Asian Leopard Cat Research Articles

A chromosome-scale fishing cat reference genome for the evaluation of potential germline risk variants.

The fishing cat, Prionailurus viverrinus, faces a population decline, increasing the importance of maintaining healthy zoo populations. Unfortunately, zoo-managed individuals currently face a high prevalence of transitional cell carcinoma (TCC), a form of bladder cancer. To investigate the genetics of inherited diseases among captive fishing cats, we present a chromosome-scale assembly, generate the pedigree of the zoo-managed population, reaffirm the close genetic relationship with the Asian leopard cat (Prionailurus bengalensis), and identify 7.4million single nucleotide variants (SNVs) and 23,432 structural variants (SVs) from whole genome sequencing (WGS) data of healthy and TCC cats. Only BRCA2 was found to have a high recurrent number of missense mutations in fishing cats diagnosed with TCC when compared to inherited human cancer risk variants. These new fishing cat genomic resources will aid conservation efforts to improve their genetic fitness and enhance the comparative study of feline genomes.

Ancestry dynamics and trait selection in a designer cat breed

The Bengal cat breed was developed from intercrosses between the Asian leopard cat, Prionailurus bengalensis, and the domestic cat, Felis catus, with a last common ancestor approximately 6 million years ago. Predicted to derive ∼94% of their genome from domestic cats, regions of the leopard cat genome are thought to account for the unique pelage traits and ornate color patterns of the Bengal breed, which are similar to those of ocelots and jaguars. We explore ancestry distribution and selection signatures in the Bengal breed by using reduced representation and whole-genome sequencing from 947 cats. The mean proportion of leopard cat DNA in the Bengal breed is 3.48%, lower than predicted from breed history, and is broadly distributed, covering 93% of the Bengal genome. Overall, leopard cat introgressions do not show strong signatures of selection across the Bengal breed. However, two popular color traits in Bengal cats, charcoal and pheomelanin intensity, are explained by selection of leopard cat genes whose expression is reduced in a domestic cat background, consistent with genetic incompatibility resulting from hybridization. We characterize several selective sweeps in the Bengal genome that harbor candidate genes for pelage and color pattern and that are associated with domestic, rather than leopard, cat haplotypes. We identify the molecular and phenotypic basis of one selective sweep as reduced expression of the Fgfr2 gene, which underlies glitter, a trait desired by breeders that affects hair texture and light reflectivity.

Identification of a novel linear B-cell epitope in porcine deltacoronavirus nucleocapsid protein.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that caused diarrhea and/or vomiting in neonatal piglets worldwide. Coronaviruses nucleocapsid (N) protein is the most conserved structural protein for viral replication and possesses good antigenicity. In this study, three monoclonal antibodies (mAbs), 3B4, 4D3, and 4E3 identified as subclass IgG2aκ were prepared using the lymphocytic hybridoma technology against PDCoV N protein. Furthermore, the B-cell epitope recognized by mAb 4D3 was mapped by dozens of overlapping truncated recombinant proteins based on the western blotting. The polypeptide 28QFRGNGVPLNSAIKPVE44 (EP-4D3) in the N-terminal of PDCoV N protein was identified as the minimal linear epitope for binding mAb 4D3. And the EP-4D3 epitope's amino acid sequence homology study revealed that PDCoV strains are substantially conserved, with the exception of the Alanine43 substitution Valine43 in the China lineage, the Early China lineage, and the Thailand, Vietnam, and Laos lineage. The epitope sequences shared high similarity (94.1%) with porcine coronavirus HKU15-155 (PorCoV HKU15), Asian leopard cats coronavirus (ALCCoV), sparrow coronavirus HKU17 (SpCoV HKU17), and sparrow deltacoronavirus. In contrast, the epitope sequences shared a very low homology (11.8 to 29.4%) with other porcine CoVs (PEDV, TGEV, PRCV, SADS-CoV, PHEV). Overall, the study will enrich the biological function of PDCoV N protein and provide foundational data for further development of diagnostic applications. KEY POINTS: • Three monoclonal antibodies against PDCoV N protein were prepared. • Discovery of a novel B-cell liner epitope (28QFRGNGVPLNSAIKPVE44) of PDCoV N protein. • The epitope EP-4D3 was conserved among PDCoV strains.

