Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.

Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins.

Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. However, little is known about its pathogenicity in pangolins. Using CT scans we show that SARSr-CoV-2 positive Malayan pangolins are characterized by bilateral ground-glass opacities in lungs in a similar manner to COVID-19 patients. Histological examination and blood gas tests are indicative of dyspnea. SARSr-CoV-2 infected multiple organs in pangolins, with the lungs the major target, and histological expression data revealed that ACE2 and TMPRSS2 were co-expressed with viral RNA. Transcriptome analysis indicated that virus-positive pangolins were likely to have inadequate interferon responses, with relative greater cytokine and chemokine activity in the lung and spleen. Notably, both viral RNA and viral proteins were detected in three pangolin fetuses, providing initial evidence for vertical virus transmission. In sum, our study outlines the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans.

Molecular characterization of a novel Spiruromorpha species in wild Chinese pangolin by mitogenome sequence analysis.

Pangolins are susceptible to a variety of gastrointestinal nematodes due to their burrowing lifestyle and feeding habits, and few parasitic nematodes have been reported. Here, a Chinese pangolin with old wounds on its leg and tail was rescued from the Heyuan City, Guangdong Province. The cox1 and SSU rRNA of the worms from the intestine of the Chinese pangolin had the highest sequence identity of 89.58% and 97.95% to the species in the infraorder Spiruromorpha. The complete mitogenome of the worm was further assembled by next-generation sequencing, with a size of 13,708 bp and a GC content of 25.6%. The worm mitogenome had the highest sequence identity of 78.56% to that of Spirocerca lupi, sharing the same gene arrangement with S. lupi and some species in other families under Spiruromorpha. However, the mitogenome between the worm and S. lupi showed differences in codon usage of PCGs, sequences of NCR, and tRNA secondary structures. Phylogenetic analysis showed that the worm mitogenome was clustered with S. lupi in the family Thelaziidae to form a separate branch. However, it is still difficult to identify the worm in the family Thelaziidae because the species in the family Thelaziidae are confused, specifically S. lupi and Thelazia callipaeda in the family Thelaziidae were separated and grouped with species from other families. Thus, the parasitic nematode from the Chinese pangolin may be a novel species in Spiruromorpha and closely related to S. lupi. This study enriches the data on gastrointestinal nematodes in the Chinese pangolin.

Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation.

BACKGROUND: Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. RESULTS: In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. CONCLUSIONS: The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

Pathogenicity and transmissibility of a novel respirovirus isolated from a Malayan pangolin.

The identification of SARS-CoV-2-like viruses in Malayan pangolins (Manis javanica) has focused attention on these endangered animals and the viruses they carry. We successfully isolated a novel respirovirus from the lungs of a dead Malayan pangolin. Similar to murine respirovirus, the full-length genome of this novel virus was 15 384 nucleotides comprising six genes in the order 3'-(leader)-NP-P-M-F-HN-l-(trailer)-5'. Phylogenetic analysis revealed that this virus belongs to the genus Respirovirus and is most closely related to murine respirovirus. Notably, animal infection experiments indicated that the pangolin virus is highly pathogenic and transmissible in mice, with inoculated mice having variable clinical symptoms and a fatality rate of 70.37 %. The virus was found to replicate in most tissues with the exception of muscle and heart. Contact transmission of the virus was 100 % efficient, although the mice in the contact group displayed milder symptoms, with the virus mainly being detected in the trachea and lungs. The isolation of a novel respirovirus from the Malayan pangolin provides new insight into the evolution and distribution of this important group of viruses and again demonstrates the potential infectious disease threats faced by endangered pangolins.

Expression and characterization of albumin fusion protein canine IFNγ-CSA in baculovirus-insect cell expression system.

In this study, canine IFNγ was fused by a flexible linker with canine serum albumin to construct the fusion protein IFNγ-CSA for the purpose to design a long-acting canine IFNγ. The fusion protein was successfully expressed in baculovirus-infected Sf9 insect cells and was purified by salting-out and ion exchange chromatography. The IFNγ-CSA fusion possessed potent anti-viral assay against vesicular stomatitis virus in cultured cells. IFNγ-CSA was also stable at 37 °C up to 72 h compared with 8 h for IFNγ alone. In vivo pharmacokinetics demonstrated a significantly longer half-life for IFNγ-CSA (15.42 h) than for canine reIFNγ (1.51 h) in KM mice. These results indicate that IFNγ-CSA expression in the baculovirus system was successful and provide a promising long-acting cytokine for veterinary clinical applications.

