Hélène Jacomy

Human Respiratory Coronavirus OC43: Genetic Stability and Neuroinvasion

ABSTRACT The complete genome sequences of the human coronavirus OC43 (HCoV-OC43) laboratory strain from the American Type Culture Collection (ATCC), and a HCoV-OC43 clinical isolate, designated Paris, were obtained. Both genomes are 30,713 nucleotides long, excluding the poly(A) tail, and only differ by 6 nucleotides. These six mutations are scattered throughout the genome and give rise to only two amino acid substitutions: one in the spike protein gene (I958F) and the other in the nucleocapsid protein gene (V81A). Furthermore, the two variants were shown to reach the central nervous system (CNS) after intranasal inoculation in BALB/c mice, demonstrating neuroinvasive properties. Even though the ATCC strain could penetrate the CNS more effectively than the Paris 2001 isolate, these results suggest that intrinsic neuroinvasive properties already existed for the HCoV-OC43 ATCC human respiratory isolate from the 1960s before it was propagated in newborn mouse brains. It also demonstrates that the molecular structure of HCoV-OC43 is very stable in the environment (the two variants were isolated ca. 40 years apart) despite virus shedding and chances of persistence in the host. The genomes of the two HCoV-OC43 variants display 71, 53.1, and 51.2% identity with those of mouse hepatitis virus A59, severe acute respiratory syndrome human coronavirus Tor2 strain (SARS-HCoV Tor2), and human coronavirus 229E (HCoV-229E), respectively. HCoV-OC43 also possesses well-conserved motifs with regard to the genome sequence of the SARS-HCoV Tor2, especially in open reading frame 1b. These results suggest that HCoV-OC43 and SARS-HCoV may share several important functional properties and that HCoV-OC43 may be used as a model to study the biology of SARS-HCoV without the need for level three biological facilities.

Recovery of a Neurovirulent Human Coronavirus OC43 from an Infectious cDNA Clone

ABSTRACT This study describes the assembly of a full-length cDNA clone of human coronavirus (HCoV)-OC43 in a bacterial artificial chromosome (BAC). The BAC containing the full-length infectious cDNA (pBAC-OC43FL) was assembled using a two-part strategy. The first step consisted in the introduction of each end of the viral genome into the BAC with accessory sequences allowing proper transcription. The second step consisted in the insertion of the whole HCoV-OC43 cDNA genome into the BAC. To produce recombinant viral particles, pBAC-OC43FL was transfected into BHK-21 cells. Recombinant virus displayed the same phenotypic properties as the wild-type virus, including infectious virus titers produced in cell culture and neurovirulence in mice.

Neuroprotective Effect of Apolipoprotein D against Human Coronavirus OC43-Induced Encephalitis in Mice

Apolipoprotein D (apoD) is a lipocalin upregulated in the nervous system after injury or pathologies such as Alzheimers disease, Parkinsons disease, and multiple sclerosis. We previously demonstrated that apoD protects against neuropathology by controlling the level of peroxidated lipids. Here, we further investigated the biological function of apoD in a mouse model of acute encephalitis. Our results show that apoD transcript and protein are upregulated during acute encephalitis induced by the human coronavirus OC43 (HCoV-OC43) infection. The apoD upregulation coincides with glial activation, and its expression returns to normal levels when the virus is cleared, concomitantly to a resolved glial reactivity. In addition, the overexpression of human apoD in the neurons of Thy-1/ApoD transgenic mice results in a threefold increase of the number of mice surviving to HCoV-OC43 infection. This increased survival rate is correlated with an upregulated glial activation associated with a limited innate immune response (cytokines, chemokines) and T-cell infiltration into infected brains. Moreover, the protection seems to be associated with a restricted phospholipase A2 activity. These data reveal a role for apoD in the regulation of inflammation and suggest that it protects from HCoV-OC43-induced encephalitis, most likely through the phospholipase A2 signaling pathways.