Korean Leopard Cat (Prionailurus bengalensis) population with low genetic diversity is distinct from Southeast Asian populations

Effective conservation strategy for a species can be established based on population differentiation patterns. The results of population genetic analyses have been useful in the decision of ESUs and MUs to maintain evolutionary potential and the fitness of populations. To provide a basis for establishing a conservation unit for the leopard cats (Prionailurus bengalensis), we analyzed their mtDNA cytb gene sequences from the Korean populations and compared them with those in other Asian leopard cats' populations. The nucleotide diversity of the Korean population was lowest among the all Asian populations. Analyses of the haplotypes, phylogenetic tree, AMOVA, and migration rate showed that the Northeast Asian population including the Korean population is distinct from the Southeast Asian population, moreover, the divergence time between the Northeast and the Southeast Asian populations was sufficiently long for differentiation to occur; therefore, it would be reasonable to manage the Northeast Asian population as an independent conservation unit and introducing species to Korea from Southeast Asia should be avoided. In contrast, the differentiation within Northeast Asia was not clear. Additional studies based on a greater number of animals in Northeast Asia around Korea will be needed to secure sources of genetic diversity to rescue the Korean leopard cat.

Characterization of Deltacoronavirus in Black-Headed Gulls (Chroicocephalus ridibundus) in South China Indicating Frequent Interspecies Transmission of the Virus in Birds.

Deltacoronavirus (DCoV) is a genus of coronavirus (CoV) commonly found in avian and swine, but some DCoVs are capable of infecting humans, which causes the concern about interspecies transmission of DCoVs. Thus, monitoring the existence of DCoVs in animals near communities is of great importance for epidemic prevention. Black-headed gulls (Chroicocephalus ridibundus) are common migratory birds inhabiting in most urban and rural wetlands of Yunnan Province, China, which is a typical habitat for black-headed gulls to overwinter. Whether Yunnan black-headed gulls carry CoV has never been determined. In this study, we identified three strains of DCoVs in fecal samples of Yunnan black-headed gulls by reverse-transcriptional PCR and sequenced their whole genomes. Genomic analysis revealed that these three strains shared genomic identity of more than 99%, thus named DCoV HNU4-1, HNU4-2, and HNU4-3; their NSP12 showed high similarity of amino acid sequence to the homologs of falcon coronavirus UAE-HKU27 (HKU27), houbara coronavirus UAE-HKU28 (HKU28), and pigeon coronavirus UAE-HKU29 (HKU29). Since both HKU28 and HKU29 were found in Dubai, there might be cross-border transmission of these avian DCoVs through specific routes. Further coevolutionary analysis supported this speculation that HNU4 (or its ancestors) in black-headed gulls originated from HKU28 (or its homologous strain) in houbara, which was interspecies transmission between two different avian orders. In addition, interspecies transmission of DCoV, from houbara to falcon, pigeon and white-eye, from sparrow to common-magpie, and quail and mammal including porcine and Asian leopard cat, from munia to magpie-robin, was predicted. This is the first report of black-headed gull DCoV in Asia which was highly homolog to other avian DCoVs, and the very “active” host-switching events in DCoV were predicted, which provides important reference for the study of spread and transmission of DCoVs.

DISTRIBUSI DAN PENGGUNAAN HABITAT EMPAT SPESIES FELIDAE DI TAMAN NASIONAL BUKIT BARISAN SELATAN

The polulation of Cat Family (Felidae) in Southeast Asia has declined in nature owing to its lost habitat, fragmentation one and massive hunting. Meanwhile, the research of Asiatic Golden Cats, Sunda Clouded Leopards, Marbled Cats, and Asian Leopard Cats is not much conducted. This research aimed to find out their distribution and habitat use in Southern Bukit Barisan National Park (BBSNP). The data intake employed Camera Trap, then analyzed by occupancy model with single season and single species applying R-software. It was found that Asiatic Golden Cat Occupancy Model yielded 0.75 in Intensive Protection Zone or Zona Perlindungan Intensive (IPZ) and 0.4 occupancy value in northen parts of Sourthern Bukit Barisan National Park (BBSNP). Furthermore, its Sunda Clouded Leopards resulted in 0.50 in IPZ and 0.42 occupancy value in northen parts of BBSNP. Its Asian Leopard Cats was 0.16 occupancy value in IPZ and unexecuted occupancy model in northen parts of BBSNP due to extremly low detection.