Genetic diversity and evolutionary characteristics of type 2 porcine reproductive and respiratory syndrome virus in southeastern China from 2009 to 2014.

The objective of this study was to assess the genetic diversity of porcine reproductive and respiratory syndrome virus circulating in Fujian province (southeastern China). Based on 53 ORF5 nucleotide sequences collected from nine sites, both highly pathogenic (sublineage 8.7) and lineage 1 strains were circulating in Fujian in 2009-2014 along with lineages 3 and 5.1. Notably, the lineage 1 strains were closely related to the NADC30 strain circulating in North America and were the predominant strains in 2014. In addition, we found that nonstructural protein 2 (NSP2) was the most variable nonstructural protein in Fujian isolates, with a 36-amino-acid (aa) insertion and seven different deletions detected in the 53 sequences examined. Similarly, analysis of GP5 amino acid sequences showed that the isolates were highly variable in primary neutralizing epitopes. Interesting, FJ3.2 and FJ7-2 strains have the mutation N44K, but they exhibited high replication and high titers in MARC-145 and PAM cells. The complete genome sequences determined for 12 type 2 isolates were 82.1-99.3% identical and were 15,016-15,407 nucleotides (nt), in length excluding the poly(A) tail. The strains also shared 88.2-99.4% identity with strain VR2332 (the prototype North American strain), 83.4-99.2% identity with strain JXA1 (the prototype high-pathogenicity Chinese strain), 88.2-97.1% identity with strain CH-1a (the prototype classical Chinese strain), and 82.9-97.1% identity with strain NADC30 (the prototype NADC30-like strain). Strikingly, phylogenetic and molecular evolutionary analyses indicated that strain FJW05 is a spontaneous recombinant between a circulating lineage 1 virus and the vaccine strain JXA1-R, which is derived from the highly pathogenic strain JXA-1. Collectively, the data highlight the epidemiology of porcine reproductive and respiratory syndrome in Fujian and may aid in selecting a suitable vaccine for use on pig farms.

First complete genome sequence of parainfluenza virus 5 isolated from lesser panda.

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.

Isolation, genome analysis and comparison of a novel parainfluenza virus 5 from a Siberian tiger (Panthera tigris).

Paramyxoviruses are important pathogens affecting various animals, including mammals and humans. Parainfluenza virus 5 (PIV5)-a member of the family Paramyxoviridae-is a major threat to the health of mammals and humans. However, studies on terrestrial wild animals infected with PIV5 are scanty. In this study, we utilized reverse transcription PCR to detect PIV5 infection in the visceral organ tissues of a Siberian tiger (Panthera tigris ssp. altaica) with vomiting, diarrhea, and dyspnea before its death. A novel PIV5 (named SR strain) with a slowly progressive cytopathic effect was isolated in Vero cells and validated using a transmission electron microscope. Full-length sequencing and analysis revealed that the whole genome of the PIV5 SR strain contained 15,246 nucleotides (nt) and seven non-overlapping genes (3'-N-V/P-M-F-SH-HN-L-5') encoding eight proteins. Phylogenetic analysis of three PIV5 strains identified in the same zoo confirmed that PIV5 strains SR and ZJQ-221 shared the closest genetic relationship as they were clustered in the same branch, while the recently found Siberian tiger strain SZ2 kept a certain distance and formed a relatively unique branch. Furthermore, mutations of nt and amino acids (aa) between strains ZJQ-221, SR, and SZ2 were identified. In summary, we report the identification and genomic characterization of a novel PIV5 strain SR isolated in a Siberian tiger, which may help future research on interspecific transmission mechanisms.

A pangolin-origin SARS-CoV-2-related coronavirus: infectivity, pathogenicity, and cross-protection by preexisting immunity.

