Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection.

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.

Potential differences in cleavage of the S protein and type-1 interferon together control human coronavirus infection, propagation, and neuropathology within the central nervous system.

Human coronaviruses (HCoV) are respiratory pathogens which have been known since the 1960's. In December 2019, a new betacoronavirus, SARS-CoV-2, was reported and is responsible for one of the biggest pandemics of the last two centuries. Similar to the HCoV-OC43 strain, available evidence suggests SARS-CoV-2 neuroinvasion associated with potential neurological disorders. Coronavirus infection of the central nervous system (CNS) is largely controlled by a viral factor, the spike glycoprotein (S) and a host factor, innate immunity. However, the interaction between these two factors remains elusive. Proteolytic cleavage of the S protein can occur at the interface between receptor binding (S1) and fusion (S2) domains (S1/S2), as well as in a position adjacent to a fusion peptide within S2 (S2'). Herein, using HCoV-OC43 as a surrogate for SARS-CoV-2, we report that both S protein sites are involved in neurovirulence and are required for optimal CNS infection. Whereas efficient cleavage at S1/S2 is associated with decreased virulence, the potentially cleavable putative S2' site is essential for efficient viral infection. Furthermore, type 1 interferon (IFN 1)-related innate immunity also plays an important role in the control of viral spread towards the spinal cord, by preventing infection of ependymal cells. Our results underline the link between the differential S cleavage and IFN 1 in the prevention of viral spread, to control the severity of infection and pathology in both immunocompetent and immunodeficient mice. Taken together, these results point towards two potential therapeutic anti-viral targets: cleavage of the S protein in conjunction with efficient IFN 1-related innate immunity to prevent or at least reduce neuroinvasion, neural spread, and potential associated neurovirulence of human coronaviruses.ImportanceHuman coronaviruses (HCoV) are recognized respiratory pathogens. The emergence of the novel pathogenic member of this family in December 2019 (SARS-CoV-2, which causes COVID-19) poses a global health emergency. As with other coronaviruses reported previously, invasion of the human central nervous system (CNS), associated with diverse neurological disorders, was suggested for SARS-CoV-2. Herein, using the related HCoV-OC43 strain, we show that the viral spike protein constitutes a major neurovirulence factor and that type 1 interferon (IFN 1), in conjunction with cleavage of S protein by host proteases, represent important host factors that participate in the control of CNS infection.To our knowledge, this is the first demonstration of a direct link between cleavage of the S protein, innate immunity and neurovirulence. Understanding mechanisms of viral infection and spread in neuronal cells is essential to better design therapeutic strategies, and to prevent infection by human coronaviruses such as SARS-CoV-2 in human CNS especially in the vulnerable populations such as the elderly and immune-compromised individuals.

Human Coronaviruses and Other Respiratory Viruses: Underestimated Opportunistic Pathogens of the Central Nervous System?

Respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. However, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). Respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. Furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza A and SARS-CoV, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. For a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (CNS). Viruses infecting human CNS cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. Like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the CNS as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to CNS cells (virus-induced neuropathology). The etiological agent of several neurological disorders remains unidentified. Opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. Herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in CNS infection, with a special emphasis on human coronaviruses.

Axonal Transport Enables Neuron-to-Neuron Propagation of Human Coronavirus OC43.

