Inviting an author to review:
Find an author and click ‘Invite to review selected article’ near their name.
- Record: found
- Abstract: found
- Article: found
Middle East Respiratory Syndrome Coronavirus Antibody Reactors Among Camels in Dubai, United Arab Emirates, in 2005
research-article
Publication date (Electronic):
24 January 2014
Journal:
Transboundary and Emerging Diseases
Publisher:
BlackWell Publishing Ltd
Read this article at
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
We tested, using a low starting dilution, sequential serum samples from dromedary camels, sheep and horses collected in Dubai from February/April to October of 2005 and from dromedary camels for export/import testing between Canada and USA in 2000–2001. Using a standard Middle East respiratory syndrome coronavirus (MERS-CoV) neutralization test, serial sera from three sheep and three horses were all negative while sera from 9 of 11 dromedary camels from Dubai were positive for antibodies supported by similar results in a MERS-CoV recombinant partial spike protein antibody ELISA. The two negative Dubai camels were both dromedary calves and remained negative over the 5 months studied. The six dromedary samples from USA and Canada were negative in both tests. These results support the recent findings that infection with MERS-CoV or a closely related virus is not a new occurrence in camels in the Middle East. Therefore, interactions of MERS-CoV at the human–animal interface may have been ongoing for several, perhaps many, years and by inference, a widespread pandemic may be less likely unless significant evolution of the virus allow accelerated infection and spread potential in the human population.
Related collections
Most cited references14
- Record: found
- Abstract: found
- Article: not found
Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.
A previously unknown coronavirus was isolated from the sputum of a 60-year-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clinical data, virus isolation, and molecular identification are presented. The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
- Record: found
- Abstract: found
- Article: found
Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans
Sander van Boheemen, Miranda de Graaf, Chris Lauber … (2013)
Introduction Coronaviruses (CoVs) infect and cause disease in a wide variety of species, including bats, birds, cats, dogs, pigs, mice, horses, whales, and humans (1, 2). Recent studies suggest that bats act as a natural reservoir for coronaviruses (3–8). Coronaviruses may cause respiratory, enteric, hepatic, or neurological diseases with highly variable severity in their hosts. Until 2003, only two coronaviruses were known to infect humans. Human coronaviruses (HCoVs) HCoV-229E and HCoV-OC43 were identified in the 1960s as the causative agents of—generally mild—respiratory illnesses (9, 10). In 2002 to 2003, a previously unknown coronavirus—severe acute respiratory syndrome coronavirus (SARS-CoV)—caused a widespread outbreak of respiratory disease in humans, resulting in approximately 800 deaths and affecting around 30 countries (11–14). As a consequence of the renewed interest in coronaviruses after the SARS outbreak, two additional human coronaviruses were discovered after 2003: HCoV-NL63 in 2004 (15, 16) and HCoV-HKU1 in 2005 (17). A recent analysis of a large collection of human nasopharyngeal specimens using a Coronaviridae-wide primer set suggested that HCoV-229E, -OC43, -NL63, and -HKU1 are the only coronaviruses circulating in the human population (18). Coronaviruses are enveloped single-stranded positive-sense RNA viruses with genomes of 25 to 32 kb, and the group includes the largest known genomes among the RNA viruses (1, 19). The coronaviruses form a subfamily (Coronavirinae) within the family Coronaviridae of the order Nidovirales. The International Committee on Taxonomy of Viruses (ICTV) has recognized four genera within the Coronavirinae subfamily: Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, which were previously referred to as coronavirus groups 1, 2, and 3, and Deltacoronavirus (20). Coronaviruses are assigned to a genus on the basis of rooted phylogeny and calculation of pairwise evolutionary distances for seven highly conserved domains in the replicase polyprotein (1, 21) (C. Lauber and A. E. Gorbalenya, unpublished data). HCoV-229E and HCoV-NL63 are viruses belonging to the genus Alphacoronavirus (1). Four monophyletic lineages (A through D) with no formal taxonomic standing, some of them encompassing multiple virus species, are commonly recognized within the genus Betacoronavirus. Lineage A includes HCoV-OC43 and HCoV-HKU1 and lineage B SARS-CoV, all of which belong to different species. Lineages C and D include viruses detected only in bats, such as Rousettus bat coronavirus HKU9 (BtCoV-HKU9) (lineage D), Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5) (both lineage C) (1). The genetic diversity of coronaviruses is likely facilitated by a high frequency of RNA recombination and the ability of their unusually large RNA genomes to both gain and lose domains (1, 22, 23). These factors are believed to have promoted the emergence of viruses with novel traits that are able to adapt to new hosts and ecological niches, sometimes causing zoonotic events. For the present study, we report and analyze the complete genome sequence of the recently identified HCoV-EMC/2012, which was isolated from the sputum of a 60-year-old man who died in a hospital in Jeddah, Saudi Arabia, after developing acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) in June 2012 (24). This virus appears to be closely related to the HCoV detected in a second patient who was transported from a hospital in Qatar to a hospital in London, 3 months after hospitalization of the first patient (25). These two cases of human infection with very similar or identical coronaviruses alarmed health authorities globally, as it was a reminder of the potential threat of coronaviruses to human health that was first highlighted by the SARS outbreak of 2003 (25). The sequence analysis of a small reverse transcription-PCR (RT-PCR) fragment that was first amplified from the HCoV-EMC/2012 genome revealed the highest similarity to two betacoronaviruses circulating in bats, BtCoV-HKU4 and -HKU5 (24). Here we present the complete genome sequence of the newly isolated HCoV-EMC/2012, accompanied by a detailed annotation of its genome organization and expression strategy. Furthermore, comparative genomic analysis and state-of-the-art classification and phylogenetic analyses were applied to determine the position of the novel agent with respect to previously characterized coronaviruses. We conclude that the HCoV-EMC/2012 genome organization and expression indeed most closely resemble those of BtCoV-HKU4 and -HKU5. However, based on our analysis and in line with the ICTV guidelines for the demarcation of coronavirus species, HCoV-EMC/2012 clearly qualifies to be recognized as the prototype of a novel species, which would thus constitute the first human coronavirus in lineage C of the genus Betacoronavirus. RESULTS Sequencing of the HCoV-EMC/2012 genome. Using a combination of approaches, including deep sequencing, cycle sequencing on a more traditional capillary sequencer, and determination of the genomic termini by rapid amplification of cDNA ends (RACE), the complete genome sequence of HCoV-EMC/2012 was determined from material that had been subjected to passage in cell culture 6 times. The data from the Roche 454 GS Junior deep-sequencing run yielded a total of 90,808 sequence reads, of which 87,256 were specific for HCoV-EMC/2012. Genome coverage ranged from 1 to 5,697 reads at single nucleotide positions, with an average of 1,006 reads in the deep-sequencing run. Based on the contigs assembled from these initial data, primers approximately 800 nucleotides (nt) apart were designed to amplify PCR fragments with 100-nt overlaps covering the entire virus genome (see Table S1 in the supplemental material for primer sequences). These amplicons were sequenced using Sanger sequencing, and a total of 104 sequence runs were assembled—along with the original 90,808 deep-sequencing reads—into a single contiguous sequence of 30,119 nt, including the first 12 nt of the 3′ poly(A) tail. Although 454 sequencing resulted in a higher single-read error rate than Sanger sequencing, the high coverage in the first data set largely corrected for these errors. Occasionally, the correct number of bases in homopolymer stretches was difficult to determine, which is a typical problem in 454 sequencing. Nevertheless, there was excellent agreement between the deep-sequencing data and the confirmatory Sanger sequencing. The final consensus sequence was submitted to GenBank (see below). This sequence contains only two ambiguous positions, nt 11623 and 27162. The variation at position 11623 translates