R. J. de Groot

Middle East Respiratory Syndrome Coronavirus (MERS-CoV); Announcement of the Coronavirus Study Group

During the summer of 2012, in Jeddah, Saudi Arabia, a hitherto unknown coronavirus (CoV) was isolated from the sputum of a patient with acute pneumonia and renal failure ([1][1], [2][2]). The isolate was provisionally called human coronavirus Erasmus Medical Center (EMC) ([3][3]). Shortly thereafter

Evidence for a coiled-coil structure in the spike proteins of coronaviruses

Abstract The amino acid sequences of the spike proteins from three distantly related coronaviruses have been deduced from cDNA sequences. In the C-terminal half, an homology of about 30% was found, while there was no detectable sequence conservation in the N-terminal regions. Hydrophobic “heptad” repeat patterns indicated the presence of two α-helices with predicted lengths of 100 and 50 Å, respectively. It is suggested that, in the spike oligomer. these α-helices form a complex coiled-coil, resembling the supersecondary structures in two other elongated membrane proteins, the haemagglutinin of influenza virus and the variable surface glycoprotein of trypanosomes.

Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo.

The non‐structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2‐containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2‐containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis.

PERSISTENCE AND EVOLUTION OF FELINE CORONAVIRUS IN A CLOSED CAT-BREEDING COLONY

Abstract Feline coronavirus (FCoV) persistence and evolution were studied in a closed cat-breeding facility with an endemic serotype I FCoV infection. Viral RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the feces and/or plasma of 36 of 42 cats (86%) tested. Of 5 cats, identified as FCoV shedders during the initial survey, 4 had detectable viral RNA in the feces when tested 111 days later. To determine whether this was due to continuous reinfection or to viral persistence, 2 cats were placed in strict isolation and virus shedding in the feces was monitored every 2–4 days. In 1 of the cats, virus shedding continued for up to 7 months. The other animal was sacrificed after 124 days of continuous virus shedding in order to identify the sites of viral replication. Viral mRNA was detected only in the ileum, colon, and rectum. Also in these tissues, FCoV-infected cells were identified by immunohistochemistry. These findings provide the first formal evidence that FCoV causes chronic enteric infections. To assess FCoV heterogeneity in the breeding facility and to study viral evolution during chronic infection, FCoV quasispecies sampled from individual cats were characterized by RT-PCR amplification of selected regions of the viral genome followed by sequence analysis. Phylogenetic comparison of nucleotides 7–146 of ORF7b to corresponding sequences obtained for independent European and American isolates indicated that the viruses in the breeding facility form a clade and are likely to have originated from a single founder infection. Comparative consensus sequence analysis of the more variable region formed by residues 79–478 of the S gene revealed that each cat harbored a distinct FCoV quasispecies. Moreover, FCoV appeared to be subject to immune selection during chronic infection. The combined data support a model in which the endemic infection is maintained by chronically infected carriers. Virtually every cat born to the breeding facility becomes infected, indicating that FCoV is spread very efficiently. FCoV-infected cats, however, appear to resist superinfection by closely related FCoVs.

Feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene.

Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV), a virulent mutant of apathogenic feline enteric coronavirus (FECV). We analysed the 3c gene--a proposed virulence marker--in 27 FECV- and 28 FIPV-infected cats. Our findings suggest that functional 3c protein expression is crucial for FECV replication in the gut, but dispensable for systemic FIPV replication. Whilst intact in all FECVs, the 3c gene was mutated in the majority (71.4 %) of FIPVs, but not in all, implying that mutation in 3c is not the (single) cause of FIP. Most cats with FIP had no detectable intestinal feline coronaviruses (FCoVs) and had seemingly cleared the primary FECV infection. In those with detectable intestinal FCoV, the virus always had an intact 3c and seemed to have been acquired by FECV superinfection. Apparently, 3c-inactivated viruses replicate not at all--or only poorly--in the gut, explaining the rare incidence of FIP outbreaks.