Human coronaviruses (HCoVs) are recognized respiratory pathogens for which accumulating evidence indicates that in vulnerable patients the infection can cause more severe pathologies. HCoVs are not always confined to the upper respiratory tract and can invade the central nervous system (CNS) under still unclear circumstances. HCoV-induced neuropathologies in humans are difficult to diagnose early enough to allow therapeutic interventions. Making use of our already described animal model of HCoV neuropathogenesis, we describe the route of neuropropagation from the nasal cavity to the olfactory bulb and piriform cortex and then the brain stem. We identified neuron-to-neuron propagation as one underlying mode of virus spreading in cell culture. Our data demonstrate that both passive diffusion of released viral particles and axonal transport are valid propagation strategies used by the virus. We describe for the first time the presence along axons of viral platforms whose static dynamism is reminiscent of viral assembly sites. We further reveal that HCoV OC43 modes of propagation can be modulated by selected HCoV OC43 proteins and axonal transport. Our work, therefore, identifies processes that may govern the severity and nature of HCoV OC43 neuropathogenesis and will make possible the development of therapeutic strategies to prevent occurrences.IMPORTANCE Coronaviruses may invade the CNS, disseminate, and participate in the induction of neurological diseases. Their neuropathogenicity is being increasingly recognized in humans, and the presence and persistence of human coronaviruses (HCoV) in human brains have been proposed to cause long-term sequelae. Using our mouse model relying on natural susceptibility to HCoV OC43 and neuronal cell cultures, we have defined the most relevant path taken by HCoV OC43 to access and spread to and within the CNS toward the brain stem and spinal cord and studied in cell culture the underlying modes of intercellular propagation to better understand its neuropathogenesis. Our data suggest that axonal transport governs HCoV OC43 egress in the CNS, leading to the exacerbation of neuropathogenesis. Exploiting knowledge on neuroinvasion and dissemination will enhance our ability to control viral infection within the CNS, as it will shed light on underlying mechanisms of neuropathogenesis and uncover potential druggable molecular virus-host interfaces.

The OC43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and CNS pathology.

The OC43 strain of human coronavirus (HCoV-OC43) is an ubiquitous respiratory tract pathogen possessing neurotropic capacities. Coronavirus structural envelope (E) protein possesses specific motifs involved in protein-protein interaction or in homo-oligomeric ion channel formation, which are known to play various roles including in virion morphology/assembly and in cell response to infection and/or virulence. Making use of recombinant viruses either devoid of the E protein or harboring mutations either in putative transmembrane domain or PDZ-binding motif, we demonstrated that a fully functional HCoV-OC43 E protein is first needed for optimal production of recombinant infectious viruses. Furthermore, HCoV-OC43 infection of human epithelial and neuronal cell lines, of mixed murine primary cultures from the central nervous system and of mouse central nervous system showed that the E protein is critical for efficient and optimal virus replication and propagation, and thereby for neurovirulence.

Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes.

Arthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti. The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells.

Infection of Ixodes ricinus by Borrelia burgdorferi sensu lato in peri-urban forests of France.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. In Europe, it is transmitted by Ixodes ticks that carry bacteria belonging to the Borrelia burgdorferi sensu lato complex. The objective of this work was to explore eco-epidemiological factors of Lyme borreliosis in peri-urban forests of France (Sénart, Notre-Dame and Rambouillet). We investigated whether the introduction of Tamias sibiricus in Sénart could alter the density of infected ticks. Moreover, the density and tick infection were investigated according to the tree species found in various patches of Sénart forest. For this purpose, ticks were sampled during 3 years. In the Sénart forest, the density of nymph and adult ticks showed no significant difference between 2008, 2009 and 2011. The nymph density varied significantly as a function of the month of collection. Regarding the nymphs, a higher rate of infection and infected density were found in 2009. Plots with chipmunks (C) presented a lower density of both nymphs and adult ticks than plots without chipmunks (NC) did. A higher rate of infection of nymphs with Borrelia was seen in C plots. The prevalence of the various species of Borrelia was also found to vary between C and NC plots with the year of the collect. The presence of chestnut trees positively influenced the density of both nymphs and adults. The infected nymph density showed a significant difference depending on the peri-urban forest studied, Sénart being higher than Rambouillet. The prevalence of Borrelia species also differed between the various forests studied. Concerning the putative role that Tamias sibiricus may play in the transmission of Borrelia, our results suggest that its presence is correlated with a higher rate of infection of questing ticks by Borrelia genospecies and if its population increases, it could play a significant role in the risk of transmission of Lyme borreliosis.

Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43.

Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183-241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis. IMPORTANCE: Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.

Human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis.

Among the various respiratory viruses infecting human beings, coronaviruses are important pathogens, which usually infect the upper respiratory tract, where they are mainly associated with common colds. However, in more vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, and various types of respiratory distress syndrome. The respiratory involvement of human coronaviruses has been clearly established since the 1960s. Nevertheless, for almost three decades now, data reported in the scientific literature has also demonstrated that, like it was described for other human viruses, coronaviruses have neuroinvasive capacities since they can spread from the respiratory tract to the central nervous system (CNS). Once there, infection of CNS cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. Neuroinvasive coronaviruses could damage the CNS as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuroimmunopathology) and/or viral replication, which directly induces damage to CNS cells (virus-induced neuropathology). Given all these properties, it has been suggested that these opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of neurologic diseases for which the etiology remains poorly understood. Herein, we present host and viral factors that participate in the regulation of the possible pathogenic processes associated with CNS infection by human coronaviruses and we try to decipher the intricate interplay between virus and host target cells in order to characterize their role in the virus life cycle as well as in the capacity of the cell to respond to viral invasion.

Neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans.

In humans, viral infections of the respiratory tract are a leading cause of morbidity and mortality worldwide. Several recognized respiratory viral agents have a neuroinvasive capacity since they can spread from the respiratory tract to the central nervous system (CNS). Once there, infection of CNS cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. Among the various respiratory viruses, coronaviruses are important pathogens of humans and animals. Human Coronaviruses (HCoV) usually infect the upper respiratory tract, where they are mainly associated with common colds. However, in more vulnerable populations, such as newborns, infants, the elderly, and immune-compromised individuals, they can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, respiratory distress syndrome, or even severe acute respiratory syndrome (SARS). The respiratory involvement of HCoV has been clearly established since the 1960s. In addition, for almost three decades now, the scientific literature has also demonstrated that HCoV are neuroinvasive and neurotropic and could induce an overactivation of the immune system, in part by participating in the activation of autoreactive immune cells that could be associated with autoimmunity in susceptible individuals. Furthermore, it was shown that in the murine CNS, neurons are the main target of infection, which causes these essential cells to undergo degeneration and eventually die by some form of programmed cell death after virus infection. Moreover, it appears that the viral surface glycoprotein (S) represents an important factor in the neurodegenerative process. Given all these properties, it has been suggested that these recognized human respiratory pathogens could be associated with the triggering or the exacerbation of neurological diseases for which the etiology remains unknown or poorly understood.

Clone 13-infected Aedes aegypti salivary components inhibit Rift Valley fever virus pathogenicity.

Rift Valley fever virus (RVFV) continues to cause large outbreaks among humans and domestic animals in Africa. RVFV Clone 13, a naturally attenuated clone, is a promising vaccine which was used during the 2009-2010 outbreak in South Africa and played a key role in the control of the disease. In this work, we infected Aedes aegypti mosquitoes with RVFV Clone 13 and prepared salivary gland extracts (SGE). C57BL/6-NRJ male mice were infected with a mixture of SGE infected by Clone 13 and the ZH548 RVFV strain. With the injection of increasing doses of Clone 13-infected SGE, all mice were protected. Our results suggest Clone 13 infected SGE contain unique antiviral components able to counteract the replication of RVFV when injected into vertebrates.

Human respiratory coronaviruses : neuroinvasive, neurotropic and potentially neurovirulent pathogens.

