Fatal canine distemper virus infection of giant pandas in China

We report an outbreak of canine distemper virus (CDV) infection among endangered giant pandas (Ailuropoda melanoleuca). Five of six CDV infected giant pandas died. The surviving giant panda was previously vaccinated against CDV. Genomic sequencing of CDV isolated from one of the infected pandas (giant panda/SX/2014) suggests it belongs to the Asia-1 cluster. The hemagglutinin protein of the isolated virus and virus sequenced from lung samples originating from deceased giant pandas all possessed the substitutions V26M, T213A, K281R, S300N, P340Q, and Y549H. The presence of the Y549H substitution is notable as it is found at the signaling lymphocytic activation molecule (SLAM) receptor-binding site and has been implicated in the emergence of highly pathogenic CDV and host switching. These findings demonstrate that giant pandas are susceptible to CDV and suggest that surveillance and vaccination among all captive giant pandas are warranted to support conservation efforts for this endangered species.

Histopathological analysis. Late in the course of infection, giant pandas exhibited clinical signs of nasal hyperkeratosis (Fig. 1a) and footpad hyperkeratosis (Fig. 1b) which are characteristic of CDV infection in other animals 18 . Severe pneumonia with dark-red congestion was observed in affected lungs along with small white patches on the surface of the lungs of one CDV-infected giant panda named Fengfeng (Fig. 1c). Lung samples from Fengfeng were examined for histopathological analysis, whereas other samples (e.g ., brain, spleens) were not assessed due to cellular autolysis of tissues collected at necropsies performed >12 hours of after death. Histological analyses of lung tissue from Fengfeng showed interstitial pneumonia with congestion, multinuclear macrophage infiltration in the alveoli, and widening of alveolar septa (Fig. 1d). Histological observations were consistent with those previously reported in the lungs of cynomolgus monkeys and red fox (Vulpes vulpes) infected by CDV 14,19 . Phylogenetic Analysis. The complete viral genome of the CDV isolate giant panda/SX/2014 was sequenced (Gen Bank accession no. KP793921) and showed highest nucleotide identity to the PS strain of CDV isolated from a dog (98.7% identity, Gen Bank accession no. JN896331) and the CDV-RD-JL strain of CDV isolated from a raccoon dog (Nyctereutes procyonoides, 98.6% identity, Gen Bank accession no. KJ848781) (Fig. 2a). Phylogenetic analysis and multiple sequence alignments based on the H gene sequence revealed that giant panda/SX/2014 belongs to the Asia-1 cluster. The H gene sequence of giant panda/SX/2014 was 99.1% identical to the SD(08)1 and LN(07)1 CDV strains (Gen Bank accession no. FJ810215 and EU325730) isolated from a fox and raccoon dog in Shandong and Liaoning province in China, respectively (Fig. 2b) 20 . The Y549H substitution is involved in H protein binding to the signaling lymphocytic activation molecule (SLAM) receptor and has been hypothesized to contribute to the emergence of highly pathogenic CDV and host range expansion (Table 3).