into a Val or Gly uncertainty at amino acid (aa) 3782 of pp1a/pp1ab. Position 27162 was either a G or an A, with the A creating a premature stop codon for translation of open reading frame 5 (ORF5) (see Discussion). The verification of our consensus sequence awaits the availability of a second HCoV-EMC/2012 virus isolate or original specimen. The overall content of G and C residues in the HCoV-EMC/2012 genome was 41%, which is similar to values reported for other coronaviruses (37% to 42%) (14). Genome organization and expression strategy. Coronavirus genomes are polycistronic positive-stranded RNAs (Fig. 1A), of which the 5′-proximal three-fourths are occupied by the large replicase open reading frames ORF1a and ORF1b. These are translated from the genomic mRNA to produce polyproteins pp1a and, following −1 ribosomal frameshifting, pp1ab, which are subsequently cleaved into 15 or 16 nonstructural proteins (nsps) (19, 23, 26). The region downstream of ORF1b is characterized by containing a variable number of smaller genes, always including those encoding the spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins. These genes are translated from subgenomic (sg) mRNAs that form a 5′- and 3′-coterminal nested set with the viral genome. Subgenomic mRNAs are composed of a common 5′ leader sequence that is identical to the genomic 5′ region and a variable part of the 3′ quarter of the genome, with different sg mRNAs making different ORFs available for translation. The complement of the leader and “body” segments of the sg mRNAs are assumed to be joined during discontinuous negative-strand RNA synthesis. This step produces the templates for sg mRNA synthesis and is directed by a base-pairing interaction between conserved transcription-regulatory sequences (TRSs) (27–29). Such TRSs are found at the 3′ end of the leader sequence (leader TRS) and at different positions upstream of genes in the genomic 3′-proximal domain (body TRSs). The synthesis of subgenome-length negative-stranded RNAs is directed by the complement of a body TRS at the 3′ end of a nascent minus-strand base pairing with the leader TRS, with the extent of sequence complementarity being an important determinant of the level at which a given sg mRNA is produced. FIG 1 Genome organization and expression of HCoV-EMC/2012. (A) The coding part of the genome and terminal untranslated regions are depicted, respectively, by a gray background and horizontal lines. Rectangles indicate ORFs and their locations in three reading frames. The dashed lines in ORF1a and ORF5 indicate base ambiguities observed during sequencing. Triangles represent sites in the replicase polyproteins pp1a and pp1ab that are predicted to be cleaved by papain-like proteinases (gray) or the 3C-like cysteine proteinase (black). Cleavage products are numbered nsp1 to nsp16, according to the convention established for other coronaviruses (23). The −1 ribosomal frameshift site (RFS) in the ORF1a/ORF1b overlap region is indicated. The location of the leader TRS (transcription-regulatory sequences) (L) and seven body TRSs (numbered) are highlighted by black dots. All coordinates correspond to the scale shown at the bottom. (B) Sequence comparison of leader TRS region and seven body TRSs. The fully conserved TRS core sequence AACGAA is highlighted. Nucleotides in the body TRSs are written in uppercase letters if the complementary nucleotide can base pair with the corresponding residue in the leader TRS region (including G-U base pairs). TRS starting coordinates in the HCoV-EMC/2012 genome are shown at the left; for the body TRSs, the numbers of (potential) base pairs with the leader TRS region are shown at the right. Inspection of the genome sequence of HCoV-EMC/2012 revealed the two large, partially overlapping replicase open reading frames ORF1a and ORF1b, as well as (at least) nine downstream ORFs (Fig. 1A). The ORF1a sequence encodes the two protease domains conserved in all other coronaviruses, a papain-like protease (PL2pro) in nsp3 and a 3C-like protease (3CLpro; also known as the “main protease”) in nsp5. Sequence comparison with other coronaviruses allowed us to predict the putative pp1a/pp1ab cleavage sites and annotate the resulting nsp1 through -16 (Table 1). According to sequence conservation analyses performed with other coronaviruses, open reading frames ORF2, -6, -7, and -8a are predicted to encode the four canonical structural proteins of coronaviruses, the envelope proteins S, E, and M and the N protein, respectively (Fig. 1A; see also Fig. S1 in the supplemental material). A leader TRS and seven putative body TRSs could be readily identified, with the sequence 5′ AACGAA 3′ forming the conserved TRS core and potential TRS duplexes during leader-body joining ranging from 14 to 19 matches over a 22-nt window that includes the core of the leader TRS (Fig. 1B). From this analysis, it can be predicted that seven subgenomic mRNAs carrying a 67-nt common leader sequence would be produced in HCoV-EMC/2012-infected cells, with sizes ranging from ~4.7 kb for mRNA2 to ~1.7 kb for mRNA8. Experimental studies are needed to confirm the correct identification of the TRSs in the genomes of HCoV-EMC/2012 and related lineage C betacoronaviruses. TABLE 1 Cleavage products of the replicase polyproteins of HCoV-EMC/2012 Cleavage product Position in polyprotein pp1a/pp1ab a Protein size(no. of amino acids) Putative functional domain(s) b nsp1 1Met-Gly193 193 nsp2 194Asp-Gly853 660 nsp3 854Ala-Gly2740 1887 ADRP, PL2pro, TM1 nsp4 2741Ala-Gln3247 507 TM-2 nsp5 3248Ser-Gln3553 306 3CLpro nsp6 3554Ser-Gln3845 292 TM-3 nsp7 3846Ser-Gln3928 83 nsp8 3929Ala-Gln4127 199 Putative primase nsp9 4128Asn-Gln4237 110 nsp10 4238Ala-Gln4377 140 nsp11 4378Ser-Leu4391 14 nsp12 4378Ser-Gln5310 933 RdRp nsp13 5311Ala-Gln5908 598 ZD, HEL1 nsp14 5909Ser-Gln6432 524 ExoN, NMT nsp15 6433Gly-Gln6775 343 NendoU nsp16 6776Ala-Arg7078 303 OMT a Amino acids of the replicase proteins pp1a and pp1ab were numbered with the assumption that a −1 ribosomal frameshift occurs to express ORF1b, as in other coronaviruses (see text); the use of the slippery sequence UUUAAAC is predicted to result in a peptide bond between Asn4385 and Arg4386 in pp1ab. b The major transmembrane domains and a selection of the most conserved domains with enzymatic activities that have been characterized functionally and/or structurally in coronaviruses are listed. Abbreviations: PL2pro, papain-like proteinase 2; ADRP, ADP-ribose 1″-phosphatase; TM, transmembrane domain; 3CLpro, 3C-like cysteine proteinase; RdRp, RNA-dependent RNA polymerase; ZD, putative zinc-binding domain; HEL1, superfamily 1 helicase; ExoN, 3′-to-5′ exonuclease; NMT, N7-methyltransferase; NendoU, nidoviral endoribonuclease specific for U; OMT, S-adenosylmethionine-dependent ribose 2′-O-methyltransferase. Furthermore, mRNA4 and -8 are predicted to be functionally bicistronic, with ribosomal leaky scanning being the likely translation initiation mechanism for both ORF4b and ORF8b. The ORF4b AUG codon is not preceded by a separate body TRS, and the 241-nt sequence separating the 5′ ends of ORF4a and ORF4b is entirely devoid of AUG codons. The AUG codon of the current ORF8b, an internal ORF that is overlapped by the N protein gene (ORF8a) and is present in all betacoronaviruses, is the third AUG codon on mRNA8, but sequence analysis and comparison with the BtCov-HKU4 and -HKU5 sequences (8) suggests that the 5′ end of ORF8b may have become truncated relatively recently (see Discussion). Twenty-two additional putative ORFs of 150 to 432 nt in length were detected throughout the genome of HCoV-EMC/2012, overlapping the major ORFs. In contrast to the ORFs shown in Fig. 1A, these 22 additional ORFs are not positioned (immediately) downstream of a body TRS, and hence it is unlikely that they are expressed. The synthesis of the replicase pp1ab polyprotein of HCoV-EMC/2012 involves −1 programmed ribosomal frameshifting, with nt 13427 to 13433 predicted to form the conserved “slippery sequence” (5′ UUUAAAC 3′) in the ORF1a/ORF1b overlap region that is typical for coronaviruses (30). The frameshift region is followed by a predicted RNA hairpin, formed by nucleotides at positions 13439 to 13450 base pairing with those at 13462 to 13473, with potential RNA pseudoknot formation occurring by base pairing of the loop of the hairpin (nt 13452 to 13460) with a downstream complementary sequence (nt 13506 to 13514). As is common in coronavirus genomes, nontranslated sequences are found only at the genomic termini, with the 5′ and 3′ untranslated regions (278 and 300 nt, respectively) having sizes similar to those found in other family members. The only other apparently untranslated region in the genome that is larger than 50 nucleotides concerns the intergenic region between ORF5 and -6 (nt 27515 to 27589). This region appears to be conserved between HCoV-EMC/2012, BtCoV-HKU4, and BtCoV-HKU5, with