In humans, viral infections of the respiratory tract are a leading cause of morbidity and mortality worldwide. Among the various respiratory viruses, coronaviruses are important ubiquitous pathogens of humans and animals. Since the late 1960's, human coronaviruses (HCoV) are recognized pathogens of the upper respiratory tract, being mainly associated with mild pathologies such as the common cold. However, in vulnerable populations, (newborns, infants, the elderly and immune-compromised individuals), they can affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, respiratory distress syndrome or even severe acute respiratory syndrome (SARS). For almost three decades now, the scientific literature has also demonstrated that HCoV are neuroinvasive and neurotropic: neurons are often the target cell in the central nervous system (CNS), inducing neurodegeneration and eventually death. Moreover, HCoV can contribute to an overactivation of the immune system that could lead to autoimmunity in the CNS of susceptible individuals. Given all these properties, it has been suggested that HCoV could be associated with the triggering or the exacerbation of human neurological diseases for which the etiology remains unknown or poorly understood.

Aedes mosquito saliva modulates Rift Valley fever virus pathogenicity.

BACKGROUND: Rift Valley fever (RVF) is a severe mosquito-borne disease affecting humans and domestic ruminants. Mosquito saliva contains compounds that counteract the hemostatic, inflammatory, and immune responses of the host. Modulation of these defensive responses may facilitate virus infection. Indeed, Aedes mosquito saliva played a crucial role in the vector's capacity to effectively transfer arboviruses such as the Cache Valley and West Nile viruses. The role of mosquito saliva in the transmission of Rift Valley fever virus (RVFV) has not been investigated. OBJECTIVE: Using a murine model, we explored the potential for mosquitoes to impact the course of RVF disease by determining whether differences in pathogenesis occurred in the presence or absence of mosquito saliva and salivary gland extract. METHODS: C57BL/6NRJ male mice were infected with the ZH548 strain of RVFV via intraperitoneal or intradermal route, or via bites from RVFV-exposed mosquitoes. The virus titers in mosquitoes and mouse organs were determined by plaque assays. FINDINGS: After intraperitoneal injection, RVFV infection primarily resulted in liver damage. In contrast, RVFV infection via intradermal injection caused both liver and neurological symptoms and this route best mimicked the natural infection by mosquitoes. Co-injections of RVFV with salivary gland extract or saliva via intradermal route increased the mortality rates of mice, as well as the virus titers measured in several organs and in the blood. Furthermore, the blood cell counts of infected mice were altered compared to those of uninfected mice. INTERPRETATION: Different routes of infection determine the pattern in which the virus spreads and the organs it targets. Aedes saliva significantly increases the pathogenicity of RVFV.

Guillain-Barre syndrome: first description of a snake envenomation aetiology.

BACKGROUND: Guillain-Barre syndrome (GBS) is considered as an acute, immune-mediated polyradiculoneuropathy with different clinical phenotypes arising after viral or bacterial infections, vaccination or surgery. However, in 40% of GBS patients the aetiology remains unknown. In this manuscript, we report the occurrence of GBS in a patient bitten by a snake (Vipera aspis) for which a cross-reaction was shown between GM2 ganglioside and glycosidic epitopes of venom proteins. METHODS: The venom of the snake implied in the patient's envenomation was collected. Its composition was characterised by ELISA and SELDI-TOF MS. Cross-reactivities between venom proteins and GM2 gangliosides were identified by Western blot after immunoabsorption of patient's serum with increasing amounts of purified GM2. Enzymatic deglycosylation of the venom was performed to determine the specificity of the patient's serum cross-reaction. FINDINGS: We proved the absence of neurotoxicity of the viper venom. The patient's serum presented specific cross-reactions with several glycosylated venom proteins. After deglycolysation of these proteins, the patient's serum cross-reactivity was abolished. Furthermore, we compared the immune response to venom proteins of sera from two groups of patients. The first group showed IgM reactivity against GM2 ganglioside associated with GBS, and cross-reacted with venom proteins. The second group presented an IgM reactivity against CMV, without neurological disorders, and reacted with neither venom proteins nor gangliosides. INTERPRETATION: Our study proved the auto-immunological aetiology of GBS in our patient based on molecular mimicry mechanisms between venom proteins and GM2 ganglioside.