key: cord-348867-c0xpzd4d authors: Zhai, Jun-Qiong; Zhai, Shao-Lun; Lin, Tao; Liu, Jian-Kui; Wang, He-Xing; Li, Bing; Zhang, He; Zou, Shu-Zhan; Zhou, Xia; Wu, Meng-Fan; Chen, Wu; Luo, Man-Lin title: First complete genome sequence of parainfluenza virus 5 isolated from lesser panda date: 2017-01-30 journal: Arch Virol DOI: 10.1007/s00705-017-3245-0 sha: doc_id: 348867 cord_uid: c0xpzd4d Parainfluenza virus 5 (PIV5) is widespread in mammals and humans. Up to now, there is little information about PIV5 infection in lesser pandas. In this study, a PIV5 variant (named ZJQ-221) was isolated from a lesser panda with respiratory disease in Guangzhou zoo in Guangdong province, southern China. The full-length genome of ZJQ-221 was found to be 15,246 nucleotides and consisted of seven non-overlapping genes encoding eight proteins (i.e., NP, V, P, M, F, SH, HN and L). Sequence alignment and genetic analysis revealed that ZJQ-221 shared a close relationship with a PIV5 strain of canine-origin (1168-1) from South Korea. The findings of this study confirm the presence of PIV5 in lesser panda and indicate this mammal as a possible natural reservoir. Furthermore they highlight the urgent need to strengthen viral surveillance and control of PIV5 in zoo animals. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-017-3245-0) contains supplementary material, which is available to authorized users. Members of the Paramyxoviridae family are the causative agents of mumps and measles in humans, Newcastle disease in poultry, peste des petits ruminants in sheep and goats, as well as distemper in carnivorous animals [1] [2] [3] [4] . Parainfluenza virus 5 (PIV5) belongs to the genus Jun-Qiong Zhai, Shao-Lun Zhai and Tao Lin contributed equally to this study. Rubulavirus in the family Paramyxoviridae (http://www. ictvonline.org/virusTaxonomy.asp?taxnode_id= 20151063), and was originally known as simian virus 5 (SV5) or canine parainfluenza virus (CPIV) [5] [6] [7] . PIV5 has a negative sense, single-stranded RNA genome of 15,246 nucleotides with a virion diameter of 50-200 nm that appears circular or polymorphous under the electron microscope [8] [9] [10] . PIV5 has eight viral proteins (i.e., NP, V, P, M, F, SH, HN and L). The V and P proteins are encoded by the V/P gene sharing the same genomic region [8] . PIV5 has a close genetic relationship with other members of genus Rubulavirus, including simian virus 41, human parainfluenza virus 2, human parainfluenza virus 4, mumps virus, mapuera virus and porcine rubulavirus [11] [12] [13] [14] [15] [16] . Recently, paramyxoviruses have been found to have a global presence and to be prevalent in several countries [17] [18] [19] . Novel paramyxoviruses that can spread among different species have resulted in outbreaks in animals [20, 21] . Hosts susceptible to PIV5 include humans, pigs, dogs, cattle, cats, hamsters and guinea pigs [22] [23] [24] [25] . PIV5 can cause central nervous and respiratory diseases in infected hosts [25, 26] . Up to now, there is little information about PIV5 in lesser pandas. In this study, a novel variant of PIV5 (designated as ZJQ-221) was isolated from a lesser panda with respiratory disease in Guangzhou zoo in Guangdong province, southern China. Furthermore, the complete genome sequence of ZJQ-221 was amplified and characterized. During the years of 2014-2016, fourteen representative clinical samples from ornamental animals (including four lesser pandas, one northeast tiger, one south China tiger, four Panthera leo and four ring-tailed lemurs) were collected from Guangzhou zoo in Guangdong province, southern China. Clinical manifestations of the animals included coughing with thin nasal fluid and a slightly elevated temperature. One lesser panda died, and its necropsy identified the presence of lobular pneumonia in the lungs. Nasal swabs and lung samples were stored at -80°C until use. Fourteen samples were tested for the possible presence of three respiratory-related pathogens (including PIV5, canine distemper virus, and coronavirus) by RT-PCR according to previous studies [27, 28] . The results revealed the positive presence of PIV5 nucleotides in the lesser panda samples. The lung samples from the lesser panda were then homogenized in 10% (w/v) sterile phosphatebuffered saline (PBS, pH 7.4), and centrifuged at 10,000 9 g for 5 min at 4°C. The supernatants were filtered aseptically (0.22 lm pore size) and the