Table_1_Co-circulation of a Novel Dromedary Camel Parainfluenza Virus 3 and Middle East Respiratory Syndrome Coronavirus in a Dromedary Herd With Respiratory Tract Infections.DOCX

Since the emergence of Middle East Respiratory Syndrome (MERS) in 2012, there have been a surge in the discovery and evolutionary studies of viruses in dromedaries. Here, we investigated a herd of nine dromedary calves from Umm Al Quwain, the United Arab Emirates that developed respiratory signs. Viral culture of the nasal swabs from the nine calves on Vero cells showed two different types of cytopathic effects (CPEs), suggesting the presence of two different viruses. Three samples showed typical CPEs of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in Vero cells, which was confirmed by partial RdRp gene sequencing. Complete genome sequencing of the three MERS-CoV strains showed that they belonged to clade B3, most closely related to another dromedary MERS-CoV isolate previously detected in Dubai. They also showed evidence of recombination between lineages B4 and B5 in ORF1ab. Another three samples showed non-typical CPEs of MERS-CoV with cell rounding, progressive degeneration, and detachment. Electron microscopy revealed spherical viral particles with peplomers and diameter of about 170nm. High-throughput sequencing and metagenomic analysis showed that the genome organization (3'-N-P-M-F-HN-L-5') was typical of paramyxovirus. They possessed typical genome features similar to other viruses of the genus Respirovirus, including a conserved motif 323FAPGNYALSYAM336 in the N protein, RNA editing sites 5'-717AAAAAAGGG725-3', and 5'-1038AGAAGAAAGAAAGG1051-3' (mRNA sense) in the P gene with multiple polypeptides coding capacity, a nuclear localization signal sequence 245KVGRMYSVEYCKQKIEK261 in the M protein, a conserved sialic acid binding motif 252NRKSCS257 in the HN protein, conserved lengths of the leader (55nt) and trailer (51nt) sequences, total coding percentages (92.6–93.4%), gene-start (AGGANNAAAG), gene-end (NANNANNAAAAA), and trinucleotide intergenic sequences (CTT, mRNA sense). Phylogenetic analysis of their complete genomes showed that they were most closely related to bovine parainfluenza virus 3 (PIV3) genotype C strains. In the phylogenetic tree constructed using the complete L protein, the branch length between dromedary camel PIV3 (DcPIV3) and the nearest node is 0.04, which is >0.03, the definition used for species demarcation in the family Paramyxoviridae. Therefore, we show that DcPIV3 is a novel species of the genus Respirovirus that co-circulated with MERS-CoV in a dromedary herd in the Middle East.

自2012年中东呼吸综合征（Middle East Respiratory Syndrome, MERS）首次被发现以来，单峰驼体内病毒的发掘与进化研究便呈蓬勃增长之势。本研究针对阿联酋乌姆盖万（Umm Al Quwain）的9头出现呼吸道症状的单峰驼幼崽队列展开调查。对这9头幼崽的鼻拭子进行Vero细胞（Vero cells）病毒培养后，观察到两种不同类型的细胞病变效应（CPE），提示存在两种不同的病毒。其中3份样本在Vero细胞上呈现出典型的中东呼吸综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）细胞病变效应，经RNA依赖的RNA聚合酶（RdRp）基因片段测序验证确认为MERS-CoV。对这3株MERS-CoV毒株的全基因组测序结果显示，它们属于B3进化分支，与此前在迪拜检出的另一株单峰驼MERS-CoV分离株亲缘关系最近；同时，这些毒株在开放阅读框1ab（ORF1ab）区域存在B4与B5谱系间的重组证据。另外3份样本则呈现出非典型的MERS-CoV细胞病变效应，表现为细胞变圆、进行性变性及脱落。电子显微镜观察可见球形病毒粒子，带有包膜刺突，直径约170nm。高通量测序与宏基因组分析显示，该病毒的基因组结构为3'-N-P-M-F-HN-L-5'，符合副黏病毒科（Paramyxoviridae）病毒的典型特征。该病毒具备呼吸道病毒属（Respirovirus）其他病毒共有的典型基因组特征，包括N蛋白中保守的基序323FAPGNYALSYAM336，P基因中具备多多肽编码能力的RNA编辑位点5'-717AAAAAAGGG725-3'与5'-1038AGAAGAAAGAAAGG1051-3'（mRNA正义链），M蛋白中的核定位信号序列245KVGRMYSVEYCKQKIEK261，HN蛋白中保守的唾液酸结合基序252NRKSCS257，以及保守的前导序列（55nt）与尾随序列（51nt）长度、总编码比例（92.6%–93.4%）、基因起始序列（AGGANNAAAG）、基因终止序列（NANNANNAAAAA）与三核苷酸基因间序列（CTT，mRNA正义链）。对其全基因组的系统发育分析显示，该病毒与牛副流感病毒3型（bovine parainfluenza virus 3, PIV3）基因型C毒株亲缘关系最近。在基于全长L蛋白构建的系统发育树中，单峰驼PIV3（Dromedary camel PIV3, DcPIV3）与最近节点的分支长度为0.04，高于副黏病毒科物种界定标准中0.03的阈值。综上，本研究表明单峰驼PIV3（DcPIV3）为呼吸道病毒属（Respirovirus）的一个全新物种，该病毒与中东呼吸综合征冠状病毒（MERS-CoV）在中东地区的单峰驼群中共循环流行。