sequence identities ranging from 63% to 84%, but we have no explanation for this observation thus far. Phylogenetic relations and taxonomic position of HCoV-EMC/2012. Phylogenetic trees were inferred using nucleotide sequences for ORF1ab (Fig. 2A) and a 332-nt fragment from ORF1b (Fig. 2B) encoding the most conserved part of the RNA-dependent RNA polymerase (RdRp) domain, which is commonly targeted in virus discovery studies. The first tree was produced for a representative set of coronaviruses for which complete genome sequences are available. In the second tree, we also included coronaviruses for which only partial genome sequences are known, particularly that of P.pipi/VM314/2008/NLD (31) which produced the best match with HCoV-EMC/2012. In both trees, HCoV-EMC/2012 clearly groups within lineage C of the genus Betacoronavirus, relatively close to BtCoV-HKU4 and BtCoV-HKU5. However, based on the 332-nt fragment from ORF1b, HCoV-EMC/2012 is more closely related to bat-derived isolate VM314/2008 (GenBank accession number GQ259977), which was isolated from Pipistrellus bats in The Netherlands 4 years ago. Phylogenetic trees were also constructed based on amino acid sequences, using coronavirus-wide conserved domains of replicative proteins in pp1ab (Fig. 3A) as well as using conserved parts of structural proteins (Fig. 3B). In both trees, HCoV-EMC/2012 clusters with betacoronaviruses, supporting its classification as a member of the genus Betacoronavirus. FIG 2 Phylogenetic trees for HCoV-EMC/2012 and selected other coronaviruses. Unrooted maximum likelihood phylogenies inferred from the nucleotide sequences of full-length ORF1ab (A) or a 332-nt fragment from the RdRp-encoding domain of ORF1b (B) are shown. HCoV-EMC/2012 and 20 viruses representing the recognized species diversity of coronaviruses were included, with bat-derived isolate VM314/2008 also included in the analysis presented in panel B (31). The viruses and corresponding species used are Alphacoronavirus 1 (Alpha-CoV1), Human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63), Miniopterus bat coronavirus 1 (BtCoV-1AB), Miniopterus bat coronavirus HKU8 (BtCoV-HKU8), Porcine epidemic diarrhea virus (PED), Rhinolophus bat coronavirus HKU2 (BtCoV-HKU2), Scotophilus bat coronavirus 512 (BtCoV-512), Betacoronavirus 1 (Beta-CoV1), Human coronavirus HKU1 (HCoV-HKU1), Murine coronavirus (MHV), Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), Rousettus bat coronavirus HKU9 (BtCoV-HKU9), Severe acute respiratory syndrome-related coronavirus (SARS-CoV), Avian coronavirus (IBV), Beluga whale coronavirus SW1 (BWCoV-SW1), Bulbul coronavirus HKU11 (ACoV-HKU11), Thrush coronavirus HKU12 (ACoV-HKU12), and Munia coronavirus HKU13 (ACoV-HKU13). Bootstrap values above 50 are shown. Arcs and symbols indicate the four coronavirus genera. The scale bar represents the number of nucleotide substitutions per site. FIG 3 Phylogenetic trees for HCoV-EMC/2012 and selected other coronaviruses. Unrooted maximum likelihood phylogenies based on coronavirus-wide conserved protein domains in replicase pp1ab (A) or on the conserved parts of structural proteins S2, E, M, and N (B) for HCoV-EMC/2012 and 20 viruses representing the recognized species diversity of coronaviruses are shown (see Fig. 2 legend for names and abbreviations). Branch support values are based on the Shimodaira-Hasegawa-like procedure and are in the range of zero to one; only nonoptimal values smaller than one are shown. Arcs and symbols indicate the four coronavirus genera. The scale bars represent average numbers of substitutions per amino acid position. ICTV assigns newly identified members of the family Coronaviridae to a subfamily and genus on the basis of rooted phylogeny and calculation of pairwise evolutionary distances for seven replicase domains (1, 21). To establish whether HCoV-EMC/2012 indeed prototypes a new species, amino acid sequence alignments were generated for each of these domains and concatenated, after which the sequence identity of HCoV-EMC/2012 with closely related strains was calculated. For this purpose, the full genomes of 9 strains, derived from 3 species, belonging to Betacoronavirus lineage C were available (Table 2). Amino acid sequence identity between conserved replicase domains of HCoV-EMC/2012 and those of other lineage C viruses ranged from 57% (ADP-ribose 1″-phosphatase [ADRP]) to 94% (helicase [Hel]). Overall amino acid sequence identities to BtCoV-HKU4 and BtCoV-HKU5 strains across the conserved domains were around 75% and 76.7%, respectively. These percentages are well below the threshold of 90% amino acid sequence identity that is used for coronavirus species identification by the ICTV. The distance between HCoV-EMC/2012 and members of these two species is as large as that observed upon interspecies comparison of other species pairs, for example, Murine coronavirus versus Human coronavirus HKU1 or Porcine epidemic diarrhea virus versus Scotophilus bat coronavirus 512 (Fig. 3). Consequently, we propose that HCoV-EMC/2012 prototypes a novel species of lineage C of the genus Betacoronavirus. TABLE 2 Percent amino acid sequence identity between conserved domains of the replicase polyprotein of HCoV-EMC/2012 and established betacoronaviruses a Virus strain % amino acid sequence identity with conserved domain of the indicated HCoV-EMC/2012 replicase polyprotein b ADRP 3CLpro RdRp Hel ExoN NendoU O-MT All domains BtCoV-HKU4.1 57.4 81.0 90.0 92.1 85.4 72.6 83.4 75.1 BtCoV-HKU4.2 57.5 81.0 90.0 92.1 85.4 72.6 83.4 75.1 BtCoV-HKU4.3 57.4 81.0 90.0 92.1 85.4 72.6 83.4 75.1 BtCoV-HKU4.4 57.5 81.0 89.9 92.1 85.4 72.6 83.4 74.9 BtCoV/133/2005 57.6 80.7 89.9 91.6 86.4 72.0 83.4 74.9 BtCoV-HKU5.1 57.6 82.6 92.1 93.8 91.7 79.7 85.3 76.7 BtCoV-HKU5.2 57.6 82.0 92.2 93.8 91.7 80.0 85.3 76.7 BtCoV-HKU5.3 57.2 82.0 92.2 93.8 91.7 80.0 85.3 76.7 BtCoV-HKU5.5 57.3 82.0 92.2 93.8 91.7 80.0 85.3 76.7 a Accession numbers used are as follows: for BtCoV-HKU4 strains, EF065505, EF065506, EF065507, and EF065508; for BtCoV/133/2005, DQ648794; and for BtCoV-HKU5 strains, EF065509, EF065510, EF065511, and EF065512. b For abbreviations, see Table 1. Genome similarities between coronavirus HCoV-EMC/2012 and coronaviruses BtCoV-HKU4 and BtCoV-HKU5. BtCoV-HKU4 and BtCoV-HKU5 (8) are the closest relatives of HCoV-EMC/2012 for which full-length genome sequences are available (see above). Accordingly, comparison of the genomes of these three viruses revealed important similarities, including the organization of the “accessory protein genes,” ORF3a through ORF5, residing between the S protein gene and those encoding the E, M, and N proteins. Upon annotating this region of the BtCoV-HKU4 and BtCoV-HKU5 genomes, Woo et al. (8) identified the body TRSs for sg mRNA3, mRNA4, and mRNA5 but unfortunately did not follow standard coronavirus nomenclature, naming the downstream open reading frames ORF3a through ORF3d (encoding ns3a through ns3d) rather than ORF3, ORF4a, ORF4b, and ORF5 (Fig. 1A). The similarities of all ORFs and proteins of HCoV-EMC/2012, BtCoV-HKU4, and BtCoV-HKU5 were calculated, and percentages of sequence identity are summarized in Table 3. The lowest percentages of sequence identity to BtCoV-HKU4 and BtCoV-HKU5 were observed for ORF3 at the nucleotide level (46.4% and 46.0%, respectively) and for ORF4b at the amino acid level (23.5% and 25.9%, respectively). The highest percentages of sequence identity to BtCoV-HKU4 and BtCoV-HKU5 were observed for the E ORF at the nucleotide level (74.6% and 75.1%, respectively) and for the M ORF at the amino acid level (82.6% and 82.2%, respectively). These data further supported the characterization of HCoV-EMC/2012 as a close relative of BtCoV-HKU4 and BtCoV-HKU5. TABLE 3 Percent identity between open reading frames of coronavirus HCoV-EMC/2012 and coronaviruses BtCoV-HKU4 and BtCoV-HKU5 at the nucleotide and amino acid levels Annotationin HCoV-EMC/2012 Annotation inBtCoV-HKU4and BtCoV-HKU5 a % identityto BtCoV-HKU4 b % identityto BtCoV-HKU5 b nt aa nt aa ORF1ab ORF1ab 70.6 72.2 70.7 73.8 S S 66.3 66.1 63.8 63.5 ORF3 NS3a 46.4 34.9 46.0 31.4 ORF4a NS3b 51.5 37.5 47.8 38.0 ORF4b NS3c 35.1 23.5 45.2 25.9 ORF5 NS3d 56.6 46.9 58.1 54.2 E E 74.6 69.5 75.1 68.2 M M 72.8 82.6 73.0 82.2 N N 67.2 71.8 66.7 67.8 ORF8b Undescribed 45.3 32.1 48.0 33.8 a Annotations used for HCoV-EMC/2012 differ from those used for BtCoV-HKU4 and BtCoV-HKU5 (10). b Accession numbers used for BtCoV-HKU4 and BtCoV-HKU5 were EF065505 and EF065509. DISCUSSION Coronaviruses have been known for quite some time as viruses that cause a variety of diseases in humans and animals (32, 33). The discovery of a coronavirus as the causative agent of SARS revived the interest in coronaviruses and resulted in a rapid increase of the number of identified coronaviruses, as well as of the number of full coronavirus genome sequences. Until this study, lineage C of the genus Betacoronavirus (formerly