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Coronavirus spike proteins mediate host-cell-attachment and virus entry. Virus replication takes place within the host cell cytosol, whereas assembly and budding occur at the endoplasmic reticulum-Golgi intermediate compartment. In this study we demonstrated that the last 39 amino acid stretches of Alphacoronavirus spike cytoplasmic domains of the human coronavirus 229E, NL63, and the porcine transmissible gastroenteritis virus TGEV interact with tubulin alpha and beta chains. In addition, a partial co-localization of TGEV spike proteins with authentic host cell β-tubulin was observed. Furthermore, drug-induced microtubule depolymerization led to changes in spike protein distribution, a reduction in the release of infectious virus particles and less amount of spike protein incorporated into virions. These data demonstrate that interaction of Alphacoronavirus spike proteins with tubulin supports S protein transport and incorporation into virus particles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two "morphotypes" of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

Unlabelled: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection., Importance: Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

A novel betacoronavirus, human coronavirus (HCoV-EMC), has recently been detected in humans with severe respiratory disease. Further characterization of HCoV-EMC suggests that this virus is different from severe acute respiratory syndrome coronavirus (SARS-CoV) because it is able to replicate in multiple mammalian cell lines and it does not use angiotensin-converting enzyme 2 as a receptor to achieve infection. Additional research is urgently needed to better understand the pathogenicity and tissue tropism of this virus in humans. In their recent study published in mBio, Kindler et al. shed some light on these important topics (E. Kindler, H. R. Jónsdóttir, M. Muth, O. J. Hamming, R. Hartmann, R. Rodriguez, R. Geffers, R. A. Fouchier, C. Drosten, M. A. Müller, R. Dijkman, and V. Thiel, mBio 4[1]:e00611-12, 2013). These authors report the use of differentiated pseudostratified human primary airway epithelial cells, an in vitro model with high physiological relevance to the human airway epithelium, to characterize the cellular tropism of HCoV-EMC. More importantly, the authors demonstrate the potential use of type I and type III interferons (IFNs) to control viral infection.

The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

Pathogens of bacterial and viral origin hijack pathways operating in eukaryotic cells in many ways in order to gain access into the host, to establish themselves and to eventually produce their progeny. The detailed molecular characterization of the subversion mechanisms devised by pathogens to infect host cells is crucial to generate targets for therapeutic intervention. Here we review recent data indicating that coronaviruses probably co-opt membranous carriers derived from the endoplasmic reticulum, which contain proteins that regulate disposal of misfolded polypeptides, for their replication. In addition, we also present models describing potential mechanisms that coronaviruses could employ for this hijacking.

Genes 4 and 5 of mouse hepatitis virus (MHV) are known to encode nonstructural proteins ns4, ns5a, and ns5b, whose function is unknown. In this study, we demonstrated that one of the MHV strains, MHV-S, did not synthesize mRNA 4 and made a smaller mRNA 5. Sequence analysis showed that the transcription initiation site for gene 4 of MHV-S was mutated from the consensus UCUAAAC to UUUAAAC, consistent with the idea that mutations in this region abolish mRNA synthesis. Furthermore, within gene 5 there were deletions totaling 307 nucleotides which deleted almost all of open reading frame 5a, but preserved open reading frame 5b of gene 5. Comparison of the growth of MHV-S with other MHV strains in DBT cells revealed no significant growth defect in MHV-S. These results suggest that ns4 and ns5a are not essential for viral replication in tissue culture cells, and thus join gene 2 and the hemagglutinin-esterase (HE) gene as nonessential viral genes in MHV.

A porcine respiratory coronavirus (PRCV) was inoculated by aerosol into nine hysterectomy-derived and colostrum-deprived pigs at the age of one week. They were killed at different times after inoculation and tissues were sampled for virus isolation and immunofluorescence. Results indicate that virus replicated to high titres in the respiratory tract. Replication mainly occurred in alveolar cells but also in epithelial cells of nasal mucosa, trachea, bronchi, bronchioli, in alveolar macrophages and in tonsils. After primary replication in the respiratory tract, viraemia occurred. Virus also reached the gastrointestinal tract after swallowing. Subsequently, PRCV was observed to replicate in the ileum. The infection spread within a few days from the ileum to the duodenum. Replication in the small intestine remained limited to a few cells located in or underneath the epithelial layer of villi and, or, crypts. The cell type could not be identified. Virus was isolated from mesenteric lymph nodes in all pigs, but immunofluorescence was not observed. Results show that small changes in molecular structure between transmissible gastroenteritis virus and PRCV resulted in important changes in host cell tropism.

