key: cord-0823556-92bvmiak authors: Mat-Rahim, Nor-Aziyah; Rashid, Tengku Rogayah Tengku Abdul; Suppiah, Jeyanthi; Thayan, Ravindran; Yusof, Apandi Mohd; Sa'at, Zainah title: Nucleocapsid gene analysis from an imported case of Middle East respiratory syndrome coronavirus, Malaysia date: 2015-07-31 journal: Asian Pacific Journal of Tropical Disease DOI: 10.1016/s2222-1808(15)60833-7 sha: 91518c8dd2557ee864fc6cc0debcaf20899a457d doc_id: 823556 cord_uid: 92bvmiak Abstract Objective To describe the complete nucleocapsid (N) gene region of Middle East respiratory syndrome coronavirus (MERS-CoV) from imported case in Malaysia and the relations with human- and camel-derived MERS-CoV. Methods Combination of throat and nasal swab specimens was subjected to viral RNA extraction. For screening, the extracted RNA was subjected to real-time RT-PCR targeting upstream of E gene, open reading frame 1b and open reading frame 1a. For confirmation, the RNA was subjected to RT-PCR targeting partial part of the RNA-dependent RNA polymerase and nucleocapsid, followed by amplification of complete N gene region. Nucleotide sequencing of the first Malaysian case of MERS-CoV was performed following the confirmation with real-time RT-PCR detection. Results Initial analysis of partial RNA-dependent RNA polymerase and N gene revealed that the nucleotides had high similarity to Jeddah_1_2013 strain. Analysis of complete N gene region (1 242 nucleotides) from the case showed high similarity and yet distinct to the nucleotide sequences of camel-derived MERS-CoV. Conclusions From the finding, there are possibilities that the patient acquired the infection from zoonotic transmission from dromedary camels. Betacoronavirus and was first isolated from a patient with severe respiratory disease and renal failure in 2012. As of 13 June 2014, a total of 697 laboratory-confirmed cases with 210 deaths have been reported worldwide [1] . All these cases were reported among people either from or have travelled from the Arab Peninsular. Most people infected with MERS-CoV and developed severe respiratory illnesses with symptoms of fever, cough, and shortness of breath. About 30% of the cases have succumbed to their illness and majority of those who died had underlying medical condition such as diabetes, cancer, and chronic lung, heart and kidney disease [2] . For screening, the extracted RNA was subjected to rRT-PCR targeting upE, ORF1b and ORF1a [4, 5] , which were optimized for SuperScript III Platinum One-Step RT-PCR System with Platinum Taq Polymerase (Invitrogen, USA). For confirmation, the RNA was subjected to RT-PCR targeting partial part of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) [5] , followed by amplification of complete N gene region designed for subsequent phylogenetic analysis (Table 1) . Table 1 Primers used for amplification and sequencing of the Malaysian case. For the amplification of partial RdRp gene region, primer RdRpSeq-Fwd and RdRpSeq-Rev were employed [5] , while primer NSeq-Fwd and NSeq-Rev were used for amplification of partial N gene region [5] . The RT-PCR human-derived MERS-CoV from 2012 to 2014 and 3 camel-derived MERS-CoV sequences are shown in Figure 1 . Maximum-likelihood analysis of N gene region was constructed by using the Tamura-Nei model. Each branch showed, in Figure 1 , the GenBank accession number followed by description of the sequence used. It is noted that the nucleotide sequence from the Malaysian case is clustered together with human-derived MERS-CoV sequences isolated from human cases in 2014, among them are those reported in Greece and USA. The sequences of the camel-derived MERS-CoV group together in their own cluster, however, close to those of human-derived sequences isolated in 2014. The sequence analysis of N gene region from the first case of MERS-CoV in Malaysia was reported. Comparative analysis of the N gene region from the Malaysian case and camel-derived MERS-CoV sequence showed more than 99% identities. The finding is concerted with results of studies performed in dromedary camel from Qatar [8] , Saudi Arabia [9, 10] and Oman [11] , which showed that the camel-derived and human-derived MERS-CoV were highly similar yet distinct. This is also reflected in the phylogenetic analysis; the camel-derived and human-derived sequences were independently clustered. Based on the travel history of the Malaysian case, the infection may be acquired after being exposed to infected camels, via patting the camel especially their nostril region and consuming the raw, unpasteurized camel milk [3] . Similar observation as the Malaysian case has been reported by Memish et al. [9] , which substantiate the cross-species transmission to humans, with the possibility of dromedary camels being the primary reservoir hosts of MERS-CoV [8] [9] [10] [11] . Juvenile camels may play a big role in the viral transmission, since the younger were shown to shed more virus compared to the old ones [9, 10, 12] . The transmission from camels to human was postulated to occur through very close contact with high infectious dose of virus via nasal route [11] . Other possible route of transmission is via consumption of raw milk from infected camels, which was supported by a report showing that MERS-CoV could survive slightly longer in raw camel milk in comparison to other species [13] . MERS-CoV in human emerged only recently, although it has been shown to be circulating in camels since 1992 or even earlier [10] . Even though higher frequency of MERS-CoV infection was observed in the camels of the Arabian Peninsula, low number of infections was recorded in human. Thus, further study to determine the exact mode of transmission from camel-to-human and human-to-human is important to mitigate the spread of this virus. We declare that we have no conflict of interest. KJ156952/Human/Saudi Arabia/Riyadh KJ156944/Human/Saudi Arabia/Riyadh KF600620/Human/Saudi Arabia/Bisha 1/2012 KF600612/Human/Saudi Arabia/Riyadh 1/2012 KJ156869/Human/Saudi Arabia/Riyadh Middle East respiratory syndrome coronavirus (MERS-CoV)-update. Geneva: World Health Organization Middle East respiratory syndrome (MERS): symptoms and complications Laboratory-confirmed case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in Malaysia: preparedness and response Detection of a novel human coronavirus by real-time reversetranscription polymerase chain reaction Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools MEGA6: molecular evolutionary genetics analysis version 6.0 Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation Human infection with MERS coronavirus after exposure to infected camels, Saudi Arabia Middle East respoiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels MERS coronavirus in dromedary camel herd, Saudi Arabia Stability of Middle East respiratory syndrome coronavirus in milk The authors would like to thank the Director General of Health