Virus spillover remains a major challenge to public health. A panel of SARS-CoV-2-related coronaviruses have been identified in pangolins, while the infectivity and pathogenicity of these pangolin-origin coronaviruses (pCoV) in humans remain largely unknown. Herein, we comprehensively characterized the infectivity and pathogenicity of a recent pCoV isolate (pCoV-GD01) in human cells and human tracheal epithelium organoids and established animal models in comparison with SARS-CoV-2. pCoV-GD01 showed similar infectivity to SARS-CoV-2 in human cells and organoids. Remarkably, intranasal inoculation of pCoV-GD01 caused severe lung pathological damage in hACE2 mice and could transmit among cocaged hamsters. Interestingly, in vitro neutralization assays and animal heterologous challenge experiments demonstrated that preexisting immunity induced by SARS-CoV-2 infection or vaccination was sufficient to provide at least partial cross-protection against pCoV-GD01 challenge. Our results provide direct evidence supporting pCoV-GD01 as a potential human pathogen and highlight the potential spillover risk.

Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Heteroplasmic mitochondrial genomes of a Raillietina tapeworm in wild Pangolin.

BACKGROUND: Raillietina species belong to the family Davaineidae, which parasitizes in a wide variety of mammals and birds, causing stunted growth, lethargy, emaciation, and digestive tract obstruction. However, only a limited number of Raillietina species have been identified in wild animals. METHODS: We analyzed and annotated the complete mitochondrial (mt) genome of a worm from the intestine of a wild pangolin using Illumina sequencing of whole genomic DNA. RESULTS: These findings showed the presence of two mtDNA sequences in Raillietina sp., designated as mt1 and mt2, with the lengths of 14,331 bp and 14,341 bp, respectively. Both the mts genomes of Raillietina sp. comprised 36 genes, containing 12 protein-coding genes (PCGs), 2 ribosomal RNAs, and 22 transfer RNAs. Gene arrangements of both mt genomes of Raillietina sp. were similar to those of most flatworms, except for taeniids, which shift positions between tRNAL1 and tRNAS2 genes. Twenty of 22 tRNA secondary structures of Raillietina sp. had a typical cloverleaf structure similar to Raillietina tetragona. Sequence differences between the mt1 and mt2 genomes were 4.4%, and this difference arises from the mtDNA heteroplasmic mutations. Moreover, heteroplasmic mtDNA mutations were detected in PCGs, tRNAs, rRNAs, NCRs, and intergenes, but the highest proportion of heteroplasmy of 79.0% was detected in PCGs, indicating the occurrence of mtDNA heteroplasmy in Raillietina sp. To our knowledge, this is the first report of mtDNA heteroplasmy in tapeworm parasites. Phylogenetic analyses of 18S rRNA, ITS2, and 12 PCG sequences demonstrated that the worm was clustered with other Raillietina species in the Davaneidae family. CONCLUSIONS: We found a novel Raillietina species in wild pangolin with the existence of mitochondrial DNA heteroplasmy. Thus, these findings provide insights into the heterogeneity of the mt genome in parasitic cestodes, and mt genome data contributes to the understanding of pangolin-parasitic cestodes in terms of their molecular biology, epidemiology, diagnosis, and taxonomy.

Molecular detection of a novel Ancylostoma sp. by whole mtDNA sequence from pangolin Manis javanica.