On farms, where the porcine respiratory coronavirus (PRCV) is enzootic, pigs usually become infected between 5 and 10 weeks of age while losing their maternal antibodies. It was examined whether PRCV replicates in the small intestine in such pigs. This point is important since intestinal replication with PRCV might induce immunity against TGEV not only by stimulating mucosal intestinal immunity, but also by the induction of a lactogenic IgA response at later age via the gut-mammary link. Five week old pigs with and without maternal antibodies were inoculated by aerosol or directly into the intestinal lumen. In aerosol inoculated pigs, virus replication was observed to high titres in the respiratory tract. Replication occurred in epithelial cells of nasal mucosa, trachea, bronchi bronchioli and alveoli and in alveolar macrophages. Small amounts of virus produced in the respiratory tract were ingested, but no intestinal replication of PRCV was demonstrated. Differences were not observed in virus titre and sites of replication in seronegative pigs compared to those in pigs with maternal antibodies. Upon inoculation of 10(5) or 10(7) TCID50 directly into the lumen of the cranial jejunum, no intestinal replication could be demonstrated.

Three strains of human coronavirus (HCV) OC43 were compared for their ability to cause enteric infections and to induce interferon alpha (IFN alpha) using the Caco-2 human colon carcinoma cell line which exhibits spontaneous epithelial differentiation in vitro. MRC-5 cell culture grown stocks were prepared from: 1. CV Paris, a strain of OC43 recovered from an outbreak of necrotizing enterocolitis in newborns. 2. CV Mb, a neurotropic strain of OC43 which exhibits strict neuronal specificity in murine neuronal cell cultures. 3. CV Rd, a strain of OC43 which grows to a high titer in human rhabdomyosarcoma (RD) cells. Immunofluorescent staining for nucleocapsid antigen and plaque assay in MRC-5 cells was used to detect viral replication. BG-9 (human foreskin) cells challenged with vesicular stomatitis virus were used to detect IFN alpha production by human peripheral blood monocytes (PBMC) stimulated by virus infected Caco-2 cells. Caco-2 cells infected with virus at a multiplicity of infection of 0.5 yielded 10(4.6) and 10(4.4) plaque forming units/ml (pfu/ml) with CV Rd and CV Paris respectively, while CV Mb yielded only 10(3) pfu/ml. Caco-2 cells infected with CV Rd induced 64 IU/ml of IFN alpha in PBMC while these cells infected with CV Paris induced less than 2 IU/ml IFN alpha. In cells infected with CV Mb 4 IU/ml IFN alpha was detected. The results suggest that a lack of IFN alpha induction by CV Paris may be an indicator of its enteropathogenic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Avian infectious bronchitis virus (IBV) was adapted to Vero cells by serial passage. No significant inhibition of IBV replication was observed when infected Vero cells were treated with alpha-amanitin or actinomycin D. In thin sections of infected cells, assembly of IBV was observed at the rough endoplasmic reticulum (RER), and mature IBV particles were located in dilated cisternae of the RER as well as in smooth cytoplasmic vesicles. In addition to typical IBV particles, enveloped particles containing numerous ribosomes were identified at later times postinfection. Monensin, a sodium ionophore which blocks glycoprotein transport to plasma membranes at the level of the Golgi complex, was found to inhibit the formation of infectious IBV. In thin sections of infected Vero cells treated with the ionophore, IBV particles were located in dilated cytoplasmic vesicles, but fewer particles were found when compared to controls. A similar pattern of virus-specific proteins was detected in control or monensin-treated IBV-infected cells, which included two glycoproteins (170 000 and 24 000 daltons) and a polypeptide of 52 000 daltons. These results suggest that the ionophore inhibits assembly of a virus which matures at intracellular membranes.

Sialodacryoadenitis (SDA) is a naturally-occurring infection of the laboratory rat raused by the coronavirus, sialodacryoadenitis virus (SDAV). The study of SDAV has been limited because there is no widely available continuous cell line for the propagation of high titers of the virus. The purpose of this study, therefore, was to compare the ability of SDAV to replicate in the permanent cell lines, LBC, of rat origin, and the mouse cell lines. L-929 and L-2. Following 2 to 6 repeated passages of SDAV in LBC cells, the virus could be readily propagated in LBC and L-2 cells, but not in L-929 cells. Similarly, SDAV adapted to replicate directly in L-2 cells could be readily propagated in LBC, but not L-929 cells. In LBC and L-2 cells, cytopathic effect (CPE), viral antigen, viral particles, and virus infectivity could be demonstrated. Titers of up to 10(8.0) infectious viral particles/0.25 ml of culture fluid were obtained at 48 hours in L-2 cells. Titers in LBC cells were one to two logs lower. When susceptible rats were inoculated with eighth passage L-2 cell-adapted virus, they developed typical lesions of SDA. Virus could be recovered from infected tissues and propagated in L-2 cells on first passage. The ability to propagate SDAV to high titers in the widely available L-2 cell line should promote the study of this virus and facilitate its comparison with other murine coronaviruses.