Vacuolating encephalitis in mice infected by human coronavirus OC43

Abstract Involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. Human respiratory coronaviruses (HCoV) can infect neural cells, persist in human brain, and activate myelin-reactive T cells. As a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of HCoV-OC43 through the development of an experimental animal model. Virus inoculation of 21-day postnatal C57BL/6 and BALB/c mice led to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence. This acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. Damage to the CNS was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. Although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals.

Human coronavirus OC43 infection induces chronic encephalitis leading to disabilities in BALB/C mice

Abstract The notion that an infectious respiratory pathogen can damage the central nervous system (CNS) and lead to neurological disease was tested using a human respiratory coronavirus, the OC43 strain of human coronavirus (HCoV-OC43). First, primary cell cultures were used to determine the susceptibility of each type of neural cells to virus infection. Neurons were the target cells, undergoing degeneration during infection, in part due to apoptosis. Second, neuropathogenicity was investigated in susceptible mice. Intracerebral inoculation of HCoV-OC43 into BALB/c mice led to an acute encephalitis with neuronal cell death by necrosis and apoptosis. Infectious virus was apparently cleared from surviving animals, whereas viral RNA persisted for several months. Some of the animals surviving to acute encephalitis presented an abnormal limb clasping reflex and a decrease in motor activity starting several months post-infection. These results suggest that viral persistence could be associated with an increased neuronal degeneration leading to neuropathology and motor deficits in susceptible individuals.

Glutamate Excitotoxicity Is Involved in the Induction of Paralysis in Mice after Infection by a Human Coronavirus with a Single Point Mutation in Its Spike Protein

ABSTRACT Human coronaviruses (HCoV) are recognized respiratory pathogens, and some strains, including HCoV-OC43, can infect human neuronal and glial cells of the central nervous system (CNS) and activate neuroinflammatory mechanisms. Moreover, HCoV-OC43 is neuroinvasive, neurotropic, and neurovirulent in susceptible mice, where it induces chronic encephalitis. Herein, we show that a single point mutation in the viral spike (S) glycoprotein (Y241H), acquired during viral persistence in human neural cells, led to a hind-limb paralytic disease in infected mice. Inhibition of glutamate excitotoxicity using a 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propranoic acid (AMPA) receptor antagonist (GYKI-52466) improved clinical scores related to the paralysis and motor disabilities in S mutant virus-infected mice, as well as protected the CNS from neuronal dysfunctions, as illustrated by restoration of the phosphorylation state of neurofilaments. Expression of the glial glutamate transporter GLT-1, responsible for glutamate homeostasis, was downregulated following infection, and GYKI-52466 also significantly restored its steady-state expression level. Finally, GYKI-52466 treatment of S mutant virus-infected mice led to reduced microglial activation, which may lead to improvement in the regulation of CNS glutamate homeostasis. Taken together, our results strongly suggest an involvement of excitotoxicity in the paralysis-associated neuropathology induced by an HCoV-OC43 mutant which harbors a single point mutation in its spike protein that is acquired upon persistent virus infection.

Mutations in the spike glycoprotein of human coronavirus OC43 modulate disease in BALB/c mice from encephalitis to flaccid paralysis and demyelination.

The etiology of most neurodegenerative diseases of the central nervous system remains unknown and likely involves a combination of genetic susceptibility and environmental triggering factors. Given that exposure to numerous infectious pathogens occurs during childhood, and that some viral infections can lead to neurodegeneration and demyelination, it is conceivable that some viruses may act as triggering factors in neuropathogenesis. We have previously shown that the prototype OC43 strain of the common cold—associated human respiratory coronavirus has the capacity to infect human neuronal and glial cells and does persist in human brains. Moreover, it has neuroinvasive properties in susceptible BALB/c mice, where it leads to a chronic encephalitis with accompanying disabilities. Here, we show that mutations in the viral spike glycoprotein, reproducibly acquired during viral persistence in human neural cell cultures, led to a drastically modified virus-induced neuropathology in BALB/c mice, characterized by flaccid paralysis and demyelination. Even though infection by both mutated and wild-type viruses led to neuroinflammation, the modified neuropathogenesis induced by the mutated virus was associated with increased viral spread and significantly more CD4+ and CD8+ T-lymphocyte infiltration into the central nervous system, as well as significantly increased levels of the proinflammatory cytokine interleukin (IL)-6 and the chemokine CCL2 (monocyte chemoattractant protein [MCP]-1). Moreover, recombinant virus harboring the S glycoprotein mutations retained its neurotropism, productively infecting neurons. Therefore, interaction of a human respiratory coronavirus with the central nervous system may modulate virus and host factors resulting in a modified neuropathogenesis in genetically susceptible individuals.