Discussion
Here All affected giant pandas were housed in the same room or adjacent rooms, suggesting that CDV may have been transmitted between pandas via direct contact and/or respiratory droplets. The surviving panda tested positive for CDV by RT-PCR, but did not develop overt clinical signs of CDV infection and was previously vaccinated against CDV, strongly supporting the utility of CDV vaccination in giant pandas. CDV transmission to captive animals may potentially occur via direct or indirect contact with infected domestic dogs or wild carnivores. Domestic dogs were considered the likely source of infection for canine distemper in Serengeti lions in 1994 21 . In North America, wild raccoons (Procyon lotor) were thought to be the source of CDV infection in large captive cats in 1991 and 1992 9 . In Japan, raccoon dogs were considered to be the source of outbreak for canine distemper in tigers in 2009 and 2010 22 . In Denmark, it was speculated that wildlife species, such as foxes, raccoon dogs, and ferrets were the sources of CDV infection of farmed mink (Neovison vison) 23 . In China, CDV infection has been observed in domestic dogs, wild canids (fox, raccoon dogs), and non-canids (mink, monkey) demonstrating the remarkable ability of this pathogen to cross species barriers 15,24 . The reservoir source of CDV leading to the outbreak among giant pandas remains unclear. While there were no carnivores in   Table 3. Nucleotide sequence identity and amino acid differences of the CDV isolated from the giant panda as compared to other closely related isolates. # Percent identity in hemagglutinin nucleotide sequence of the CDV isolated from the giant panda when compared to other closely related isolates. * Indicates residue identical to that of FJ810215 (SD(08)1). Italics indicates mutated residues of giant panda/SX/2014 when compared to other strains. Bold highlights isolates with a histidine residue 549 of the CDV H protein.
the Shanxi Rare Wild Animal Rescue and Research Center, it is possible that domestic dogs or other susceptible wild animals carrying CDV in the area were responsible. The highly variable nature of H gene sequences among viruses belonging to the Morbillivirus genus has been exploited to characterize CDV field strains and investigate relationships among various strains. Sequencing of CDV from giant pandas revealed five unique amino acid changes (V26M, T213A, K281R, S300N, P340Q) encoded by the H gene that have not been observed previously in Asia-1 strains. Amino acid residues at positions 549 in the CDV H protein are implicated in CDV host range restriction and pathogenesis 25,26 . Notably, the H gene of CDV from giant pandas possessed a Y549H substitution which has been associated with the emergence of highly pathogenic CDV and host range expansion. Before this outbreak, the Y549H substitution had only observed in twelve CDV strains isolated from mink, fox and raccoon dogs in Shandong province and an isolate from a mink in Liaoning province in China 24 . While the Shanxi Rare Wild Animal Rescue and Research Center is located a significant distance away from these provinces (> 900 kilometers), these species may represent potential sources of CDV leading to this outbreak among giant pandas. Previous work has shown that serial passage of dog-derived CDV strain 5804 in ferrets led to acquisition of the Y549H substitution 27 . CDV isolates with a histidine residue at position 549 also showed enhanced virulence in raccoons relative to strains lacking histidine at this position 28 . We therefore speculate that the high-level of virulence associated with giant panda/SX/2014 infection of giant pandas may be related to the presence of a histidine residue at position 549 of the H protein.
The additional unique H protein amino acid substitutions identified may reflect adaptive changes facilitating CDV infection of giant pandas. As an RNA virus, CDV is capable of rapid mutation leading to viral variants that are potentially better equipped for replication in giant pandas, as has been documented for the emergence of the Y549H substitution during serial passage in ferrets 27 .
Vaccination represents an effective approach to prevent CDV infection of domestic dogs and may have utility in captive giant panda populations. Currently, there are no standard vaccination strategies in place for the prevention of infectious diseases in captive giant pandas in China. The effectiveness of CDV vaccination in giant pandas is supported by the observation that the single panda to survive CDV infection during this outbreak (Zhuzhu) was previously vaccinated against CDV and had high-titer SN antibodies. This animal did not display clinical signs despite recovery of CDV genomic material from blood and nasal swab samples, suggesting that the protective immune responses elicited by CDV vaccination were not sufficient to prevent naturally-acquired CDV infection but may have attenuated disease. Ultimately, universal CDV vaccination of captive giant pandas may be warranted but must be also weighed against potential vaccination risks. Additional studies to better understand the safety and efficacy of CDV vaccines in giant pandas are needed as CDV vaccines are more widely implemented. In a study involving two giant pandas, a commercially available canarypox-vectored CDV vaccine safely elicited SN antibody titers above a level considered to be protective against CDV disease 29  Virus detection by PCR and RT-PCR. Nasal swab, urine, fecal and blood samples from each affected panda were collected at the time of clinical disease onset. Viral DNA and RNA were isolated from samples using the AxyPrep Multisource Genomic DNA Miniprep kit (AXYGEN, Union City, USA) and RNeasy Mini kit (QIAGEN, Germantown, MD) according to manufacturer's protocols. Extracted nucleic acids were tested by RT-PCR for CDV using primers specific for CDV H gene (P1:5′-CGAGTCTTTGAGATAGGGTT-3′ and P2: 5′-CCTCCAAAGGGTTCCCATGA-3′). RT-PCR and PCR testing for other viruses threatening giant pandas (canine adenovirus, canine herpesvirus, canine coronavirus, and canine parainfluenza virus) were performed using previously reported methods 17,30 . RT-PCR testing for CDV was also performed on samples collected from the heart, liver, spleen, lungs, kidneys, intestines, and brain of each deceased giant panda, with the exception of Chengcheng for whom tissue samples were not available. Serum samples were collected from the giant pandas during the outbreak to measure SN antibody titers against CDV.
Histopathological analysis of giant pandas infected with CDV. Necropsies were performed on all deceased giant pandas at which time tissue samples were collected for histologic examination. Lung samples from the giant panda named Fengfeng, who experienced a long illness duration, were selected for histologic review using routine methods. Tissue samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin prior to analysis.
Virus isolation and sequencing. CDV was isolated from lung and spleen tissue collected from the deceased giant panda Dabao as follows. Tissue samples were inoculated onto monolayers of Vero cells expressing canine SLAM protein, which have been previously used to grow and isolate CDV 31 . The presence of CDV was confirmed by RT-PCR detection using primers specific for the CDV N gene (P3: 5′-GTGACTGCTCCTGATACTGC-3′ and P4: 5′-ACCAACTCCCATAGCATAAC-3′). The giant panda CDV isolate was named as giant panda/ SX/2014. The complete H gene of the giant panda/SX/2014 isolate and virus contained in lung samples from deceased giant pandas was amplified for sequencing by RT-PCR using H gene specific primers (P5: Scientific RepoRts | 6:27518 | DOI: 10.1038/srep27518 5′-GTCTTGCCTGATTGTCAGGC-3′ and P6: 5′-GGTTTGATTCAATCGTCGG-3′). The entire genome of the giant panda/SX/2014 CDV isolate was amplified and sequenced using a set of fifteen primer pairs to generate overlapping PCR amplicons (Table S1).
Phylogenetic analysis. Phylogenetic trees were constructed using molecular evolutionary genetics analysis MEGA 6 software (http://www.megasoftware.net/mega.php) with the neighbor-joining (NJ) method to calculate distance. Bootstrapping with 1,000 replicates was performed to determine the percentage reliability for each internal node.