filtrates (500 ll) were inoculated onto Vero cell monolayers in a 25-cm 2 cell culture flask. After viral attachment for 2 h at 37°C, unattached viruses were removed by gentle washing with PBS and the cells were maintained in Dulbecco's minimal essential medium (DMEM, Gibco), containing 2% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antimitotic, at 37°C in a 5% CO 2 atmosphere. The viral cultures were harvested when the cytopathic effects (CPE) reached 80% coverage and then stored at -80°C [25] . In order to observe the virus particles, the cell suspensions were centrifuged at 1,000 rpm for 10 min, and then fixed using 2.5 * 3% glutaraldehyde fixation fluid for about 4 h at 4°C in a refrigerator. Finally, they were submitted to the electron microscope center, South China Agricultural University for processing. RNA extraction of positive homogenates was performed using the MiniBEST Universal RNA Extraction Kit (TaKaRa), and viral RNA was reverse transcribed into first-strand cDNA with a random primer (5 0 -NNNNNN-3 0 ) using the PrimeScript first strand cDNA Synthesis Kit (TaKaRa). PCR was then used to amplify the full-length genome sequences using 12 pairs of primers listed in Table S1 . Positive PCR products were purified (MiniBEST Agarose Gel DNA Extraction Kit, TaKaRa) and cloned into the pMD19-T vector (TaKaRa). Positive recombinant plasmids were further sequenced using the Sanger sequencing method (BGI Inc., Guangzhou branch). Sequence alignment and phylogenetic analysis based on different rubulavirus species sequences (Table 1) was performed using DNAStar Lasergene 7.10 (Madison, WI, USA) and MEGA 5 (http://www.megasoftware.net) [34] . In comparison with uninfected cells (Fig. S1a) , Vero cells inoculated with PIV5-positive samples showed demonstrable CPE by passage 3 (Fig. S1b) . One viral strain (named ZJQ-221) was thus obtained. Further observation by electron microscopy found that the isolated ZJQ-221 strain was spherical and had a diameter of 50-200 nm that is similar to, and characteristic of, paramyxoviruses (Fig. S1c) . Moreover, through sequencing, the full-length genome sequence (15, In addition, the SH protein was not present in some PIV5 strains due to nucleotide substitution, such as KUN-11 (KC852177), SER (JQ743328), CC14 (KP893891) PV5-BC14 (KM067467) and AGS (KX060176), implying that the protein is not essential for PIV5 infection in pigs, dogs, calves and cells [25, 29, 30, 32, 33] . However, the ZJQ-221 strain had a SH protein and was thus different from the above-mentioned PIV5 strains. Furthermore, phylogenetic analysis ( Fig. 1) was performed based on the complete genome sequences of different species of rubulaviruses. In the phylogenetic tree, ZJQ-221 and 1168-1 shared the closest genetic relationship and were clustered in the same branch (Fig. 1) . To the best of our knowledge, very little data about PIV5 infections in zoo animals is available. In this study, we tested whether PIV5 infections are present in lesser pandas, northeast tigers, south China tigers, Panthera leo and ring-tailed lemurs. While the RT-PCR results showed that only lesser panda samples were positive for PIV5, due to the limited number of animal samples collected in the zoo, we believe that PIV5 might not be restricted to lesser pandas. In fact, a previous study showed antibodies against PIV5 exist in zoo tigers [35] . Moreover, PIV5 nucleotide sequences were also detected in nasal swab samples from northeast tiger and south China tiger collected in 2016 in the same zoo, and their F genes were close to the lesser panda-origin PIV5 strain described here (data not shown). This suggests that PIV5 is a common pathogen in zoo animals, and might play an important causal role in the respiratory diseases of zoo animals. In summary, we have identified a novel PIV5 isolate in lesser panda and performed whole genome sequencing, indicating that this mammal may act as a possible natural reservoir for this virus. This study contributes to the epidemiology and genomics of PIV5, and suggests an urgent need to strengthen viral surveillance and control of PIV5 in zoo animals. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. 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Moreover, this work was also partially supported by Ministry of Science and Technology of the People's Republic of China (Grant No. 2015GA780010), Guangdong Provincial Department of Science and