Emily Y. M. Wong

New Hepatitis E Virus Genotype in Camels, the Middle East

In a molecular epidemiology study of hepatitis E virus (HEV) in dromedaries in Dubai, United Arab Emirates, HEV was detected in fecal samples from 3 camels. Complete genome sequencing of 2 strains showed >20% overall nucleotide difference to known HEVs. Comparative genomic and phylogenetic analyses revealed a previously unrecognized HEV genotype.

Novel Betacoronavirus in Dromedaries of the Middle East, 2013

In 2013, a novel betacoronavirus was identified in fecal samples from dromedaries in Dubai, United Arab Emirates. Antibodies against the recombinant nucleocapsid protein of the virus, which we named dromedary camel coronavirus (DcCoV) UAE-HKU23, were detected in 52% of 59 dromedary serum samples tested. In an analysis of 3 complete DcCoV UAE-HKU23 genomes, we identified the virus as a betacoronavirus in lineage A1. The DcCoV UAE-HKU23 genome has G+C contents; a general preference for G/C in the third position of codons; a cleavage site for spike protein; and a membrane protein of similar length to that of other betacoronavirus A1 members, to which DcCoV UAE-HKU23 is phylogenetically closely related. Along with this coronavirus, viruses of at least 8 other families have been found to infect camels. Because camels have a close association with humans, continuous surveillance should be conducted to understand the potential for virus emergence in camels and for virus transmission to humans.

Acute Middle East Respiratory Syndrome Coronavirus infection in livestock dromedaries, Dubai, 2014

Camels carry Middle East respiratory syndrome coronavirus, but little is known about infection age or prevalence. We studied >800 dromedaries of all ages and 15 mother–calf pairs. This syndrome constitutes an acute, epidemic, and time-limited infection in camels <4 years of age, particularly calves. Delayed social separation of calves might reduce human infection risk.

New Hepatitis E Virus Genotype in Bactrian Camels, Xinjiang, China, 2013.

To the Editor: Hepatitis E virus (HEV) is a member of the family Hepeviridae, genus Orthohepevirus, which comprises 4 species, Orthohepevirus A–D. Orthohepevirus A contains 7 genotypes (HEV1–7) (1,2). HEV1 and HEV2 infect humans only; HEV3, HEV4, and HEV7 can infect humans and other mammals; and HEV5 and HEV6 have been detected in animals only. Worldwide, HEV is the most common cause of acute viral hepatitis in humans. The disease is generally selflimiting, but high death rates have been observed among HEV-infected pregnant women. Chronic HEV infection is a problem in immunocompromised patients, such as solid organ transplant recipients (3). Human HEV3 and HEV4 infections have been associated with consumption of undercooked pork or game meat (4). In 2014, we described the discovery of a novel genotype of HEV in dromedaries (Camelus dromedarius or 1-humped camels), suggesting another possible source of human HEV infection (5). This dromedary HEV was subsequently classified as a novel Orthohepevirus A genotype, HEV7 (1,2). Recently, this HEV7 genotype was also isolated from a liver transplant recipient from the Middle East with chronic HEV infection (6). The patient regularly consumed dromedary camel meat and milk, implying camelto-human transmission of the virus (6). Like the dromedary, the Bactrian camel (Camelus bactrianus or 2-humped camels) is an Old World camelid species. Thus, we hypothesize that Bactrian camels may also be reservoirs of HEV. To test this hypothesis and increase our understanding of the epidemiology of HEV in camels, we performed a molecular epidemiology study using feces samples from camels in China. During November 2012–May 2013, we collected and tested 1 feces sample each from 205 Bactrian camels on a farm in Xinjiang, China. We performed RNA extraction and reverse transcription PCR (RT-PCR) as previously described (7). We screened for HEV by PCR amplification of a 251-bp fragment of open-reading frame (ORF) 2, using primers 5′-GTTGTCTCAGCCAATGGCGA-3′ and 5′-GTAGTTTGGTCATACTCAGCAGC-3′. PCR was performed, using previously described conditions (7), with the annealing temperature set at 50°C. DNA sequencing and quantitative real-time RT-PCR were performed as previously described (7). Three samples were positive for HEV; we performed complete genome sequencing of these samples as described (online Technical Appendix, http:// wwwnc.cdc.gov/EID/article/22/12/16-0979-Techapp1.pdf) (5,7). We also performed comparative genomic analysis as previously described (1,2,8). We constructed a phylogenetic tree using the maximum-likelihood method and MEGA7 (9); bootstrap values were calculated from 1,000 trees. The optimal substitution model for each ORF was selected by MEGA7 (Figure). RT-PCR for a 251-bp fragment in ORF2 of HEV was positive for 3 (1.5%) of the 205 fecal samples; virus loads were 1.6 × 103, 2.1 × 103, and 1.8 × 104 copies/mg, respectively. Whole-genome sequencing of the 3 Bactrian camel HEV (BcHEV) strains (GenBank accession nos. KX387865–7) showed genome sizes of 7,212–7,223 bp and a G + C content of 52.7%–53.1%. Overall, nucleotides in the BcHEV genome differed by >20% compared with those in all other HEVs. Genomes of the 3 BcHEV isolates contained 3 major ORFs; genome organization was typical of and characteristics were similar to those of HEVs from other Orthohepevirus A species. Phylogenetic trees constructed using ORF1, ORF2, ORF3, and concatenated ORF1/ORF2, excluding the hypervariable region, showed that these 3 BcHEV isolates clustered with the 2 dromedary camel HEV7 strains and the HEV7 strain from the liver-transplant recipient with chronic hepatitis (Figure; online Technical Appendix Figure 1)