Wildlife Crossings at Felda Aring - Tasik Kenyir Road, Malaysia

The Felda Aring - Tasik Kenyir Road was identified as one of the most threatening roads to wildlife in Malaysia. The present study was conducted to assess the road crossing activities involving the medium- to large-mammal species due to the problem stated. The objectives of this study were to (1) predict the suitability of the road and its surroundings as the roaming areas for the Asian elephant (Elephas maximus, n = 104) and Malayan tapir (Tapirus indicus, n = 66), (2) identify the mammalian species inhabiting the forest beside the road, (3) compare the forest’s common species [photographic capture rate index (PCRI) > 10/ detection probability (P) ≥ 0.05] with the ones utilising the road crossing structures; the viaducts and the bridges, and (4) determine the most impacted species from traffic collisions. The road and its surroundings were classified as moderately suitable to the elephant and tapir (suitability values = 0.4 - 0.8). A total of 16 mammal species were recorded at the forest edges, in which the wild pig (Sus scrofa) (PCRI = 118.96, P = 0.3719 ± 0.027), barking deer (Muntiacus muntjak) (PCRI = 68.89, P = 0.2219 ± 0.0232), sun bear (Helarctos malayanus) (PCRI = 11.13, P = 0.0507 ± 0.0159), tapir (PCRI = 11.13, P = 0.0469 ± 0.0118), elephant (PCRI = 10.7, P = 0.0787 ± 0.0195) and Malayan porcupine (Hystrix brachyura) (PCRI = 10.7, P = 0.103 ± 0.0252) were the common species utilising the crossing structures. In contrast, the Asian palm civet (Paradoxurus hermaphroditus) and leopard cat (Prionailurus bengalensis) were the most frequently hit species on the road [F(7,398) = 28.53, p < 0.0005]. The present study found that large-mammal species were utilising the crossing structures at a higher frequency, whereas more medium-mammal species were involved in traffic collisions.

Genetic Characterization of Feline Parvovirus Isolate Fe-P2 in Korean Cat and Serological Evidence on Its Infection in Wild Leopard Cat and Asian Badger.

Feline parvovirus (FPV) is a small, non-enveloped, single-stranded DNA virus that infects cats. We recently isolated a feline parvovirus Fe–P2 strain from a dead stray cat in Iksan, 2017. Its partial genomic sequence (4,643 bases) was obtained, and phylogenetic analysis based on the VP2 nucleotide sequence showed that the FPV Fe-P2 strain was closely related to the FPV isolate Gigucheon in cat, 2017 (MN400978). In addition, we performed a serum neutralization (SN) test with the FPV isolates in various mammalian sera. These were from raccoon dog, water deer, Eurasian otter, Korean hare, leopard cat, and Asian badger, which were kindly provided by Chungnam Wild Animal Rescue Center. Notably, serological evidence of its infection was found in Asian badger, Meles leucurus (2/2) and leopard cat, Prionailurus bengalensis (5/8) through SN tests, whereas there was no evidence in raccoon dog, water deer, Eurasian otter, and Korean hare based on the collected sera in this study. These findings might provide partial evidence for the possible circulation of FPV or its related viruses among wild leopard cat and Asian badger in Korea. There should be additional study to confirm this through direct detection of FPVs in the related animal samples.

Ultracontinuous Single Haplotype Genome Assemblies for the Domestic Cat (Felis catus) and Asian Leopard Cat (Prionailurus bengalensis).

