A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

High-Quality Genomes of Pangolins: Insights into the Molecular Basis of Scale Formation and Adaption to Myrmecophagous Diet.

Pangolins are one of nature's most fascinating species being scales covered and myrmecophagous diet, yet relatively little is known about the molecular basis. Here, we combine the multi-omics, evolution, and fundamental proteins feature analysis of both Chinese and Malayan pangolins, highlighting the molecular mechanism of both myrmecophagous diet and scale formation, representing a fascinating evolutionary strategy to occupy the unique ecological niches. In contrast to conserved organization of epidermal differentiation complex, pangolin has undergone large scale variation and gene loss events causing expression pattern and function conversion that contribute to cornified epithelium structures on stomach to adapt myrmecophagous diet. Our assemblies also enable us to discover large copies number of high glycine-tyrosine keratin-associated proteins (HGT-KRTAPs). In addition, highly homogenized tandem array, amino content, and the specific expression pattern further validate the strong connection between the molecular mechanism of scale hardness and HGT-KRTAPs.

The reference genome and transcriptome of the limestone langur, Trachypithecus leucocephalus, reveal expansion of genes related to alkali tolerance.

BACKGROUND: Trachypithecus leucocephalus, the white-headed langur, is a critically endangered primate that is endemic to the karst mountains in the southern Guangxi province of China. Studying the genomic and transcriptomic mechanisms underlying its local adaptation could help explain its persistence within a highly specialized ecological niche. RESULTS: In this study, we used PacBio sequencing and optical assembly and Hi-C analysis to create a high-quality de novo assembly of the T. leucocephalus genome. Annotation and functional enrichment revealed many genes involved in metabolism, transport, and homeostasis, and almost all of the positively selected genes were related to mineral ion binding. The transcriptomes of 12 tissues from three T. leucocephalus individuals showed that the great majority of genes involved in mineral absorption and calcium signaling were expressed, and their gene families were significantly expanded. For example, FTH1 primarily functions in iron storage and had 20 expanded copies. CONCLUSIONS: These results increase our understanding of the evolution of alkali tolerance and other traits necessary for the persistence of T. leucocephalus within an ecologically unique limestone karst environment.

Genomic Mechanisms of Physiological and Morphological Adaptations of Limestone Langurs to Karst Habitats.

Knowledge of the physiological and morphological evolution and adaptation of nonhuman primates is critical to understand hominin origins, physiological ecology, morphological evolution, and applications in biomedicine. Particularly, limestone langurs represent a direct example of adaptations to the challenges of exploiting a high calcium and harsh environment. Here, we report a de novo genome assembly (Tfra_2.0) of a male François's langur (Trachypithecus francoisi) with contig N50 of 16.3 Mb and resequencing data of 23 individuals representing five limestone and four forest langur species. Comparative genomics reveals evidence for functional evolution in genes and gene families related to calcium signaling in the limestone langur genome, probably as an adaptation to naturally occurring high calcium levels present in water and plant resources in karst habitats. The genomic and functional analyses suggest that a single point mutation (Lys1905Arg) in the α1c subunit of the L-type voltage-gated calcium channel Cav1.2 (CACNA1C) attenuates the inward calcium current into the cells in vitro. Population genomic analyses and RNA-sequencing indicate that EDNRB is less expressed in white tail hair follicles of the white-headed langur (T. leucocephalus) compared with the black-colored François's langur and hence might be responsible for species-specific differences in body coloration. Our findings contribute to a new understanding of gene-environment interactions and physiomorphological adaptative mechanisms in ecologically specialized primate taxa.

DNA methylation and transcriptome analysis reveal epigenomic differences among three macaque species.

Macaques (genus Macaca) are the most widely distributed non-human primates, and their evolutionary history, gene expression profiles, and genetic differences have been extensively studied. However, the DNA methylomes of macaque species are not available in public databases, which hampers understanding of epigenetic differences among macaque species. Epigenetic modifications can potentially affect development, physiology, behavior, and evolution. Here, we investigated the methylation patterns of the Tibetan macaque (M. thibetana; TM), Chinese rhesus macaque (M. mulatta lasiota; CR), and crab-eating macaque (M. fascicularis; CE) through whole-genome bisulfite sequencing from peripheral blood. We compared genome-wide methylation site information for the three species. We identified 12,128 (CR vs. CE), 59,165 (CR vs. TM), and 39,751 (CE vs. TM) differentially methylated regions (DMRs) in the three macaques. Furthermore, we obtained the differentially expressed genes (DEGs) among the three macaque species. The differences between CR and CE were smaller at both the methylome and transcriptome levels than compared with TM (CR vs. TM and CE vs. TM). We also found a change in the density of single nucleotide mutations in DMRs relative to their flanking regions, indicating a potential mechanism through which genomic alterations may modulate methylation landscapes, thereby influencing the transcriptome. Functional enrichment analyses showed the DMR-related genes were enriched in developmental processes and neurological functions, such as the growth hormone-related pathway, insulin secretion pathway, thyroid hormone synthesis pathway, morphine addiction, and GABAergic synapses. These differences may be associated with variations in physiology and habitat among the macaques. Our study provides one of the first genome-wide comparisons of genetic, gene expression, and epigenetic variations across different macaques. Our results should facilitate further research on comparative genomic and genetic differences in macaque species.

HiOmics: A cloud-based one-stop platform for the comprehensive analysis of large-scale omics data.