known as subgroup 2c) included virus isolates from bats. Here, we determined and analyzed the complete genome sequence of a previously unknown lineage C betacoronavirus that was isolated from the sputum of a 60-year-old male suffering from acute pneumonia and renal failure in the Kingdom of Saudi Arabia whose death was probably a consequence of this infection (24). The sequencing of the full HCoV-EMC/2012 genome was greatly facilitated by the advent of high-throughput techniques. Using an optimized random amplification deep-sequencing approach, approximately 90% of the virus genome was covered with high accuracy in a single run. Using the data from this first run, primers could be designed to perform conventional Sanger sequencing for confirmation. This combination of techniques allowed the determination of the complete virus genome within a few days, without a requirement for prior knowledge of the virus genome under investigation. The error rate in 454 deep sequencing was generally higher than in Sanger sequencing, but the high coverage across the HCoV-EMC/2012 virus genome (up to 5,697 reads per nucleotide position) corrected for most of the incorrect base callings. The sequence obtained using the 454 platform aligned almost perfectly with that obtained by Sanger sequencing, with the exception of two nucleotide positions. The deep-sequencing data revealed variation at position 11623 (U or G), with G occurring in 44% of the reads, suggesting that ORF1a-encoded residue 3782 can be either valine (codon GUC) or glycine (codon GGC). The valine codon was the more abundant codon at this position in HCoV-EMC/2012, and valine is also present in most other betacoronaviruses. At position 27162, both G and A were detected in different runs, with an A in 45% of the reads. This G-to-A substitution introduces a premature stop codon (UGG to UAG) in ORF5. The virus stock that we sequenced was derived from passage of the virus from a sputum specimen six times in Vero cell culture. Hence, the observed sequence variants may reflect either natural heterogeneity or emerging genetic changes that occurred during virus passage in cell culture. Additional HCoV-EMC/2012 virus isolates or patient materials are currently not available to verify these genome sequence ambiguities at positions 11623 and 27162. Adaptation to cell culture leading to a loss of functionality of genes, and in particular in relation to the so-called “accessory protein genes,” has previously been described for a variety of coronaviruses, including SARS-CoV (2, 34). These genes, like ORF3 through ORF5 of HCoV-EMC/2012, are dispersed between the structural protein genes (35) and in some cases may even overlap such a gene, as in the case of the ORF overlapping the N protein gene in betacoronaviruses (Fig. 1A) (23, 36). The origin of most accessory protein genes remains unclear, although for some, acquisition by recombination with cellular or heterologous viral sequences seems plausible (37, 38). Accessory gene functions have been probed by reverse genetics (knockout mutants) for a variety of coronaviruses, including SARS coronavirus (39), which established that they are not essential for replication in cell culture systems. In animal models, on the other hand, profound effects on pathogenesis after the inactivation (or transfer to a heterologous coronavirus) of accessory protein genes have been previously described (40–42). In some cases, accessory gene products have been implicated in immune evasion, e.g., by interfering with cellular innate immune signaling (43). The apparent absence of selection pressure on coronavirus accessory protein genes during cell culture passage may explain the relatively high frequency with which loss of functionality appears to occur. The detection of an internal termination codon in part of the HCoV-EMC/2012 ORF5 sequences (45% of the reads) may constitute another example of such an event, which would lead to the truncation of the ORF5 protein after 107 amino acids. This would resemble a 29-nt deletion that occurred in the SARS-CoV genome, which resulted in the truncation of ORF8 (34, 44), and a 45-nt in-frame deletion in ORF7b of the same virus that emerged upon cell culture passage (23). Our analysis identified a potential ORF underlying the N protein gene (ORF8a), which is a common feature in betacoronaviruses. This ORF was not previously described for BtCoV-HKU4 and BtCoV-HKU5 (8) but is conserved in the genome sequences of both viruses (see Fig. S1J in the supplemental material). Remarkably, in HCoV-EMC/2012, both the 5′ and 3′ parts of the ORF appear to have been truncated. In BtCoV-HKU4 and BtCoV-HKU5, the ORF8b AUG codon would be the second AUG on sg mRNA8, making leaky ribosomal scanning a likely mechanism for translation initiation. In HCoV-EMC/2012, however, this AUG codon (positions 28606 to 28608) seems to have been mutated to AUA. Conservation of the sequence immediately downstream of this position, which is now formally upstream of ORF8b in HCoV-EMC/2012, was observed with BtCoV-HKU4 and BtCoV-HKU5, suggesting that the putative loss of this AUG codon may also have been a relatively recent event. In the 3′ part of ORF8b, sequence alignment of HCoV-EMC/2012 with BtCoV-HKU4 and BtCoV-HKU5 suggests that the former acquired a premature termination codon at positions 29099 to 29101 (UAA). Although we cannot at present assess the timing of these events in HCoV-EMC/2012 evolution, due to the lack of alternative samples for this species, the presumed loss of ORF8b functionality may also be a consequence of virus passage in cell culture. To classify newly identified coronaviruses as the prototype of a novel virus species, it is required that the amino acid sequence identity in the conserved replicase domains in all intervirus pairwise comparisons is below the 90% threshold (1). Here, we propose HCoV-EMC/2012 to represent a novel species of the betacoronavirus genus, since the amino acid sequence identities between HCoV-EMC/2012 and its closest relatives BtCoV-HKU4 and BtCoV-HKU5 in the seven conserved domains of ORF1ab were 75% and 77%, respectively. These viruses were originally detected in Asia in lesser bamboo bats (Tylonycteris pachypus) and Japanese house bats (Pipistrellus abramus), respectively (8). This proposed classification will remain provisional until approved by ICTV. The ICTV guidelines for coronavirus species demarcation require the availability of a (nearly) complete genome sequence prior to virus classification. However, there is considerable correlation between the results based on full-genome sequence analysis and those determined using the most conserved part of the ORF1b-encoded RdRp domain, which is commonly used in screening for new coronaviruses. In 2010, this partial sequence was reported for a betacoronavirus (VM314/2008) that was isolated 2 years earlier from a Pipistrellus pipistrellus bat in The Netherlands. This virus was provisionally classified a betacoronavirus based on a 332-nt fragment from the RdRp-encoding domain of ORF1b (31), which shares 88% nucleotide sequence identity with HCoV-EMC/2012, the highest identity with any coronavirus sequence available in the public domain. Although this high similarity is not sufficient to resolve the taxonomic relation between HCoV-EMC/2012 and isolate VM314/2008, it suggests that they may both belong to the same coronavirus species. Establishing the genome sequence of VM314/2008, or closely related viruses, is urgently required to verify this hypothesis. Based on the genetic relation between HCoV-EMC/2012 and bat coronaviruses, it is tempting to speculate that HCoV-EMC/2012 emerged from bats—either directly or via an intermediate animal host, possibly Pipistrellus bats. This bat species is known to be present in the Kingdom of Saudi Arabia and neighboring countries. Although most infections of human coronaviruses are relatively mild, the infection by HCoV-EMC/2012 with fatal outcome, and a similar severe case of an infection with a closely related coronavirus in London (25), is a reminder that certain coronaviruses may cause severe and sometimes fatal infections in humans. It is important to develop an animal model that can be used to fulfill Koch’s postulates for the novel virus, by demonstrating that the isolated virus can indeed cause the observed disease. The availability of the HCoV-EMC/2012 genome sequence will facilitate the development of a variety of diagnostic assays that can be used to study the prevalence and clinical impact of HCoV-EMC/2012 infections in humans. The first generation of assays for this purpose has recently been described (45). We anticipate that the availability of this full-length virus genome sequence will be valuable for the development of additional applied and fundamental research. MATERIALS AND METHODS Virus propagation. Patient material had been subjected to passage in Vero cells four times in the Dr. Soliman