In addition to including one of the most popular companion animals, species from the cat family Felidae serve as a powerful system for genetic analysis of inherited and infectious disease, as well as for the study of phenotypic evolution and speciation. Previous diploid-based genome assemblies for the domestic cat have served as the primary reference for genomic studies within the cat family. However, these versions suffered from poor resolution of complex and highly repetitive regions, with substantial amounts of unplaced sequence that is polymorphic or copy number variable. We sequenced the genome of a female F1 Bengal hybrid cat, the offspring of a domestic cat (Felis catus) x Asian leopard cat (Prionailurus bengalensis) cross, with PacBio long sequence reads and used Illumina sequence reads from the parents to phase >99.9% of the reads into the 2 species’ haplotypes. De novo assembly of the phased reads produced highly continuous haploid genome assemblies for the domestic cat and Asian leopard cat, with contig N50 statistics exceeding 83 Mb for both genomes. Whole-genome alignments reveal the Felis and Prionailurus genomes are colinear, and the cytogenetic differences between the homologous F1 and E4 chromosomes represent a case of centromere repositioning in the absence of a chromosomal inversion. Both assemblies offer significant improvements over the previous domestic cat reference genome, with a 100% increase in contiguity and the capture of the vast majority of chromosome arms in 1 or 2 large contigs. We further demonstrated that comparably accurate F1 haplotype phasing can be achieved with members of the same species when one or both parents of the trio are not available. These novel genome resources will empower studies of feline precision medicine, adaptation, and speciation.

Behavior and health issues in Bengal cats as perceived by their owners: A descriptive study

The Bengal cat is a recently established hybrid cat breed that was created by crossbreeding the domestic shorthair cat (Felis silvestris catus) and the wild Asian leopard cat (Prionailurus bengalensis). In spite of its popularity, research on behavior and health issues in this breed remains limited. A questionnaire was sent to Bengal cat owners in Flanders and Wallonia (Belgium), and the Netherlands to document the suitability of the breed as a pet from the point of view of the owners by exploring the presence of behavioral and health issues that may provoke the cat’s relinquishment. It included questions about their decision to own a Bengal cat, the cat demographics and living environment, potential undesirable behaviors, and health issues. The owners of 60.5% (n = 155) of cats chose this breed due to a combination of looks and character. For most cats (99.2% n = 254), the respondents looked for information about the breed before acquiring a cat. Breed-typical health conditions, as described by International Cat Care, were reported in 9.9 % (n = 24) of cats, with being overweight as the most common one. The most frequent behaviors were climbing (89.5%, n = 229), vocalizing (88.7%, n = 227), playing with water (79.7%, n = 204), and hunting (78.9%, n = 202). These were, however, rarely considered problematic by the owners. The most frequent behaviors often classified as problematic by the owners were destructive behaviors (33.2%, n = 85), followed by pica (16.4%, n = 42), aggression toward animals (16%, n = 41), and urination outside the litter tray (13.3%, n = 34). There were no significant differences between cats from early and later generations nor between cats with and without outdoor access. The fact that most owners looked for information about the breed before acquiring the cat and that the most frequently displayed behaviors were not considered problematic by their owners could translate into a lower incidence of relinquishments. Nevertheless, some of those behaviors may still be indicative of welfare issues, independently of the owner’s perception. Further and more in-depth research is needed to understand the potential issues of keeping Bengal cats as pets.

Diet, prey selection, and activity of Asian golden cats and leopard cats in northern Laos

AbstractThe Asian golden cat (Catopuma temminckii) occurs in small, declining, and highly fragmented populations throughout Southeast Asia, whereas the smaller leopard cat (Prionailurus bengalensis) is common and widespread. In contrast to leopard cats, little is known about the ecology of Asian golden cats, and resource partitioning between these species has not been studied. We used DNA-confirmed scats, camera-trap data, and prey surveys, to determine the diet, prey selection, and activity, of Asian golden cats and leopard cats in a protected area in northern Laos. The two felids had different diets: Asian golden cats consumed mostly ungulates (35% biomass consumed), murid rodents (23%), and carnivores (15%), whereas leopard cats consumed mostly murid rodents (79%). Asian golden cats were not random in their consumption of ungulates, because muntjac (Muntiacus spp.) were selectively consumed over larger ungulates, indicating muntjac were preyed upon rather than scavenged. Dietary overlap between the two felid species was moderate (R0 = 0.60), and the dietary niche breadth of Asian golden cats (B = 8.44) was nearly twice as high as that of leopard cats (4.54). The mean (± SD) scat diameter was greater for Asian golden cats (2.1 ± 0.3 cm) than leopard cats (1.8 ± 0.2 cm), although diameters of leopard cat scats were considerably larger than previously assumed for this species. The felid species differed in their activity patterns, because Asian golden cats were diurnal, whereas leopard cats were nocturnal, although they did not differ in their use of elevation, suggesting there was no habitat segregation. Overall, leopard cats appeared to coexist with Asian golden cats, a potential predator and competitor, by exhibiting dietary and temporal partitioning. Our results showed that muntjac were important prey of Asian golden cats, suggesting the management of muntjac might be important for conserving populations of Asian golden cats.