Rat coronavirus readily propagated and induced marked cytopathic effect in a rat cell line, LBC cell culture, which provided a sensitive, practical assay system for viral infectivity and neutralizing antibody, and a satisfactory source of the virus.

Coronaviruses have been isolated from cases of neonatal calf diarrhoea in many parts of the world and were revealed in the respiratory tract recently, during a study of calf pneumonia. In experiments on gnotobiotic calves an enteric coronavirus replicated in the nasal epithelium and a respiratory isolate was shed in faeces. The enteric coronavirus prevented subsequent infection with either homologous enteric or the respiratory isolate. A dual tropism for this agent was emphasized by the detection of coronavirus antigen in intestinal and respiratory epithelium of naturally-infected calves.

Sialodacryoadenitis virus of rat readily propagated and induced marked cytopathic effect in a rat cell line, LBC cell culture, which provides a sensitive, practical assay system for viral infectivity and neutralizing antibody, and a satisfactory source of the virus.

A lethal infection by neurotropic JHM strain of mouse hepatitis virus was prevented by the local adoptive transfer of three virus-specific Lyt-1+2-, L3T4+ T cell clones. The transfer of Lyt-1+ T cells specific for an unrelated antigen (hen egg lysozyme) did not protect. Protection required I region compatibility between the T cells and the recipients, and was reversed either by irradiation of the cells before transfer or by pretreatment of the recipients with cyclophosphamide. Adoptive transfer prevented death due to JHM virus infection but did not result in altered antiviral immunoglobulin synthesis or the suppression of viral replication in the central nervous system (CNS). The data presented implicate a local DTH response in the protection of the host from lethal infection of the CNS by a neurotropic virus, but clearly imply that other antiviral effector mechanisms are necessary for the suppression of viral replication.

Bovine embryonic lung (BEL) cells, grown in 8-chamber cell-culture slides, were inoculated with 2.5, 25, and 250 TCID50 of neonatal calf diarrhea coronavirus (NCDCV)/chamber. The NCDCV inocula were prepared in serum-free, trypsin-free regular medium (RM), and in serum-free medium containing 1 microgram (TM1), 5 microgram (TM5), and 10 microgram (TM10) of trypsin/ml; the the cells were maintained under identical media. Cytopathic effect (CPE), hemadsorption of rat RBC, formation of syncytia, appearance of immunofluorescing cells, and release of infectious virus was followed from postinoculation hours 4 to 100. Maintaining the BEL cells in medium containing trypsin up to 10 microgram/ml did not affect them adversely. The BEL cells appeared resistant to infection with NCDCV in RM by CPE, syncytia, immunofluorescing cells, or infectious virus-release parameters. In TM1, TM5, and TM10, all parameters were positive for NCDCV replication. They manifested and developed in positive correlation with increasing trypsin concentration from 1 microgram to 10 microgram/ml. Release of infectious virions preceded all other parameters of virus replication. Replication of NCDCV was demonstrated earlier by hemadsorption and immunofluorescing cells than by syncytia or CPE. The TM5 and TM10 increased the susceptibility of BEL cells to NCDCV by more than a millionfold, as measured by the titer of released virus in these media (greater than 1-(6) TCID50/ml) as compared with those in RM (infectious virus not demonstrated).

The in vitro adaptation of enteric coronaviruses is difficult. In our Laboratories, the multiplication of the COI bovine strain (F15 origin), on Vero cells is commonly practised. Since other laboratory tests (hemagglutination inhibition, cytopathic effect and immunofluorescence) are not totally reliable, electron microscopic studies present definite advantages in the interpretation of viral synthesis, as demonstrated at the 11th passage in our study. We were able to observe the following: --that the rate of infected and productive cells was important; --that the stages of viral morphogenesis were well established; --that the viral synthesis was prolific and regular and could be witnessed by important intracytoplasmic storage. The results of this study prove that the choice of our cell system is very satisfactory.