Fakeeh Hospital, Jeddah, Saudi Arabia. Subsequently, in the Erasmus Medical Center, Rotterdam, The Netherlands, LLC-MK2 cells were inoculated with HCoV-EMC/2012 in minimal essential medium (MEM-Eagle) with Earle’s salts (BioWhittaker, Verviers, Belgium), supplemented with 2% serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine. Vero cells were inoculated with virus in Dulbecco’s modified Eagle medium (BioWhittaker) supplemented with 1% serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine. After inoculation, the cultures were incubated at 37°C in a CO2 incubator and checked daily for cytopathic changes. Three days after inoculation, supernatant from Vero cells was collected and used for virus genome characterization. Arbitrarily primed PCR and virus genome sequencing. To characterize the viral genome, we used a random amplification deep-sequencing approach as the first step. Virus-containing supernatant was centrifuged for 10 min at 3,000 rpm to remove cellular debris. This supernatant was then filtered through a 0.45-µm-pore-size centrifugal filter unit (Millipore, Amsterdam, The Netherlands) to minimize bacterial contamination. Omnicleave endonuclease (Epicenter Biotechnologies, Madison, WI) was used to remove any free DNA and RNA, according to the manufacturer’s protocol. Subsequently, viral RNA was extracted from the purified, infected cell culture supernatant using a High Pure RNA isolation kit (Roche Diagnostics, Almere, The Netherlands). To remove contaminating mammalian rRNA, a Ribo-Zero RZH110424 rRNA removal kit (Epicenter Biotechnologies, Madison, WI) was used according to the manufacturer’s protocol. RNA was reverse transcribed using circular permuted primers (46) that were extended with random hexamer sequences, namely, CCCACCACCAGAGAGAAAN(6), ACCAGAGAGAAACCCACCN(6), GAGAAACCCACCACCAGAN(6), GGAGGCAAGCGAACGCAAN(6), AAGCGAACGCAAGGAGGCN(6), and ACGCAAGGAGGCAAGCGAN(6). Per reaction, reverse transcription mixtures contained 6 µl RNA, 1 µl primer (20 pmol), 0.5 µl (20 U) RNase inhibitor (Promega, Leiden, The Netherlands), 1 µl (10 mM each) deoxynucleoside triphosphates (Roche), and 5 µl water. After a 5 min incubation at 65°C for optimal primer hybridization to the template, 4 µl (10×) first-strand buffer, 1 µl (200 U/µl) SuperScript III reverse transcriptase (Invitrogen, Bleiswijk, The Netherlands), 1 µl (0.1 M) dithiothreitol (DTT), and 0.5 µl (20 U) RNase inhibitor (Promega) were added to the mixture in a 20-µl volume. To obtain cDNA, the reverse transcription mixture was sequentially incubated at 25°C for 5 min and at 42°C for 1 h. After 3 min at 95°C and 2 min on ice, 1 µl Klenow DNA polymerase (5 U) (New England BioLabs Inc., Ipswich, MA) was added and the mixture was sequentially incubated at 25°C for 5 min, 37°C for 1 h, and 75°C for 20 min to obtain double-stranded cDNA. The cDNA was purified using a MinElute PCR purification kit (Qiagen, Venlo, The Netherlands) according to the instructions of the manufacturer. To amplify the purified cDNA, a PCR with the individual circular permuted primers without the random hexamer was performed. The PCR mixture contained 2 µl primer (40 pmol), 2 µl purified cDNA, 1.25 µl (10 mM each) deoxynucleoside triphosphate (Roche), 5 µl (10×) PfuUltra II Rxn buffer, and 1 µl (2.5 U) PfuUltra II DNA polymerase (Stratagene, Amsterdam, The Netherlands). Water was added to reach a final volume of 50 µl. The PCR mixture was incubated at 95°C for 2 min and then for 40 cycles of 95°C for 20 s, 56°C for 1 min, and 72°C for 2 min, followed by a final extension at 72°C for 10 min. Fragments were purified using a MinElute PCR purification kit (Qiagen) according to the instructions of the manufacturer. Amplified fragments were sequenced using a 454/Roche GS Junior sequencing platform. A fragment library was created according to the manufacturer’s protocol without DNA fragmentation (GS FLX Titanium rapid library preparation; Roche), selecting for fragments larger than 100 bp. The emulsion-based PCR (emPCR) (amplification method Lib-L) and GS Junior sequencing run were performed according to the instructions of the manufacturer (Roche). The sequence reads were trimmed at 30 nt from the 3′ and 5′ ends to remove all primer sequences. Sequence reads were assembled into contigs using CLC Genomics 5.5.1 software (CLC Bio, Aarhus, Denmark). Using this deep-sequencing approach, approximately 90% of the virus genome sequence was obtained. As a second step, specific primers were designed to amplify overlapping fragments of approximately 800 bp by RT-PCR. These PCR products were purified from agarose gels and sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) and a 3130XL genetic analyzer (Applied Biosystems), according to the instructions of the manufacturers. The genomic 5′- and 3′-terminal sequences were determined using a FirstChoice RLM-RACE kit (Ambion, Bleiswijk, The Netherlands). Virus classification. Newly identified members of the family Coronaviridae are generally assigned by the ICTV to a subfamily and genus on the basis of rooted phylogeny and calculation of pairwise evolutionary distances for seven replicase polyprotein domains (1): the ADP-ribose 1″-phosphatase (ADRP) in nsp3, the coronavirus 3C-like (3 CL) protease (3CLpro, or “main protease”) in nsp5, the RNA-dependent RNA polymerase (RdRp) in nsp12, the helicase (Hel) in nsp13, the exoribonuclease (ExoN) in nsp14, the nidoviral endoribonuclease specific for U (NendoU) in nsp15, and the ribose-2′-O-methyltransferase (O-MT) in nsp16. Amino acid sequence alignments were generated for each of these domains using ClustalW within the BioEdit (version 7.0.5.3) (47) program and concatenated, after which the sequence identity of HCoV-EMC/2012 with closely related strains was calculated. For this purpose, the full genomes of 9 strains, derived from 3 species, belonging to Betacoronavirus lineage C were available. To support virus classification, protein-based phylogenetic trees were generated. Multiple amino acid alignments, including sequences of HCoV-EMC/2012 and one representative of each of the 20 recognized species of the subfamily Coronavirinae, were produced for the following proteins, using the Viralis platform (48) followed by manual correction: ADRP, the N-terminal part of PLP2, TM1, Y domain, nsp4 to nsp16, and the C-terminal part of the spike (S) protein (S2), envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. From each protein alignment, the most informative blocks (49) were extracted using the BAGG program (50), and only these strongly conserved alignment regions were used for further analyses. Two concatenated alignments were used. The first included replicase pp1ab protein regions (4,110 aa positions, gap content of 0.9%), and the second included regions in the C-terminal domain of the S protein (S2) and the E, M, and N proteins (1,127 aa positions, gap content of 3.9%). ProtTest version 3.2 (51) was used to select the best-fitting model of protein evolution. For both datasets, the LG model with rate heterogeneity (4 categories) ranked top among 112 models tested, with a relative weight of 0.98 under the Bayesian information criterion (BIC) and 0.74 under the corrected Akaike information criterion (AICc) for the first data set and 0.97/0.75 (BIC/AICc) for the second data set. Hence, this model was applied for maximum likelihood phylogeny reconstruction using PhyML version 3.0 (52). Phylogenetic reconstruction. Nucleotide sequences were aligned using the ClustalW software running within the BioEdit (version 7.0.5.3) (47) program and MAFFT version 6 (53). Maximum likelihood phylogenetic trees with 100 bootstrap replicates were estimated under the general time-reversible model (GTR) + I + Γ4 and the transversion model (TVM) + I + Γ4 (determined by ModelTest [54]), using PhyML 3.0 software (52). For both the 332-nt ORF1ab alignment that included isolate VM314/2008 and the alignment of the complete ORF1ab, the GTR + I + Γ4 model ranked top among 65 models tested, with relative weights of 0.8185 and 1.000 under AIC, respectively. Nucleotide sequence accession number. The final HCoV-EMC/2012 consensus sequence was submitted to GenBank under accession number JX869059. SUPPLEMENTAL MATERIAL Figure S1 Alignments of (poly)proteins encoded by the genome of HCoV-EMC/2012 and its two closest relatives, BtCoV-HKU4 and BtCoV-HKU5. Expert-curated multiple alignments for pp1ab (A), S protein (B), ORF3 protein (C), ORF4a protein (D), ORF4b protein (E), ORF5 protein (F), E protein (G), M protein (H), N protein (I), and the ORF8b product (J) are shown. (A) Amino acid residues in pp1ab between which cleavage by viral proteinases is predicted to occur are highlighted in gray. Download Figure S1, DOC file, 0.1 MB. Figure S1, DOC file, 0.1 MB Table S1 Primers designed to amplify ~800-nucleotide PCR fragments with 100-nucleotide overlaps covering the entire HCoV-EMC/2012 genome. Table S1, DOCX file, 0.1 MB.