Identification of a Novel Linear B-Cell Epitope on the Nucleocapsid Protein of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV), first identified in 2012, is a swine enteropathogen now found in many countries. The nucleocapsid (N) protein, a core component of PDCoV, is essential for virus replication and is a significant candidate in the development of diagnostics for PDCoV. In this study, monoclonal antibodies (mAbs) were generated and tested for reactivity with three truncations of the full protein (N1, N2, N3) that contained partial overlaps; of the five monoclonals chosen tested, each reacted with only the N3 truncation. The antibody designated 4E88 had highest binding affinity with the N protein and was chosen for in-depth examination. The 4E88 epitope was located to amino acids 308-AKPKQQKKPKK-318 by testing the 4E88 monoclonal for reactivity with a series of N3 truncations, then the minimal epitope, 309-KPKQQKKPK-317 (designated EP-4E88), was pinpointed by testing the 4E88 monoclonal for reactivity with a series of synthetic peptides of this region. Homology analysis showed that the EP-4E88 sequence is highly conserved among PDCoV strains, and also shares high similarity with sparrow coronavirus (HKU17), Asian leopard cat coronavirus (ALCCoV), quail coronavirus (UAE-HKU30), and sparrow deltacoronavirus (SpDCoV). Of note, the PDCoV EP-4E88 sequence shared very low similarity (<22.2%) with other porcine coronaviruses (PEDV, TGEV, PRCV, SADS-CoV, PHEV), demonstrating that it is an epitope that can be used for distinguishing PDCoV and other porcine coronavirus. 3D structural analysis revealed that amino acids of EP-4E88 were in close proximity and may be exposed on the surface of the N protein.

Origin, evolution, and virulence of porcine deltacoronaviruses in the United States.

ABSTRACTA novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.

Who's behind that mask and cape? The Asian leopard cat's Agouti (ASIP) allele likely affects coat colour phenotype in the Bengal cat breed.

Coat colours and patterns are highly variable in cats and are determined mainly by several genes with Mendelian inheritance. A 2-bp deletion in agouti signalling protein (ASIP) is associated with melanism in domestic cats. Bengal cats are hybrids between domestic cats and Asian leopard cats (Prionailurus bengalensis), and the charcoal coat colouration/pattern in Bengals presents as a possible incomplete melanism. The complete coding region of ASIP was directly sequenced in Asian leopard, domestic and Bengal cats. Twenty-seven variants were identified between domestic and leopard cats and were investigated in Bengals and Savannahs, a hybrid with servals (Leptailurus serval). The leopard cat ASIP haplotype was distinguished from domestic cat by four synonymous and four non-synonymous exonic SNPs, as well as 19 intronic variants, including a 42-bp deletion in intron 4. Fifty-six of 64 reported charcoal cats were compound heterozygotes at ASIP, with leopard cat agouti (APbe) and domestic cat non-agouti (a) haplotypes. Twenty-four Bengals had an additional unique haplotype (A2) for exon 2 that was not identified in leopard cats, servals or jungle cats (Felis chaus). The compound heterozygote state suggests the leopard cat allele, in combination with the recessive non-agouti allele, influences Bengal markings, producing a darker, yet not completely melanistic coat. This is the first validation of a leopard cat allele segregating in the Bengal breed and likely affecting their overall pelage phenotype. Genetic testing services need to be aware of the possible segregation of wild felid alleles in all assays performed on hybrid cats.