Metagenomic analysis of viromes of dromedary camel fecal samples reveals large number and high diversity of circoviruses and picobirnaviruses

Abstract The recent discovery of Middle East Respiratory Coronavirus and another novel dromedary camel coronavirus UAE-HKU23 in dromedaries has boosted interest in search of novel viruses in dromedaries. In this study, fecal samples of 203 dromedaries in Dubai were pooled and deep sequenced. Among the 7330 assembled viral contigs, 1970 were assigned to mammalian viruses. The largest groups of these contigs matched to Picobirnaviridae, Circoviridae, Picornaviridae, Parvoviridae, Astroviridae and Hepeviridae. Many of these viral families were previously unknown to dromedaries. In addition to the high abundance of contigs from Circoviridae (n=598 with 14 complete genomes) and Picobirnaviridae (n=1236), a high diversity of contigs from these two families was found, with the 14 Circoviridae complete genomes forming at least five clusters and contigs from both genogroup I and genogroup II potentially novel picobirnaviruses. Further studies comparing the incidence of these viral families in healthy and sick dromedaries will reveal their pathogenic potential.

Polyphyletic origin of MERS coronaviruses and isolation of a novel clade A strain from dromedary camels in the United Arab Emirates

Little is known regarding the molecular epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV) circulating in dromedaries outside Saudi Arabia. To address this knowledge gap, we sequenced 10 complete genomes of MERS-CoVs isolated from 2 live and 8 dead dromedaries from different regions in the United Arab Emirates (UAE). Phylogenetic analysis revealed one novel clade A strain, the first detected in the UAE, and nine clade B strains. Strain D998/15 had a distinct phylogenetic position within clade A, being more closely related to the dromedary isolate NRCE-HKU205 from Egypt than to the human isolates EMC/2012 and Jordan-N3/2012. A comparison of predicted protein sequences also demonstrated the existence of two clade A lineages with unique amino acid substitutions, A1 (EMC/2012 and Jordan-N3/2012) and A2 (D998/15 and NRCE-HKU205), circulating in humans and camels, respectively. The nine clade B isolates belong to three distinct lineages: B1, B3 and B5. Two B3 strains, D1271/15 and D1189.1/15, showed evidence of recombination between lineages B4 and B5 in ORF1ab. Molecular clock analysis dated the time of the most recent common ancestor (tMRCA) of clade A to March 2011 and that of clade B to November 2011. Our data support a polyphyletic origin of MERS-CoV in dromedaries and the co-circulation of diverse MERS-CoVs including recombinant strains in the UAE.

Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23.

Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1.