- Record: found
- Abstract: found
- Article: found
Antibodies against MERS Coronavirus in Dromedary Camels, United Arab Emirates, 2003 and 2013
Benjamin Meyer, Marcel A. Müller, Victor M Corman … (2014)
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen associated with severe respiratory symptoms and renal failure in infected patients ( 1 , 2 ). Globally, 156 laboratory-confirmed cases of infection with MERS-CoV, including 65 deaths, were reported as of early November 2013. All human cases were linked to the Arabian Peninsula (Saudi Arabia, Jordan, Oman, Qatar, Kuwait, and the United Arab Emirates). Imported cases were detected in countries in Europe and Africa (United Kingdom, Germany, Italy, France, and Tunisia) ( 3 ). Transmission patterns, including the putative zoonotic source of the virus, remain unclear. Hypotheses include frequent zoonotic infections with limited subsequent human-to-human transmission chains and existence of a self-sustained epidemic in humans ( 4 ). A recent study found evidence to support the existence of epidemiologically unlinked cases in a large outbreak in the al-Hasa region, Saudi Arabia ( 5 ). It was speculated that zoonotic introductions of MERS-CoV from an unknown reservoir might occur at high rates, in addition to obvious human-to-human transmission. Coronaviruses (CoV) are positive-sense RNA viruses. Viruses in the genera Alphacoronavirus and Betacoronavirus are associated with mammals and show a particularly high level of diversification in bats. Viruses in the genera Gammacoronavirus and Deltacoronavirus are mostly avian-associated viruses ( 6 , 7 ). MERS-CoV belongs to Betacoronavirus phylogenetic lineage C that, in addition to MERS-CoV, contains 2 distinct bat-associated CoV species (HKU4 and HKU5) ( 1 , 8 ). Insectivorous bats of the family Vespertilionidae were recently shown to carry viruses that are probably conspecific with MERS-CoV ( 9 ). However, the limited rate of contact between humans and insectivorous bats makes a continuous and frequent acquisition of MERS-CoV from bats an unlikely scenario. In a manner similar to observations regarding severe acute respiratory syndrome CoV (SARS-CoV), an intermediate reservoir host might exist from which human infections are acquired. Dromedary camels from different regions in Africa and the Arabian Peninsula have been shown to have antibodies against MERS-CoV ( 10 , 11 ). Animals from the Arabian Peninsula had high neutralizing serum activities overall and reciprocal antibody titers ≤320–1,280, which support recent infection with MERS-CoV or a highly related virus. Thus, dromedary camels might serve as intermediate hosts. However, detailed serologic studies in countries with actual incidence of MERS-CoV infections in humans have not been conducted. Serologic analysis of CoVs is challenging because of cross-reactivity between CoVs infecting the same host and the broad distribution of CoVs in diverse mammalian species ( 6 , 7 , 12 – 14 ). Antibodies directed against some of the major antigens of different CoVs are known to cross-react in standard serologic assays ( 15 , 16 ). Potential cross-reactivity is a diagnostic challenge because camelids are known to be infected with bovine CoV (BCoV), a distinct betacoronavirus of phylogenetic lineage A unrelated to the MERS-CoV ( 17 , 18 ). As an additional challenge, camel immunoglobulins lack a light chain peptide, which affects specific physical properties, such as altered size and stability, compared with immunoglobulins of other mammals ( 19 , 20 ). The influence of this feature on serologic assays has not been thoroughly investigated. Thus, serologic assays should be applied with caution, and different assay formats should be tested concurrently. We reported a 2-staged approach for MERS-CoV serologic analysis in humans ( 15 , 16 ). Expanding upon these studies, we used in the present study a recombinant MERS-CoV spike protein immunofluroescence assay (rIFA) augmented by a validated protein microarray ( 10 , 21 ), followed by MERS-CoV–specific neutralization assay, to screen 651 dromedary camel serum samples from the United Arabian Emirates. Cross-reactivity against clade A betacoronaviruses was assessed by using a immunofluorescence assay (IFA) and a BCoV-specific neutralization assay. Serum samples obtained in 2003 and 2013 were compared to obtain information for the time in which MERS-related CoV has been circulating in camels. Methods Sampling A total of 651 dromedary camel (Camelus dromedarius) serum samples were systematically sampled in Dubai, United Arab Emirates and the surrounding area in 2003 (collection 4, n = 151) and in 2013 (collections 1A, 1B, 2, and 3; n = 500). The total number of camels in that area was 360,000 in 2010 ( 22 ). Fecal samples were also available for collections 1A and 1B (n = 182), all obtained in 2013. Animals in collection 1B were born and raised at the Dubai Central Veterinary Research Laboratory, which tests ≈70,000 camels per year ( 23 ) and had no contact with other camels. Camels in collection 2 were racing camels (age range 2–8 years), and camels in collection 3 were adult livestock camels originally purchased from Saudi Arabia, Sudan, Pakistan and Oman. Dromedary camel blood was obtained for routine health screening by jugular vein puncture according to standard veterinary procedures by trained personnel. For most serum samples, animal owners requested sample codes to be anonymous. All samples obtained during 2003 and 2013 were stored at −80°C until further analysis. For comparison, 16 serum samples from C. bactrianus camels in zoologic gardens in Germany were included in the study. All serum samples were shipped in agreement with German import regulations. Recombinant Spike IFA For screening purposes, an rIFA was used ( 15 , 24 ). In brief, Vero B4 cells were transfected with pCG1 eukaryotic expression vector that contained the complete spike sequence of MERS-CoV or human CoV-OC43. Cells were fixed 24-h post-transfection with ice-cold acetone/methanol and stored dry at 4°C. Serum samples were applied at a dilution of 1:80 for 1 h at 37°C, which was optimal for reducing nonspecific reactions and maintaining sensitivity. Secondary detection was conducted by using a goat anti-llama IgG fluorescein isothiocyanate–conjugated antibody. For some negative serum samples, dilutions of 1:20 and 1:40 were also tested. Spike Protein Microarray A confirmatory assay based on a protein microarray was performed as described ( 10 , 21 ) by using the spike S1 subunits of MERS-CoV, human CoV-OC43, and SARS-CoV. Serum samples were used at 1:20 dilutions on microarray chips. Relative light units were determined by using secondary cyanine 5–conjugated goat anti-llama IgG. MERS-CoV Conventional IFA A MERS-CoV IFA with infected Vero cells was conducted as described ( 15 ) by using commercially available MERS-CoV IFA slides (EUROIMMUNAG, Lübeck Germany). Serum samples were used at dilutions of 1:20–1:5,120. Secondary detection was conducted by using goat anti-llama fluorescein isothiocyanate–labeled IgG (1:200 dilution; Agrisera, Vännas, Sweden). Serum Neutralization Test Serum neutralization tests were conducted as described ( 10 ) by using Vero B4 (MERS-CoV) or PT (BCoV) cells. To reduce volumes of serum needed, all neutralization tests were performed in a 96-well format. Reactions contained 50 PFUs of MERS-CoV (EMC/2012 strain) or BCoV (Nebraska strain) in 25 μL of medium mixed 1:1 with camel serum diluted in 25 μL serum-free Dulbecco minimum essential medium. The starting dilution was 1:40. After incubation for 1 h at 37°C, each well was infected for 1 h at 37°C with a 50 μL virus–serum mixture. Supernatants were removed and fresh complete Dulbecco minimum essential medium was