Cell Cycle Synchronization of Skin Fibroblast Cells in Four Species of Family Felidae

This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 μm). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 μm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.

An autosomal genetic linkage map of the domestic cat, Felis silvestris catus

We report on the completion of an autosomal genetic linkage (GL) map of the domestic cat ( Felis silvestris catus). Unlike two previous linkage maps of the cat constructed with a hybrid pedigree between the domestic cat and the Asian leopard cat, this map was generated entirely with domestic cats, using a large multi-generational pedigree ( n = 256) maintained by the Nestlé Purina PetCare Company. Four hundred eighty-three simple tandem repeat (STR) loci have been assigned to linkage groups on the cat's 18 autosomes. A single linkage group spans each autosome. The length of the cat map, estimated at 4370 cM, is long relative to most reported mammalian maps. A high degree of concordance in marker order was observed between the third-generation map and the 1.5 Mb-resolution radiation hybrid (RH) map of the cat. Using the cat 1.9 × whole-genome sequence, we identified map coordinates for 85% of the loci in the cat assembly, with high concordance observed in marker order between the linkage map and the cat sequence assembly. The present version represents a marked improvement over previous cat linkage maps as it ( i) nearly doubles the number of markers that were present in the second-generation linkage map in the cat, ( ii) provides a linkage map generated in a domestic cat pedigree which will more accurately reflect recombination distances than previous maps generated in a hybrid pedigree, and ( iii) provides single linkage groups spanning each autosome. Marker order was largely consistent between this and the previous maps, though the use of a hybrid pedigree in the earlier versions appears to have contributed to some suppression of recombination. The improved linkage map will provide an added resource for the mapping of phenotypic variation in the domestic cat and the use of this species as a model system for biological research.

Comparative Analysis of Complete Genome Sequences of Three Avian Coronaviruses Reveals a Novel Group 3c Coronavirus

In this territory-wide molecular epidemiology study of coronaviruses (CoVs) in Hong Kong involving 1,541 dead wild birds, three novel CoVs were identified in three different bird families (bulbul CoV HKU11 [BuCoV HKU11], thrush CoV HKU12 [ThCoV HKU12], and munia CoV HKU13 [MuCoV HKU13]). Four complete genomes of the three novel CoVs were sequenced. Their genomes (26,396 to 26,552 bases) represent the smallest known CoV genomes. In phylogenetic trees constructed using chymotrypsin-like protease (3CL(pro)), RNA-dependent RNA polymerase (Pol), helicase, spike, and nucleocapsid proteins, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 formed a cluster distantly related to infectious bronchitis virus and turkey CoV (group 3a CoVs). For helicase, spike, and nucleocapsid, they were also clustered with a CoV recently discovered in Asian leopard cats, for which the complete genome sequence was not available. The 3CL(pro), Pol, helicase, and nucleocapsid of the three CoVs possessed higher amino acid identities to those of group 3a CoVs than to those of group 1 and group 2 CoVs. Unique genomic features distinguishing them from other group 3 CoVs include a distinct transcription regulatory sequence and coding potential for small open reading frames. Based on these results, we propose a novel CoV subgroup, group 3c, to describe this distinct subgroup of CoVs under the group 3 CoVs. Avian CoVs are genetically more diverse than previously thought and may be closely related to some newly identified mammalian CoVs. Further studies would be important to delineate whether the Asian leopard cat CoV was a result of interspecies jumping from birds, a situation analogous to that of bat and civet severe acute respiratory syndrome CoVs.

