Acute and persistent infection of human neural cell lines by human coronavirus OC43

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.     

Infection of primary cultures of human neural cells by human coronaviruses 229E and OC43.

We evaluated the ability of human coronaviruses to infect primary cultures of human neural cells. Double immunofluorescence with antibodies to virus and cell markers showed infection of fetal astrocytes and of adult microglia and astrocytes by strain OC43. RNA amplification revealed infection of fetal astrocytes, adult microglia, and a mixed culture of adult oligodendrocytes and astrocytes by strain 229E. Infectious virus was released only from fetal astrocytes, with higher titers for OC43. Human coronaviruses have the capacity to infect some cells of the central nervous system, although infection of adult cells appears abortive.     

The effect of climbing chalk powder on the infectivity of human coronavirus OC43

There does not appear to be any studies in the published literature on the stability of SARS-CoV-2 in climbing chalk powder (magnesium carbonate and/or calcium carbonate), which has been hypothesized to pose a potential risk of fomite transmission of coronavirus disease 2019 (COVID-19) within climbing gyms. The aim of this study was to determine the infectivity of a model human coronavirus HCoV-OC43 in the presence of climbing chalk powder on a dry plastic surface. The stability of HCoV-OC43 on a plastic surface dusted with climbing chalk powders (magnesium carbonate, calcium carbonate or a blended chalk) was determined by titration on BHK-21 fibroblast cells. No chalk and no virus controls were included. HCoV-OC43 was stable on the plastic surface for 48 h. The stability of HCoV-OC43 was significantly (P ≤ 0·05) reduced in the presence of magnesium carbonate, calcium carbonate and the chalk blend; the infectivity was reduced by ≥2·29 log₁₀ 50% tissue culture infective dose (TCID₅₀) immediately upon on contact and by ≥2·46 log₁₀ TCID₅₀ within 1 h of contact. These findings suggest that the infectivity of coronaviruses is reduced by climbing chalk, limiting the risk of potential fomite transmission.     

Elucidation of the stability and functional regions of the human coronavirus OC43 nucleocapsid protein

Human coronavirus OC43 (HCoV‐OC43) is one of the causes of the “common cold” in human during seasons of cold weather. The primary function of the HCoV‐OC43 nucleocapsid protein (N protein) is to recognize viral genomic RNA, which leads to ribonucleocapsid formation. Here, we characterized the stability and identified the functional regions of the recombinant HCoV‐OC43 N protein. Circular dichroism and fluorescence measurements revealed that the HCoV‐OC43 N protein is more highly ordered and stabler than the SARS‐CoV N protein previously studied. Surface plasmon resonance (SPR) experiments showed that the affinity of HCoV‐OC43 N protein for RNA was approximately fivefold higher than that of N protein for DNA. Moreover, we found that the HCoV‐OC43 N protein contains three RNA‐binding regions in its N‐terminal region (residues 1–173) and central‐linker region (residues 174–232 and 233–300). The binding affinities of the truncated N proteins and RNA follow the order: residues 1–173–residues 233–300 > residues 174–232. SPR experiments demonstrated that the C‐terminal region (residues 301–448) of HCoV‐OC43 N protein lacks RNA‐binding activity, while crosslinking and gel filtration analyses revealed that the C‐terminal region is mainly involved in the oligomerization of the HCoV‐OC43 N protein. This study may benefit the understanding of the mechanism of HCoV‐OC43 nucleocapsid formation.     

Antigenic relationships among proteins of bovine coronavirus, human respiratory coronavirus OC43, and mouse hepatitis coronavirus A59

Antisera prepared against each of three single and one pair of major structural proteins of the bovine coronavirus (Mebus strain) were used in immunoblotting studies to measure cross-reactivity with the structural proteins of the human coronavirus OC43 and the mouse hepatitis coronavirus A59. We conclude that the bovine coronavirus is comprised of four major structural proteins, gp190 (normally present as 120- and 100-kilodalton subunits), gp140, pp52, and gp26. The human coronavirus OC43 has an antigenically homologous counterpart of similar molecular mass to each of these proteins. The mouse hepatitis coronavirus A59 has an antigenically homologous counterpart to only three of these proteins: gp190, pp52 and gp26. There is no counterpart in the mouse virus to the 140-kilodalton glycoprotein, the apparent hemagglutinin of the bovine coronavirus.     

Human respiratory coronavirus OC43: genetic stability and neuroinvasion

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.     