added. Assays were terminated by fixation with 8% paraformaldehyde for 30 min and stained with crystal violet after 3 days. Neutralization titers were defined as serum dilutions reducing cytopathic effects in 2 parallel wells. Detection of Virus Nucleic Acid Viral RNA was extracted from serum and fecal samples by using the MagNA Pure System (Roche, Basel, Switzerland) and an input volume of 100 μL of serum or fecal material suspended 1:10 in phosphate-buffered saline buffer. The elution volume was 100 μL for serum and fecal suspensions. To identify CoV-specific nucleic acids, 2 generic CoV PCRs were performed as described ( 25 – 27 ), followed by subsequent Sanger sequencing of the amplified DNA. Results To characterize reactivity of camel serum samples with MERS-CoV in different assay formats, we chose 11 camel serum samples with weak and strong reactivity predetermined by using a simple IFA. The 11 serum samples were titrated in a 2-fold dilution series in all applied assays. The reactivity pattern of the MERS-CoV spike protein (MERS-S) was compared against that of the human CoV-OC43 spike protein (OC43-S). As in our previous study ( 10 ), human CoV-OC43 was used instead of BCoV in these initial experiments because it is serologically indistinguishable from BCoV and is not subject to handling restrictions of German Animal Diseases Protection Act ( 28 ). Overall titers against MERS-S were higher than those against OC43-S, and several serum samples reacted exclusively against 1 of the 2 viruses (Table 1), suggesting the absence of general cross-reactivity between spike proteins of both viruses by IFA. Typical patterns of reactivity observed for camel serum samples are shown in the Figure, panel A. Table 1 Validation of serologic assays for coronaviruses with differentially reactive dromedary serum samples, United Arab Emirates, 2013* Serum no. rIFA titer†‡ Protein array (RFU) ‡§ vIFA titer†‡ Neutralization test titer¶# MERS-S OC43-S MERS-S1 OC43-S1 SARS-S1 MERS-CoV MERS-CoV BCoV 1 – – 2,555 3,868 2,606 – – 40 2 – 320 2,770 18,896 2,776 – – 80 3 – 640 3,950 65,535 2,751 – – 160 4 320 – 65,535 3,921 1,726 640 40 – 5 >10,240 320 65,535 7,247 2,306 >5,120 2,560 160 6 5,120 640 65,535 5,069 2,098 2,560 640 160 7 >10,240 160 65,535 7,179 2,198 >5,120 640 40 8 5,120 320 65,535 55,826 2,412 >5,120 1,280 160 9 5,120 >5,120 65,535 65,535 2,087 >5,120 1,280 320 10 >10,240 320 65,535 22,695 2,303 >5,120 1,280 320 11 5,120 1,280 65,535 28,391 2,858 >5,120 640 40 *rIFA, recombinant immunofluorescence assay (antigen used was complete spike protein); RFU, relative fluorescence units; vIFA, Middle East respiratory syndrome coronavirus–based immunofluorescence assay (antigen used complete virus); MERS-S, spike protein from Middle East respiratory syndrome coronavirus; OC43-S, spike protein from human coronavirus OC34; SARS-S, spike protein from severe acute respiratory syndrome virus; MERS-CoV, Middle East respiratory syndrome coronavirus; BCoV, bovine coronavirus; –, negative.
†Serum dilutions started at 1:20.
‡Assay was used for screening purposes.
§RFU 65,535 RFU. The OC43-S1 reactivity pattern across the serum panel was comparable with that for the OC43-S rIFA. As expected, all serum samples were negative against the SARS-S1 control antigen. A comparison of typical reactivity patterns in the microarray with those of the IFA is shown in the Figure, panel B. Results for the rIFA and protein microarray were highly congruent. The panel of camel serum samples was additionally tested in a commercially available IFA that used cells infected with MERS-CoV (vIFA) (EUROIMMUN AG). The use of whole virus provides additional structural and nonstructural protein antigens, including envelope, membrane, nucleocapsid, and diverse replicase proteins. However, because of conserved features of nonstructural proteins among even distantly related CoVs ( 7 , 12 ), cross-reactivity was possible with this assay ( 15 ). In the tested panel of camel serum samples, vIFA titers corresponded well to titers determined by rIFA and generally equal to or higher than titers in the rIFA (Table 1). Despite the absence of cross-reactivity between MERS-S–positive and OC43-S–positive serum samples in this test (Figure, panel A), in previous studies the vIFA showed false-positive results with human CoV-OC43–positive serum samples, in particular if used at lower dilutions, such as 1:10 or 1:20 ( 15 , 16 ). To confirm results from affinity assays with results from a functional test, we determined endpoint virus neutralization titers by using a microneutralization test against MERS-CoV and BCoV. In most animals MERS-CoV serum neutralization titers were higher than titers against BCoV (serum samples 4–11) (Table 1). High IFA titers generally corresponded with high neutralization titers, with exceptions for some BCoV antibody–positive serum samples. Divergence between affinity and neutralization assays can result from waning neutralizing antibody activity for infections that occurred long ago. Neutralization assays confirmed the absence of cross-neutralization between MERS-CoV and BCoV antibodies in either direction even at low dilutions, such as 1:40. However, sample no. 1 (Table 1) neutralized BCoV at a dilution of 1:40 despite showing negative results in all other serologic assays. This finding indicates that nonspecific neutralization activities might be encountered with camel serum samples, suggesting that higher serum dilutions should be used when conducting critical investigations such as viral reservoir studies. On the basis of the validation studies, we investigated 4 collections of serum samples from dromedary camels from the United Arab Emirates that were sampled in 2003 and 2013. For initial screening, we chose the rIFA because of its proven sensitivity and decreased chances of generating false-positive results. All 667 camel serum samples from the United Arab Emirates and Germany were initially screened at dilutions of 1:80. A total of 89.0%–100.0% of serum samples in 4 collections showed positive results (Table 2). Seroprevalence was higher for collections from exclusively adult animals (collections 3 and 4) than for a collection from young racing camels (2–8 years of age, collection 2). Clear seropositive results included 151 dromedary camel serum samples obtained in 2003 (collection 4). All 16 serum samples from German zoologic gardens were tested at the same dilution and showed no reactivity in the rIFA. Re-testing at lower dilutions of 1:20 and 1:40 confirmed absence of reactivity in these serum samples. Subcollection 1B contained serum samples from 5 animals that were born in, and had never left, a closed animal research facility in Dubai; these animals were seronegative. Table 2 MERS-CoV serologic results for dromedary serum and fecal samples, United Arab Emirates, 2003 and 2013* Collection Year No. camels/ sex Camel age Feature No. samples Serum dilution, no. (%) positive rIFA, MERS-S† Neutralization test, MERS-CoV 80 1,280 1A 2013 2/M, F A, J Paired serum and fecal samples 177 175 (98.9) 24 (13.6) 74 (41.8) 79 (44.6) 1B 2013 2/M, F A, J Animals raised at CVRL 5 0 5 (100.0) 0 0 2 2013 2/M, F 2–8 y Racing camels 100 89 (89.0) 55 (55.0) 3 (3.0) 42 (42.0) 3 2013 2/M, F A Livestock camels‡ 218 217 (99.5) 23 (10.6) 13 (6.0) 182 (83.5) 4 2003 1/F A Systematically sampled 151 151 (100.0) 35 (23.2) 30 (19.9) 86 (57.0) Total 651 632 (97.1) 142 (21.8) 120 (18.4) 389 (59.8) *MERS-CoV, Middle East respiratory syndrome coronavirus; rIFA, recombinant immunofluorescence assay with MERS-CoV spike protein; MERS-S, spike protein from MERS-CoV; A, adult; J, juvenile; CVRL, Dubai Central Veterinary Research Laboratory.
†Fluorescence signal intensity was rated as negative, +, ++, +++, and ++++.