BACKGROUND: Ancylostoma species are hematophagous parasites that cause chronic hemorrhage in various animals and humans. Pangolins, also known as scaly anteaters, are mammals that live in soil environments where they are readily exposed to soil-borne parasitic nematodes. However, only a limited number of helminth species have been identified in this animal host so far. METHODS: Ancylostoma sp. was isolated from a wild pangolin, and the complete mitochondrial (mt) genome of Ancylostoma sp. was obtained by Illumina sequencing of total genomic DNA. RESULTS: The circular complete mt genome that was assembled had a total length of 13,757 bp and comprised 12 protein-coding genes (PCGs), 22 transfer ribosomal RNAs, two ribosomal RNAs (rRNAs), two non-coding regions and one AT-rich region, but lacked the gene coding for ATPase subunit 8 (atp8). The overall AT content of the mt genome of Ancylostoma sp. was 76%, which is similar to that of other nematodes. The PCGs used two start codons (ATT and TTG) and three stop codons (TAA, TAG, and T). The nucleotide identity of the 12 PCGs ranged from 83.1% to 89.7% and had the highest sequence identity with Ancylostoma caninum among species in the Ancylostomatidae family. Also, the pangolin-derived Ancylostoma sp. lacked repeat sequences in the non-coding regions and in the unique sequence of the short non-coding regions, which differentiated it from other Ancylostoma species. In addition, phylogenetic analyses of 18S rRNA and mtDNA sequences revealed that the Ancylostoma sp. was positioned in a separate branch in the subfamily Ancylostomatinae along with other Ancylostoma species. CONCLUSIONS: The Ancylostoma sp. isolated from a pangolin in this study was identified as a possible new Ancylostoma species. The identification of this Ancylostoma sp. from pangolin enriches our knowledge of the species in the Ancylostomatidae family and provides information that will lead to a better understanding of the taxonomy, diagnostics, and biology of hookworms.

A new canine distemper virus lineage identified from red pandas in China.

Canine distemper virus (CDV) is a highly contagious virus that causes multi-systemic, sub-clinical to fatal diseases in a wide range of carnivore species. Based on the sequences of the haemagglutinin (H) gene, CDV strains have been classified into 18 major genetic lineages. In this study, we characterized the genomes of CDV isolated from the lungs of two dead red pandas in China. Histopathological and immunohistochemical analyses revealed damage due to viral infection in these lungs. The two strains showed a deep genetic distance from the other 18 recognized lineages (>4.6% at nucleotide level and >5.0% at amino acid level). The maximum clade credibility tree of the H- gene sequences showed that they belonged to an independent clade and had diverged a relatively long time ago from the Asia-4 lineage (since 1884). These results suggest that the analyzed strains belong to a new CDV lineage, which we designate as Asia-6. Our finding indicates that CDV infections in wildlife in China are complex and are a threat to endangered carnivores.

Identification of Amblyomma javanense and detection of tick-borne Ehrlichia spp. in confiscated Malayan Pangolins.

Due to habitat destruction and illegal hunting and trade, the number of pangolins has been sharply reduced. To protect pangolins from extinction, relevant departments are combined and active action have been taken. A total of 21 confiscated Malayan pangolins were rescued in 2019, but died continuously for unknown reasons. This study aimed to investigate the reasons for the death of these pangolin and rescue them. 19 of the 21 confiscated pangolins had ticks on their body integument. A total of 303 ticks were collected and identified as Amblyomma javanense (A. javanense) according to their morphology and the sequences of 16S rRNA and internal transcribed spacer 2 (ITS2). There were multi-organ damages in the dead pangolins, especially congestion and hemorrhage in lung, heart and kidney and inflammation of which were observed using HE staining. Pathogens' nucleic acid detection showed ticks were only positive for Ehrlichia spp, with 56.7% positive rate of collected ticks (127/224), which was further confirmed in tissues from dead pangolins. Our findings confirm that ehrlichiosis caused by Ehrlichia spp. from A. javanense might accelerate the confiscated pangolin's death. More attention should be payed to tick-elimination work and the diagnoses and treatment of tick-borne diseases in the follow-up rescue operation.

Covariation of the Fecal Microbiome with Diet in Nonpasserine Birds.

