vaccine with a VSV backbone and CHIKV structural proteins (Chattopadhyay et al. 2013), and a recombinant measle-virus-based chikungunya vaccine (Ramsauer et al. 2015).
Antiviral strategies in the mosquito
In response to insecticide resistance, alternative strategies are needed for controlling the vector and preventing epidemics. Previously, the primary strategy was the use of synthetic chemicals to kill adult vectors using aerial spraying. In parallel, the control of larvae has been developed by chemical application, microbial larvicides and biological agents (predator fishes that eat mosquito larvae). The obstacle to larval control is the ability of detect, access, and eliminate the breeding zone, which is a costly task. Thus, new strategies have been developed thanks to entomological and epidemiological approaches such as attractive targeted sugar baits, the bacteria Wolbachia, the sterile insect technique and genetic manipulation (Achee et al. 2019). The impact of Wolbachia in the inhibition of arbovirus in the mosquito will be discussed below (Chapter 3).
Table of contents :
1 ALPHAVIRUSES
Classifications
Taxonomy of Alphavirus
Arbovirus: an ecological group
Viral particle structure and genome organization
Non-structural proteins
Structural proteins
Replicative cycle in mammals and mosquitos
Binding and entry by endocytosis
Fusion and Viral genome release
Replication complex and assembly
2 CHIKUNGUNYA VIRUS – AN OLD-WORLD ALPHAVIRUS
Discovery
Transmission cycles and vector distribution
Epidemiology
Infectious route in competent vectors
The initial site of infection: the midgut
The final site of infection: the salivary glands
Pathogenesis and cellular tropism in vertebrate hosts
Human infection: clinical signs
Acute and chronic phases
Vaccine, antiviral treatments, and strategies in humans and mosquitos
Vaccine and antiviral treatments in humans
Antiviral strategies in the mosquito
3 CELLULAR RESPONSES IN ALPHAVIRUSES-INFECTED MAMMALS AND INSECTS
Innate immune response
In mammals
In the mosquito
Apoptosis
In mammals
3.2.1.1 The apoptosis signaling pathway
3.2.1.2 Regulation of apoptosis during alphavirus infection
3.2.1.3 Apoptosis-autophagy balance and crosstalk with other pathways In insects
3.2.2.1 The apoptotic pathway in Drosophila and Aedes
3.2.2.2 The effect of apoptosis on arbovirus outcome
4 ROLE OF P53 AND P53 ISOFORMS DURING ARBOVIRUS INFECTION IN MAMMALS AND INSECTS
Human p53
Discovery of p53
The p73/p63 and p53 family
Human p53 and p53 isoforms
Structure of p53 and p53 isoforms
Stabilization of p53
A transcription factor
Drosophila and mosquito p53
Dmp53/Dp53 and p53 isoforms in Drosophila
Recent discovery of two p53 isoforms in Aedes
Control of p53 pathways during viral infection
Function of p53 during “non-arboviral” infections
Different roles of p53 during arboviral infections: DENV, WNV and ZIKV
Modulation of p53 transcriptional activity by p53 isoforms during viral infection
OBJECTIVES
EXPERIMENTAL REPORT
CHAPTER 1) OPPOSITE EFFECT OF P53 ON CHIKV INFECTION IN A HUMAN CELL LINE AND IN VIVO IN DROSOPHILA MELANOGASTER
1 OBJECTIVES
2 MATERIAL AND METHODS
Cell lines and viruses
Generation of TP53 CRISPR-mediated knockout LHCN-M2 cell line
sgRNA design
Cloning in lentiCRISPRv2 plasmid
Lentivirus production
Stable cell line generation
Verification of gene knock-out by Western blot