Molecular Diversity and Phylogeography of the Asian Leopard Cat, Felis bengalensis, Inferred from Mitochondrial and Y-Chromosomal DNA Sequences

To investigate genetic diversity and phylogeography of the Asian leopard cat (Felis bengalensis), mitochondrial DNA (mtDNA) sequences were determined for 39 individuals from various areas. Sequences combining the complete cytochrome b gene (1,140 bp) with the partial control region (646-810 bp) were classified into 24 haplotypes: 21 types from 21 animals, one from eight animals from Tsushima Islands, one from eight animals from Iriomote Island, and one from two animals from Southeast Asia. Phylogenetic trees of the 24 haplotypes clearly showed three clades: a Northern Lineage and Southern Lineages 1 and 2. The Northern Lineage consisted of animals from Tsushima Islands, the Korean Peninsula, the continental Far East, Taiwan, and Iriomote Island. Within the Northern Lineage, genetic contacts could have occurred between geographically neighboring populations before isolation by straits. Southern Lineage 1, comprising Southeast Asian animals, showed higher genetic diversity. Southern Lineage 2 had large genetic distances from other lineages. Within the control region, the Asian leopard cats shared two to four repetitive motifs, and the number of motifs and their constitution were highly variable among individuals. The motifs were polymorphic even within individuals and could be classified into 31 types. Finally, males of mtDNA Southern Lineage 1 had either of two types of the Y-chromosomal gene ZFY, whereas all males of Northern Lineage shared only one type. Our results indicate that the diversity of southern populations is higher and that genetic differentiation among northern local populations reflects past geographical isolation.

Development of Y Chromosome Intraspecific Polymorphic Markers in the Felidae

Y chromosome haplotyping based on microsatellites and single nucleotide polymorphisms (SNPs) has proved to be a powerful tool for population genetic studies of humans. However, the promise of the approach is hampered in the majority of nonhuman mammals by the lack of Y-specific polymorphic markers. We were able to identify new male-specific polymorphisms in the domestic cat Felis catus and 6 additional Felidae species with a combination of molecular genetic and cytogenetic approaches including 1) identifying domestic cat male-specific microsatellites from markers generated from a male cat microsatellite-enriched genomic library, a flow-sorted Y cosmid library, or a Y-specific cat bacteria artificial chromosome (BAC) clone, (2) constructing microsatellite-enriched libraries from flow-sorted Y chromosomes isolated directly from focal wildcat species, and (3) screening Y chromosome conserved anchored tagged sequences primers in Felidae species. Forty-one male-specific microsatellites were identified, but only 6 were single-copy loci, consistent with the repetitive nature of the Y chromosome. Nucleotide diversity (pi) of Y-linked intron sequences (2.1 kbp) was in the range of 0 (tiger) to 9.95 x 10(-4) (marbled cat), and the number of SNPs ranged from none in the tiger to 7 in the Asian leopard cat. The Y haplotyping system described here, consisting of 4 introns (SMCY3, SMCY7, UTY11, and DBY7) and 1 polymorphic microsatellite (SMCY-STR), represents the first available markers for tracking intraspecific male lineage polymorphisms in Felidae species and promises to provide significant insights to evolutionary and population genetic studies of the species.

Detection of a Novel and Highly Divergent Coronavirus from Asian Leopard Cats and Chinese Ferret Badgers in Southern China

Since an outbreak of severe acute respiratory syndrome (SARS) was averted in 2004, many novel coronaviruses have been recognized from different species, including humans. Bats have provided the most diverse assemblages of coronaviruses, suggesting that they may be the natural reservoir. Continued virological surveillance has proven to be the best way to avert this infectious disease at the source. Here we provide the first description of a previously unidentified coronavirus lineage detected from wild Asian leopard cats (Prionailurus bengalensis) and Chinese ferret badgers (Melogale moschata) during virological surveillance in southern China. Partial genome analysis revealed a typical coronavirus genome but with a unique putative accessory gene organization. Phylogenetic analyses revealed that the envelope, membrane, and nucleoprotein structural proteins and the two conserved replicase domains, putative RNA-dependent RNA polymerase and RNA helicase, of these novel coronaviruses were most closely related to those of group 3 coronaviruses identified from birds, while the spike protein gene was most closely related to that of group 1 coronaviruses from mammals. However, these viruses always fell into an outgroup phylogenetic relationship with respect to other coronaviruses and had low amino acid similarity to all known coronavirus groups, indicating that they diverged early in the evolutionary history of coronaviruses. These results suggest that these viruses may represent a previously unrecognized evolutionary pathway, or possibly an unidentified coronavirus group. This study demonstrates the importance of systematic virological surveillance in market animals for understanding the evolution and emergence of viruses with infectious potential.