Opportunistic feeding and multiple other environment factors can modulate the gut microbiome, and bias conclusions, when wild animals are used for studying the influence of phylogeny and diet on their gut microbiomes. Here, we controlled for these other confounding factors in our investigation of the magnitude of the effect of diet on the gut microbiome assemblies of nonpasserine birds. We collected fecal samples, at one point in time, from 35 species of birds in a single zoo as well as 6 species of domestic poultry from farms in Guangzhou city to minimize the influences from interfering factors. Specifically, we describe 16S rRNA amplicon data from 129 fecal samples obtained from 41 species of birds, with additional shotgun metagenomic sequencing data generated from 16 of these individuals. Our data show that diets containing native starch increase the abundance of Lactobacillus in the gut microbiome, while those containing plant-derived fiber mainly enrich the level of Clostridium Greater numbers of Fusobacteria and Proteobacteria are detected in carnivorous birds, while in birds fed a commercial corn-soybean basal diet, a stronger inner-connected microbial community containing Clostridia and Bacteroidia was enriched. Furthermore, the metagenome functions of the microbes (such as lipid metabolism and amino acid synthesis) were adapted to the different food types to achieve a beneficial state for the host. In conclusion, the covariation of diet and gut microbiome identified in our study demonstrates a modulation of the gut microbiome by dietary diversity and helps us better understand how birds live based on diet-microbiome-host interactions.IMPORTANCE Our study identified food source, rather than host phylogeny, as the main factor modulating the gut microbiome diversity of nonpasserine birds, after minimizing the effects of other complex interfering factors such as weather, season, and geography. Adaptive evolution of microbes to food types formed a dietary-microbiome-host interaction reciprocal state. The covariation of diet and gut microbiome, including the response of microbiota assembly to diet in structure and function, is important for health and nutrition in animals. Our findings help resolve the major modulators of gut microbiome diversity in nonpasserine birds, which had not previously been well studied. The diet-microbe interactions and cooccurrence patterns identified in our study may be of special interest for future health assessment and conservation in birds.

Recombination in JXA1-R vaccine and NADC30-like strain of porcine reproductive and respiratory syndrome viruses.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most devastating swine diseases worldwide, resulting in immense economic losses. PRRS virus (PRRSV) has undergone rapid evolution since its first recognition in 1990s. In the present study, a PRRSV strain named FJXS15 causing high morbidity and mortality was isolated from piglets and sows from a farm participating in vaccination in China. Phylogenetic and molecular evolutionary analyses revealed that FJXS15 was highly similar to the JXA1-R vaccine strain (a live attenuated virus vaccine strain derived from the highly pathogenic PRRSV JXA1) in the ORF1a (nt 901-)-ORF4 (-nt 419) coding regions, as well as to FJZ03 (lineage 1, NADC30-like) in the 5'-UTR, ORF5a-ORF7 coding regions, and 3'-UTR, suggestive of a natural recombination event. Recombination analyses showed that recombination events occurred in two inter-lineage recombination events between Lineages 1 and 8 based on based on classification system (Shi et al., 2010), and two recombination breakpoints at positions 1-1092 and 13771-15537 of the sequence alignment (with reference to the VR-2332 strain). Animal experiments demonstrated that FJXS15-infected animals had more severe histopathological lung lesions than did JXA1-R-infected and control groups. A 25% mortality rate was found in FJXS15-infected piglets, which was similar to that found with other Chinese HP-PRRSV strains. Thus, the recombinant virus is a highly virulent PRRSV. Moreover, this report provides evidence for inter-subgenotypic recombination between the JXA1-R vaccine virus and a circulating Lineage 1 virus.

Outbreak and genotyping of canine distemper virus in captive Siberian tigers and red pandas.

In this study, four canine distemper virus (CDV) strains were isolated from captive Siberian tigers (Panthera tigris altaica) and red pandas (Ailurus fulgens) during two separate CDV outbreaks in a zoo in Guangdong province, China. Sequence alignment and phylogenetic analyses based on the full-length hemagglutinin (H) and fusion (F) genes showed that they were closely identical to genotype Asia-1. Prior to confirmation of CDV in Siberian tigers, to control spread of the disease, a live attenuated combination CDV vaccine was used among almost all carnivore animals except for red pandas in which another recombinant combination CDV vaccine was used. However, about two months later, CDV re-emerged and caused the death among red pandas. Based on the vaccination records, the live combination vaccine could be considered an ideal weapon against CDV in zoo carnivore animals. Although the recombinant combination CDV vaccine was safe for red pandas, its protection effectiveness remains to be further investigated. Moreover, according to the outbreak interval time and sequence characterization, we suspected that stray cats circulating in the zoo were the intermediate host, which contributed to CDV spread from stray dogs to zoo animals. This study revealed the importance of vaccination and biosecurity for zoo animals.