Complete genomic sequence of human coronavirus OC43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event

Coronaviruses are enveloped, positive-stranded RNA viruses with a genome of approximately 30 kb. Based on genetic similarities, coronaviruses are classified into three groups. Two group 2 coronaviruses, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV), show remarkable antigenic and genetic similarities. In this study, we report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759). Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome. In the region between the spike and membrane protein genes, a 290-nucleotide deletion is present, corresponding to the absence of BCoV ORFs ns4.9 and ns4.8. Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and for those of other group 2 coronaviruses. The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene. Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890. An evolutionary rate in the order of 4 x 10(-4) nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.     

Evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus OC43

The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.     

Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to murine coronaviruses A59 and JHM and human coronavirus OC43, but not to human coronavirus 229E.

Two coronaviruses (SK and SD), isolated from fresh autopsy brain tissue from two multiple sclerosis patients, were compared with known human and murine coronaviruses. In plaque neutralization assays, antisera prepared against multiple sclerosis isolates SK and SD demonstrated significant cross-reactivity to each other and to murine coronavirus A59, weak cross-reactivity to murine coronavirus JHM, but no cross-reactivity to the human coronavirus 229E. Antiserum to SK or SD failed to inhibit hemagglutination of chicken erythrocytes by the human coronavirus OC43. However, OC43 antiserum neutralized both SD and SK. Specific coronavirus polypeptides were identified and compared by immunoprecipitation and polyacrylamide gel electrophoresis. Infected and mock-infected 17Cl-1 cells were pretreated with actinomycin D and labeled with [35S]methionine. Polypeptides in Nonidet P-40 cytoplasmic extracts were immunoprecipitated with homologous and heterologous antisera. Identical polypeptides were precipitated from A59-, SD-, or SK-infected cell extracts by SD, SK, OC43, or A59 antisera. The polypeptides of human virus 229E were antigenically distinct, with the exception of weak recognition of a polypeptide of 50,000 molecular weight. We conclude that the two multiple sclerosis virus isolates SK and SD are closely related serologically to the murine coronavirus A59 and the human coronavirus OC43.     

Differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus.

Hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait, is associated with a predisposition to neoplasia. The present study describes the differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Primary cutaneous outgrowths were derived from normal appearing subepidermal biopsies of ACR phenotypes and appropriate controls. Exponentially growing cell cultures from ACR subjects and a portion of the clinically asymptomatic ACR progeny subjected to the viral probe were 100-1000 fold more susceptible to transformation than were normal skin fibroblast cultures. The virally transformed human skin fibroblasts showed a loss of anchorage dependency in carboxymethylcellulose suspension and formed tumors in athymic mice. The results suggest that skin fibroblasts obtained from individuals gine sarcoma virus.     

Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein

Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0 Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded β-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication.     

Differential susceptibility to human serum byAeromonas spp.

A total of 91 strains ofAeromonas (A. hydrophila, A. sobria, andA. caviae) of clinical origin were challenged in vitro against pooled human serum. A majority of the isolates ofA. hydrophila andA. sobria were resistant to the bactericidal activity of human serum, as opposed to the more serum-sensitiveA. caviae species. This difference in serum sensitivity may potentially explain the greater association of the former species with bacteremia and invasive disease.     

Differential susceptibility to nitric oxide-evoked apoptosis in human inflammatory cells

Apoptosis of neutrophils and their subsequent phagocytosis is critical to the successful resolution of inflammation. During inflammation, activated inflammatory cells generate reactive oxygen and nitrogen species, including nitric oxide (NO) and superoxide anion (O₂^??), which rapidly combine to generate peroxynitrite (ONOO^?). NO and ONOO^? are proapoptotic in human neutrophils. This study examines the effects of NO and ONOO^? on caspase activation and mitochondrial permeability in human neutrophils and determines the ability of these species to evoke apoptosis in human monocyte-derived macrophages (MDMs). NO or ONOO^? release from donor compounds was characterized by electrochemistry and electron paramagnetic resonance. Neutrophils and MDMs isolated from the peripheral blood of healthy volunteers were exposed to NO or ONOO^? before analysis of apoptosis by caspase activation, mitochondrial permeability, and annexin V binding. Both NO and ONOO^? induced apoptosis via rapid activation of caspases 2 and 3 in neutrophils. In contrast, only ONOO^? promoted apoptosis in MDMs, whereas a variety of NO donors were ineffective at inducing apoptosis in this cell type. We propose that human macrophages are refractory to NO-stimulated apoptosis in order that they persist long enough within the inflammatory focus to phagocytose apoptotic neutrophils, thereby ensuring successful resolution of inflammation.