Infection of knockout cell lines with CHIKV
Transfection of plasmid Flag-RIG-I 2CARD
Flow cytometry analysis
Western blot
RNA extraction and RT-qPCR
Cell viability assays
Subcellular fractionation
In vivo Drosophila melanogaster
Detection of Wolbachia by PCR and tetracycline treatment
Fly injection
Survival curve
RNA extraction of whole fly
TCID50/mL
Statistical analysis
3 RESULTS
IN MAMMALS: INNATE IMMUNE ANTIVIRAL ACTIVITY OF P53 IN HUMAN SKELETAL MUSCLE CELLS INFECTED WITH CHIKV
Infection of an LHCN-M2 cell line by CHIKV induces stabilization of p53 protein
Infection of LHCN-M2 cell line by CHIKV induces Type I interferon immune signaling response
but not cell cycle arrest and not apoptotic p53 dependent
Effect of LHCN-M2 p53 deletion on CHIKV infection and cellular outcome
Generation of p53 knockout LHCN-M2 (sgRNA_p53) and luciferase (sgRNA_luc) control cell line using CRISPR/Cas9 technology
Infection of p53 knockout LHCN-M2 with CHIKV
Effect of p53 knockout on cell viability and p53-target genes during CHIKV infection
Effect of p53 knockout on interferon Type-I signaling during CHIKV infection
Capacity of p53 knockout LHCN-M2 cells to induce the Type-I interferon signaling pathway
Effect of p53 knockout on CHIKV-induced cell death
Nuclear translocation of p53 and NF-κB during CHIKV infection
Effect of p53 knockout on the release of cytochrome c during CHIKV infection
Discussion
IN INSECTS: IN DROSOPHILA MELANOGASTER P53 EXPRESSION IMPACTS THE VIRAL REPLICATION OF CHIKV AND SINV
Detection of Wolbachia spp. in Drosophila w1118 and p53-/- strains
Survival curve of w1118 and p53-/- flies injected with CHIKV
Viral replication of SINV and CHIKV and CHIKV production in w1118 and p53-/- strains
Discussion
IN MOSQUITO CELLS: INFECTION OF MOSQUITO AEDES ALBOPICTUS AND AEDES AEGYPTI CELL LINES WITH CHIKV
RESULTS AND DISCUSSION
Effect of the origin of CHIKV production on the permissiveness and pro-apoptotic response of
mosquito cell lines
Time course of CHIKV infection in C6/36 cells and analysis of pro-apoptotic and antioxidant
p53-target genes
Conclusion
CHAPTER 2) ANALYSIS OF THE EFFECT OF P53 ISOFORMS ON CHIKV INFECTION IN MAMMALIAN CELL LINES
1 OBJECTIVES
2 MATERIAL AND METHODS
Cell lines and viruses
Generation of TP53 CRISPR-mediated knockout LHCN-M2 and U2OS cell line
Transduction of LHCN-M2 with doxycycline-inducible shRNA-Δ40p53
Generation of Doxycycline-inducible system for overexpression of Δ40p53α and Δ133p53α isoforms
2.2.2.1 Production of VSVg pseudo particles
2.2.2.2 Verification of doxycycline-inducible system for overexpression of p53 isoforms in LHCN-M2 cells
Cell viability
Immunostaining and flow cytometry analysis
3 RESULTS
Generation of an endogenous Δ40p53 isoform overexpressing cell line using CRISPR/Cas9 technology
Effect of Δ40p53 overexpression on CHIKV infection
Change in cell morphology of the LHCN-M2 Δ40p53 cell line
Effect of Δ40p53 isoform in the new generated CRISPR/Cas9 LHCN-M2 cell line on the cell viability of LHCN-M2 during CHIKV infection
Loss of effect of the Δ40p53 isoform on viral infection in the CRISPR/Cas9 U2OS cell line
Doxycycline-inducible system overexpressing Δ40p53 and Δ133p53 isoforms
Generation of the LHCN-M2 doxycycline-inducible system for the over-expression of Δ40p53 and
Δ133p53 isoforms
Effect of overexpression of p53 isoforms on CHIKV viral capsid
Discussion
GENERAL CONCLUSION
REFERENCES