Discontinuous and non‐discontinuous subgenomic RNA transcription in a nidovirus

Arteri‐, corona‐, toro‐ and roniviruses are evolutionarily related positive‐strand RNA viruses, united in the order Nidovirales. The best studied nidoviruses, the corona‐ and arteriviruses, employ a unique transcription mechanism, which involves discontinuous RNA synthesis, a process resembling similarity‐assisted copy‐choice RNA recombination. During infection, multiple subgenomic (sg) mRNAs are transcribed from a mirror set of sg negative‐strand RNA templates. The sg mRNAs all possess a short 5′ common leader sequence, derived from the 5′ end of the genomic RNA. The joining of the non‐contiguous ‘leader’ and ‘body’ sequences presumably occurs during minus‐strand synthesis. To study whether toroviruses use a similar transcription mechanism, we characterized the 5′ termini of the genome and the four sg mRNAs of Berne virus (BEV). We show that BEV mRNAs 3–5 lack a leader sequence. Surprisingly, however, RNA 2 does contain a leader, identical to the 5′‐terminal 18 residues of the genome. Apparently, BEV combines discontinuous and non‐discontinous RNA synthesis to produce its sg mRNAs. Our findings have important implications for the understanding of the mechanism and evolution of nidovirus transcription.

Sequence analysis of the 3' end of the feline coronavirus FIPV 79-1146 genome: Comparison with the genome of porcine coronavirus TGEV reveals large insertions

Abstract The genetic information, carried on mRNA 6 of feline infectious peritonitis virus (FIPV) strain 79–1146, was determined by sequence analysis of cDNA clones derived from the 3′ end of the FIPV genome. Two ORFs were found, encoding polypeptides of 11 K (ORF-1) and 22K (ORF-2). The FIPV sequence was compared to the 3′ end sequence of transmissible gastroenteritis virus (TGEV). ORF-1 has a homologous counterpart (ORF-X3) in the TGEV genome; both ORFs are located at the same position relative to the nucleocapsid gene. However, as a result of an in-frame insertion or deletion, ORF-1 is 69 nucleotides larger than ORF-X3. A similar event has occurred immediately downstream of ORF1: a 624-nucleotide segment, containing the complete ORF-2, is absent in the TGEV sequence. Most sequence similarity (98.5%) was found in the 3′ noncoding sequences. ORF-X3 and ORF-1 are preceded by the sequence AAC-TAAAC, which is assumed to be the transcription-initiation signal in FIPV and TGEV (P. A. Kapke and D. A. Brian (1986) Virology 151, 41–49). By S1 nuclease analysis, the 5′ end of FIPV RNA 6 was mapped immediately upstream of this sequence. A 700-nucleotide TGEV-specific RNA was found by cross-hybridization with an FIPV 3′ end probe, suggesting that TGEV ORF-X3 is also carried on a separate mRNA. The differences at the 3′ ends of the FIPV and TGEV genomes maybe the result of RNA recombination events.

Phylogenetic and Evolutionary Relationships among Torovirus Field Variants: Evidence for Multiple Intertypic Recombination Events

ABSTRACT Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3′ end of the HE gene, the N gene, and the 3′ nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses.

Glycosylation of the Severe Acute Respiratory Syndrome Coronavirus Triple-Spanning Membrane Proteins 3a and M

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.

The DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the yeast ubiquinol-cytochrome c reductase: a protein with an extremely high content of acidic amino acids.

We have determined the DNA sequence of the nuclear gene coding for the 17‐kd subunit VI of the ubiquinol‐cytochrome c reductase. The reading frame found encodes a putative polypeptide of 17 394 daltons. This protein is highly unusual: 38% of its residues are acidic and 14% are basic amino acids. The most notable feature in the protein sequence is a stretch of 25 consecutive acidic amino acids. The polypeptide has homology with the 9‐kd so‐called ‘hinge’ protein of beef‐heart complex III, which also has a cluster of acidic residues. Acidic amino acids are likely to be essential for the function of these proteins, since their degree of conservation is higher than that of other residues.