A six-year descriptive epidemiological study of human coronavirus infections in hospitalized patients in Hong Kong

We conducted a six-year epidemiological study on human coronaviruses (HCoVs) circulating in Hong Kong, using 8275 nasopharyngeal samples from patients with acute respiratory tract infections. HCoVs were detected in 77 (0.93%) of the samples by a pan-HCoV RT-PCR assay. The most frequently detected HCoV species was HCoV-OC43 (0.58%), followed by HCoV-229E (0.15%), HCoV-HKU1 (0.13%) and HCoV-NL63 (0.07%). HCoVs were detected throughout the study period (September 2008–August 2014), with the highest detection rate from September 2010 to August 2011 (22/1500, 1.47%). Different seasonal patterns of each HCoV species in Hong Kong were noted. HCoV-OC43 was predominant in the fall and winter, whereas HCoV-HKU1 showed peak activity in winter, with a few cases occurred in spring and summer. HCoV-229E mainly occurred in winter and spring, while HCoV-NL63 was predominant in summer and autumn. HCoVs most commonly infect the elderly and young children, with median age of 79.5 years (range, 22 days to 95 years). Intriguingly, the detection rate of HCoV-OC43 in the age group of > 80 years (26/2380, 1.09%) was significantly higher than that in the age group of 0–10 years (12/2529, 0.47%) (P < 0.05). These data provides new insight into the epidemiology of coronaviruses.

Two novel dromedary camel bocaparvoviruses from dromedaries in the Middle East with unique genomic features

The recent emergence of Middle East respiratory syndrome (MERS) coronavirus and its discovery from dromedary camels has boosted interest in the search for novel viruses in dromedaries. While bocaparvoviruses are known to infect various animals, it was not known that they exist in dromedaries. In this study, we describe the discovery of two novel dromedary camel bocaparvoviruses (DBoVs), DBoV1 and DBoV2, from dromedary faecal samples in Dubai. Among 667 adult dromedaries and 72 dromedary calves, 13.9 % of adult dromedaries and 33.3 % of dromedary calves were positive for DBoV1, while 7.0 % of adult dromedaries and 25.0 % of dromedary calves were positive for DBoV2, as determined by PCR. Sequencing of 21 DBoV1 and 18 DBoV2 genomes and phylogenetic analysis showed that DBoV1 and DBoV2 formed two distinct clusters, with only 32.6-36.3 % amino acid identities between the DBoV1 and DBoV2 strains. Quasispecies were detected in both DBoVs. The amino acid sequences of the NS1 proteins of all the DBoV1 and DBoV2 strains showed <85 % identity to those of all the other bocaparvoviruses, indicating that DBoV1 and DBoV2 are two bocaparvovirus species according to the ICTV criteria. Although the typical genome structure of NS1-NP1-VP1/VP2 was observed in DBoV1 and DBoV2, no phospholipase A2 motif and associated calcium binding site were observed in the predicted VP1 sequences for any of the 18 sequenced DBoV2, and no start codons were found for their VP1. For all 18 DBoV2 genomes, an AT-rich region of variable length and composition was present downstream to NP1. Further studies will be crucial to understand the pathogenic potential of DBoVs in this unique group of animals.

Molecular epidemiology of canine picornavirus in Hong Kong and Dubai and proposal of a novel genus in Picornaviridae.

Previously, we reported the discovery of a novel canine picornavirus (CanPV) in the fecal sample of a dog. In this molecular epidemiology study, CanPV was detected in 15 (1.11%) of 1347 canine fecal samples from Hong Kong and one (0.76%) of 131 canine fecal samples from Dubai, with viral loads 1.06×10(3) to 6.64×10(6) copies/ml. Complete genome sequencing and phylogenetic analysis showed that CanPV was clustered with feline picornavirus (FePV), bat picornavirus (BatPV) 1 to 3, Ia io picornavirus 1 (IaioPV1) and bovine picornavirus (BoPV), and this cluster was most closely related to the genera Enterovirus and Sapelovirus. The Ka/Ks ratios of all the coding regions were <0.1. According to the definition of the Picornavirus Study Group of ICTV, CanPV, FePV, BatPV 1 to 3, IaioPV1 and BoPV should constitute a novel genus in Picornaviridae. BEAST analysis showed that this genus diverged from its most closely related genus, Sapelovirus, about 49 years ago.