Novel treatment with neuroprotective and antiviral properties against a neuroinvasive human respiratory virus

ABSTRACT Human coronaviruses (HCoVs) are recognized respiratory pathogens with neuroinvasive and neurotropic properties in mice and humans. HCoV strain OC43 (HCoV-OC43) can infect and persist in human neural cells and activate neuroinflammatory and neurodegenerative mechanisms, suggesting that it could be involved in neurological disease of unknown etiology in humans. Moreover, we have shown that HCoV-OC43 is neurovirulent in susceptible mice, causing encephalitis, and that a viral mutant with a single point mutation in the viral surface spike (S) protein induces a paralytic disease that involves glutamate excitotoxicity in susceptible mice. Herein, we show that glutamate recycling via the glial transporter 1 protein transporter and glutamine synthetase are central to the dysregulation of glutamate homeostasis and development of motor dysfunctions and paralytic disease in HCoV-OC43-infected mice. Moreover, memantine, an N-methyl-d-aspartate receptor antagonist widely used in the treatment of neurological diseases in humans, improved clinical scores related to paralytic disease and motor disabilities by partially restoring the physiological neurofilament phosphorylation state in virus-infected mice. Interestingly, memantine attenuated mortality rates and body weight loss and reduced HCoV-OC43 replication in the central nervous system in a dose-dependent manner. This novel action of memantine on viral replication strongly suggests that it could be used as an antiviral agent to directly limit viral replication while improving neurological symptoms in various neurological diseases with a viral involvement. IMPORTANCE Mutations in the surface spike (S) protein of human respiratory coronavirus OC43 appear after persistent infection of human cells of the central nervous system, a possible viral adaptation to this environment. Furthermore, a single amino acid change in the viral S protein modulated virus-induced neuropathology in mice from an encephalitis to a neuropathology characterized by flaccid paralysis, which involves glutamate excitotoxicity. We now show that memantine, a drug that is used for alleviating symptoms associated with neuropathology, such as Alzheimers disease, can partially restore the physiological state of infected mice by limiting both neurodegeneration and viral replication. This suggests that memantine could be used as an antiviral agent while improving neurological symptoms in various neurological diseases with a viral involvement.

Titration of Human Coronaviruses, HCoV-229E and HCoV-OC43, by an Indirect Immunoperoxidase Assay

Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as “tissue culture infectious dose” (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues, or fluids).

Susceptibility of murine CNS to OC43 infection.

Multiple sclerosis (MS), one of the most common neurological diseases of young adults, is accompanied by inflammatory demyelination and loss of oligodendrocytes in the central nervous system (CNS). Although its etiology remains unclear, a generally accepted hypothesis is that virus infections could initiate a CNS-directed immune process in a genetically predisposed host (Oldstone 1997). Amongst various animal models of virus-induced demyelination, studies on murine hepatitis virus (MHV) have revealed that this virus is capable of causing direct oligodendrocyte cytopathology, but may also elicit a variety of immunopathological responses (Lane and Buchmeier 1997). Given that MHV causes MS-like CNS demyelination, the related human coronaviruses (HCoV) represent a logical target of investigation as a potential microbial agent involved in MS pathogenesis.