key: cord-0906458-9amjb7zs authors: St-Jean, Julien R.; Desforges, Marc; Talbot, Pierre J. title: Genetic Evolution of Human Coronavirus OC43 in Neural Cell Culture date: 2006 journal: The Nidoviruses DOI: 10.1007/978-0-387-33012-9_88 sha: 116625daed29500dd2eae93b9d47152715dad52c doc_id: 906458 cord_uid: 9amjb7zs nan for up to one-third of common colds. HCoV-OC43 possesses a genome that comprises genes encoding various structural and nonstructural proteins. Amongst these proteins, the S protein is biologically very important because it could be involved in determination of viral tropism. Indeed, it could for instance be associated with the capacity of the virus to reach the central nervous system (CNS) and possibly trigger neurological disorders. It could also confer the host species specificity observed with coronaviruses. In past years, we have shown that HCoV-OC43 is neurotropic and neuroinvasive, as it persistently infects neural cell cultures 1 and human brains. 2 Although we have suggested that OC43 could remain genetically surprisingly stable in the environment, 3 it is known that coronaviruses can adapt in cell culture or under selection pressure, for instance related to immune system evasion. Virus from persistent infections was purified at different passages. Prior to purification, virus was clarified and precipitated with polyethylene glycol (PEG) 8000 (Sigma). Accudenz (Accurate Chemicals) was used to perform gradient purification. Viral RNA was extracted using the GenElute Direct mRNA Miniprep Kit (Sigma) and reverse transcribed with MMuLVreverse transcriptase (Invitrogen). The Expand High-Fidelity Taq polymerase (Roche) was used to perform PCR. Primers specific to the HE, S and N genes were used to amplify target regions. 3 PCR amplicons were purified using the Qiaex II gel extraction kit (Qiagen) prior to sequencing, which was carried out by Bio S&T (Montréal, Québec, Canada). Prior to performing assays for modulation of tropism and infectivity, susceptibility of different cell lines to HCoV-OC43, ATCC strain, was determined ( Table 2 ). The same cell lines were then infected with virus isolated from different purifications (HRT-18 P33, P54, P110, and P155; H4 P47 and P90; H4 P56.1, P56.2, P56.3, P116.1, P116.2, and P116.3; TE-671 P38 and P79; U-87 MG P35, and MO3.13 P5, P6, and P22) in order to correlate the observed mutations with a modulation of tropism or infectivity. Supernatants were titrated using an indirect immunoperoxidase assay (IPA), as previously described. 5 Persistent infections of neural cell lines were initially performed to determine whether virus carrying mutations in genes encoding the surface protein S originated as a consequence of viral persistence. The HE protein gene and the nucleocapsid protein gene N were also sequenced in order to determine if these genes contributed to adaptation in cell culture. Viral particles released from persistently infected neural cell lines were isolated and purified by gradient centrifugation, and genomic RNA was sequenced. Results showed various mutations in the S gene but very few in HE and N genes, suggesting that the S gene is responsible for adaptation to the cellular environment, which could be associated with neurotropism, neuroinvasion, and presumably neuropathogenesis ( Table 1) . Almost every acquired mutation (Table 1 ) was conserved at subsequent passages, suggesting that they could confer an adaptive advantage and a stable phenotype to the virus. Five mutations were predominant and were found in almost all persistent infections (D24Y, S83T, H183R, Y241H, and N489H). The first four mutations are hypervariable region. To correlate the observed mutations in the S gene with viral replication and tropism, assays for modulation of tropism and infectivity 6 were performed using cell lines originating from various human tissues as well as from various animal species, for which 500 located in the putative receptor binding site, whereas the fifth one is located within the H482Y N489H P972L N413T N639N T855I F683Y K506T P973S F420S T855I N880K (I) L693F T641S A978S N489H D875H L893H A759E N768T T1086N K506N L893R S959C S898S (I) E896K D1170A T536N A965V W974L V980A S901F Q541L T975A T975P N1203 (D) W974L R757H I1227T V980A I1227T F982L E896D T1245I S1093S V986I C897G G1169G G1169D E933G M1222K E1236A F982L D1232Y S1192R P1249L T1225I I1304I P1228S * D, deletion; I, insertion. ** Passages (and purification numbers) are indicated below the cell line. susceptibility to HCoV-OC43 infection was previously determined ( Table 2 ). These analyses revealed that mutations found throughout the S gene could affect the latter viral properties in certain cell lines. Amongst the virus variants obtained following persistent infections and virus purifications, five showed extended cellular tropism and increased replication titers in vitro: U87-MG P35, H4 P47, H4 P56.3, H4 P116.1, and H4 P116.2 (data not shown). Furthermore, some variants isolated from persistent infections were more virulent in mice and could form plaques, in opposition to the ATCC HCoV-OC43 reference strain VR759 (data not shown). We have identified several mutations in the S gene of the HCoV-OC43 genome following persistent infections in different cell lines. These mutations will help us to further characterize viral adaptation during persistence and to understand mechanisms that are implicated in viral tropism and infectivity. Future studies will be carried out Almazán and 501 vector . 7, 8 using an infectious cDNA clone of the OC43 strain assembled in a BAC The construction of this clone was performed in collaboration with F. L. Enjuanes and will provide an invaluable tool to further understanding the underlying mechanisms for viral replication and tropism. In combination with the experiments coronavirus neuropathogenesis. described above, the clone will be useful in elucidating the molecular basis of human Acute and persistent infection of human neural cell lines by human coronavirus OC43 A human glial hybrid cell line differentially expressing genes subserving oligodendrocyte and astrocyte phenotype Vacuolating encephalitis in mice infected by human coronavirus OC43, host range Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome Neuroinvasion by human respiratory The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended Recovery of a neuro virulent human coronavirus OC43 from an infectious cDNA clone