‡Originally purchased from Saudi Arabia, Sudan, Pakistan, and Oman. A confirmatory microneutralization test was conducted at dilutions of 1:640 and 1:1,280 for all IFA-reactive serum samples. These high dilutions were chosen on the basis of our observation of high levels of neutralizing serum activity in camels ( 10 ). Most (59.8%, 389/651) serum samples had high neutralizing titers >1,280 (Table 2). In 18.4% (120/651) of all serum samples, neutralization titers ranged from 640 through 1,280, and 21.8% (142/651) of rIFA-positive serum samples had neutralizing titers 640 were tested by using a BCoV-specific microneutralization assay. At a dilution of 1:640, a total of 19.2% (23/120) of MERS-CoV–neutralizing serum samples had concomitant neutralizing activities against BCoV (Table 3). Of serum samples that had MERS-CoV neutralizing antibody titers >1,280, a total of 24.2% (94/389) had concomitant neutralizing activities against BCoV. Table 3 BCoV neutralization test results for MERS-CoV–positive dromedary serum samples, United Arab Emirates, 2003 and 2013* Collection No. BCoV positive/no. MERS-CoV positive (serum dilution, %) 640–1,280 >1,280 1A 15/74 (20.3) 14/79 (17.7) 1B 0 0 2 0/3 (0.0) 14/42 (33.3) 3 2/13 (15.4) 52/182 (28.6) 4 6/30 (20.0) 14/86 (16.3) Total 23/120 (19.2) 94/389 (24.2) *BCoV, bovine coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus. Fecal samples were available for 182 dromedary camels in collection 1. All samples were tested by using a subfamily Coronavirinae–specific broad-range reverse transcription PCR (RT-PCR) and a highly sensitive RT-PCR specific for genus Betacoronavirus phylogenetic lineage C. Both assays were specific for the viral RNA-dependent RNA polymerase gene. Two positive fecal samples were identified by both assays. Sequencing of amplified cDNA fragments of 182 nt and 404 nt identified sequences 99% identical with BCoV strain Mebus (GenBank accession nos. KF894801 and U00735.2). To further confirm virus identity, we amplified a region within the spike protein gene (positions 24303–24702 in BCoV strain Mebus) by using RT-PCR and sequencing it. Amplicons from both animals were 97% identical at nucleotide level with BCoV strain Mebus, indicating the presence of BCoV in camels as reported ( 10 ). We tested all serum samples in the same way by RT-PCR and obtained uniformly negative results. Discussion We have shown that dromedary camels from the United Arab Emirates, a country with human cases of MERS-CoV infection, have antibodies that can neutralize MERS-CoV at high rates. Antibodies were detected in serum samples obtained in 2013 and in serum samples obtained >10 years earlier, which indicated the long-standing presence of MERS-CoV or a closely related virus in dromedary camels in that region. Our data add to previous studies in which our group and others have reported wide antibody prevalence in camels in various regions, including Oman, Egypt, and the Canary Islands ( 10 , 11 ). A 10% lower seroprevalence in collection 2, which contained young racing camels, suggests that animals might be infected as juveniles. However, because only limited data were made available by owners, a definite statement awaits confirmation. The absence of antibodies in a control cohort from Germany might be explained by the fact that these animals belonged to a different camelid species (C. bactrianus vs. C. dromedarius). However, because MERS-CoV has a highly conserved receptor structure, we did not assign high priority to the hypothesis that the closely related camel species C. bactrianus, should be less susceptible than C. dromedarius camels to MERS-CoV ( 29 , 30 ). Differences in antibody prevalence rates might reflect a restricted geographic distribution of the virus, which corresponds to our previous finding of a relatively lower prevalence of antibodies against MERS CoV in camels from the Canary Islands, which have been isolated from their point of origin in Africa for many years ( 10 ). Therefore, MERS-CoV–like viruses in camelids might be spreading across a region covering at least the eastern Arabian Peninsula, including Oman, the United Arab Emirates, Egypt, and Morocco from where some of the antibody-positive camels described by Reusken et al. originated ( 10 ). The high rates of antibody prevalence in contemporary serum samples and samples from 2003 suggest that the virus has spread in camelids for some time. However, recognition of camelids as the bona fide reservoir for MERS-CoV has to await sequencing of camelid-associated MERS-related CoV. In this context, only animals infected with conspecific viruses can be regarded as reservoirs for a given virus. Although neutralization assays can provide evidence of infection with a virus belonging to the same serotype, no systematic studies have defined whether serotypes correlate with CoVs species. Nevertheless, for several CoV clades, serotypes defined by neutralization assay will not include >1 viral species. Members of the species Betacoronavirus 1, including CoV-OC43 and BCoV, show cross-neutralization with each other, but the closely related sister species (human CoV-HKU1) does not show cross-neutralization ( 31 ). Feline CoV (FCoV) comprises 2 subserotypes that show limited cross-reactivity but are considered 1 virus species. Transmissible gastroenteritis virus of swine shows more efficient cross-neutralization with 1 of these FCoV subserotypes than the other and is classified as 1 species with FCoV even though it is carried by a different host ( 32 ). Human CoVs 229E and NL63, which form 2 closely related sister taxa, do not show cross-neutralization and concordantly form 2 different species by genetic criteria ( 33 ). Therefore, our finding of high neutralizing antibody titers in camelids is suggestive (but not evidentiary) of the presence of viruses conspecific with MERS-CoV in camelids. Final confirmation will depend on the identification of virus sequences in camelids, which should expectably be closely related to human-specific MERS-CoV sequences. Camels probably acquired MERS-CoV at some unknown time. Potential sources include bats of the family Vespertilionidae, in which a virus with a close phylogenetic relationship with MERS-CoV has been detected ( 9 ). This virus, which is carried by vespertilionid bats of the genus Neoromicia, has been confirmed to be conspecific with MERS-CoV. Lineage C betacoronaviruses in other bat taxa have also been proposed to be related to MERS-CoV ( 34 , 35 ). However, although these viruses cluster phylogenetically with MERS-CoV, they are not conspecific with MERS-CoV on the basis of sequence distance criteria, such as that were proposed by Drexler et al. ( 36 ). In vespertilionid bats, including those in the genus Neoromicia, virus conspecific with MERS-CoV differs from human MERS-CoV, even if formally a member of the same species. The observed degree of sequence divergence between this virus and MERS-CoV makes any direct and recent transmission from bats to humans seem unlikely. Nevertheless, it cannot be excluded from available data that the virus source population in bats has not been detected. For example, a recent investigation of Rhinolophus bats in China identified viruses with close relationships to the bona fide ancestor of SARS-CoV, and viruses described in many studies yielded only conspecific yet less related viruses ( 37 ). In that study, viruses from civet cats, which are deemed to be intermediary hosts in the transition of SARS-CoV from bats to humans, were still more closely related to human SARS-CoV than even the closest bat-borne virus. If camelids should function as intermediary hosts in a similar manner, we should expect a virus in camelids that has a closer phylogenetic relationship with any bat-borne CoV and thus should be easily detectable with available RT-PCRs. Larger studies to confirm the presence of MERS-CoV in camelids should receive high priority so as to define the animal reservoir of MERS-CoV and possibly control it by such measures as vaccination or control of animal movement. However, before implementation of any control measures, whether camelids are a continuous source of infection for humans needs to be firmly established.
Author and article information
Journal
Journal ID (nlm-ta): Transbound Emerg Dis
Journal ID (iso-abbrev): Transbound Emerg Dis
Journal ID (publisher-id): tbed
Title:
Transboundary and Emerging Diseases
Publisher:
BlackWell Publishing Ltd
(Oxford, UK
)
ISSN
(Print):
1865-1674
ISSN
(Electronic):
1865-1682
Publication date
(Print):
April
2014
Publication date
(Electronic):
24
January
2014
Volume: 61
Issue: 2
Pages: 105-108
Affiliations
[1
]National Centres for Animal Disease (NCAD), Canadian Food Inspection Agency, Canadian
Science Centre for Human and Animal Health Winnipeg, MB, Canada
Author notes
Correspondence:, S. Alexandersen. National Centres for Animal Disease (NCAD), Canadian Food Inspection
Agency, Canadian Science Centre for Human and Animal Health, 1015 Arlington Street,
Winnipeg MB R3E 3M4, Canada., Tel.: +1 204 789 2102;, Fax: +1 204 789 2038;, E-mail:
soren.alexandersen@
123456inspection.gc.ca
Article
DOI: 10.1111/tbed.12212
PMC ID: 4282458
PubMed ID: 24456414
SO-VID: 07740752-310d-492e-945d-6e32a7b16b62
Copyright © © 2014 Her Majesty the Queen in Right of Canada 2014 Reproduced with the permission
of the Minister of Agriculture and Agri-food and Minister of Health
License:
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
History
Date
received
: 06
January
2014
Categories
Subject:
Rapid Communications
ScienceOpen disciplines: Infectious disease & Microbiology
Data availability:
ScienceOpen disciplines: Infectious disease & Microbiology