Analyzing the vast amount of omics data generated comprehensively by high-throughput sequencing technology is of utmost importance for scientists. In this context, we propose HiOmics, a cloud-based platform equipped with nearly 300 plugins designed for the comprehensive analysis and visualization of omics data. HiOmics utilizes the Element Plus framework to craft a user-friendly interface and harnesses Docker container technology to ensure the reliability and reproducibility of data analysis results. Furthermore, HiOmics employs the Workflow Description Language and Cromwell engine to construct workflows, ensuring the portability of data analysis and simplifying the examination of intricate data. Additionally, HiOmics has developed DataCheck, a tool based on Golang, which verifies and converts data formats. Finally, by leveraging the object storage technology and batch computing capabilities of public cloud platforms, HiOmics enables the storage and processing of large-scale data while maintaining resource independence among users.

Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice.

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin1. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

Antibiotic resistance profiles of gut microbiota across various primate species in Guangxi.

Introduction: Understanding the gut microbiota and antibiotic resistance gene (ARG) profiles in non-human primates (NHPs) is crucial for evaluating their potential impact on human health and the environment. Methods: In this study, we performed metagenomic analysis of 203 primate fecal samples, including nine NHP species and humans, to comprehensively characterize their gut microbiota and ARGs. Results: Our study reveals the prevailing phyla in primates as Firmicutes, Bacteroidetes, Euryarchaeota, and Proteobacteria. The captive NHPs exhibited higher ARG abundance compared to their wild counterparts, with tetracycline and beta-lactam resistance genes prevailing. Notably, ARG subtypes in Trachypithecus leucocephalus (T. leucocephalus) residing in karst limestone habitats displayed a more dispersed distribution compared to other species. Interestingly, ARG profiles of NHPs clustered based on geographic location and captivity status. Co-occurrence network analysis revealed intricate correlations between ARG subtypes and bacterial taxa. Procrustes analysis unveiled a significant correlation between ARGs and microbial phylogenetic community structure. Taxonomic composition analysis further highlighted differences in microbial abundance among NHPs and humans. Discussion: Our study underscores the impact of lifestyle and geographical location on NHP gut microbiota and ARGs, providing essential insights into the potential risks posed by NHPs to antibiotic resistance dissemination. This comprehensive analysis enhances our understanding of the interplay between NHPs and the gut resistome, offering a critical reference for future research on antibiotic resistance and host-microbe interactions.

Metavirome of 31 tick species provides a compendium of 1,801 RNA virus genomes.

The increasing prevalence and expanding distribution of tick-borne viruses globally have raised health concerns, but the full repertoire of the tick virome has not been assessed. We sequenced the meta-transcriptomes of 31 different tick species in the Ixodidae and Argasidae families from across mainland China, and identified 724 RNA viruses with distinctive virome compositions among genera. A total of 1,801 assembled and complete or nearly complete viral genomes revealed an extensive diversity of genome architectures of tick-associated viruses, highlighting ticks as a reservoir of RNA viruses. We examined the phylogenies of different virus families to investigate virome evolution and found that the most diverse tick-associated viruses are positive-strand RNA virus families that demonstrate more ancient divergence than other arboviruses. Tick-specific viruses are often associated with only a few tick species, whereas virus clades that can infect vertebrates are found in a wider range of tick species. We hypothesize that tick viruses can exhibit both 'specialist' and 'generalist' evolutionary trends. We hope that our virome dataset will enable much-needed research on vertebrate-pathogenic tick-associated viruses.

Comparative Gut Microbiome in Trachypithecus leucocephalus and Other Primates in Guangxi, China, Based on Metagenome Sequencing.

The Trachypithecus leucocephalus (white-headed langur) is a highly endangered, karst-endemic primate species, inhabiting the karst limestone forest in Guangxi, Southwest China. How white-headed langurs adapted to karst limestone and special dietary remains unclear. It is the first time to study the correlation between the gut microbiome of primates and special dietary, and environment in Guangxi. In the study, 150 fecal samples are collected from nine primates in Guangxi, China. Metagenomic sequencing is used to analyze and compare the gut microbiome composition and diversity between white-headed langurs and other primates. Our results indicate that white-headed langurs has a higher diversity of microbiome than other primates, and the key microbiome are phylum Firmicutes, class Clostridia, family Lachnospiraceae, and genera Clostridiates and Ruminococcus, which are related to the digestion and degradation of cellulose. Ten genera are significantly more abundant in white-headed langurs and François' langur than in other primates, most of which are high-temperature microbiome. Functional analysis reveals that energy synthesis-related pathways and sugar metabolism-related pathways are less abundant in white-headed langurs and François' langur than in other primates. This phenomenon could be an adaptation mechanism of leaf-eating primates to low-energy diet. The gut microbiome of white-headed langurs is related to diet and karst limestone environment. This study could serve as a reference to design conservation breeding, manage conservation units, and determine conservation priorities.

Human parainfluenza 3 and respiratory syncytial viruses detected in pangolins.

Pangolins have gained increasing global attention owing to their public health significance as potential zoonotic hosts since the identification of SARS-CoV-2-related viruses in them. Moreover, these animals could carry other respiratory viruses. In this study, we investigated the virome composition of 16 pangolins that died in 2018 with symptoms of pneumonia using metagenomic approaches. A total of eight whole virus sequences belonging to the Paramyxoviridae or Pneumoviridae families were identified, including one human parainfluenza virus 3, one human respiratory syncytial virus A, and six human respiratory syncytial virus B. All of these sequences showed more than 99% nucleotide identity with the virus isolated from humans at the whole-genome level and clustered with human viruses in the phylogenetic tree. Our findings provide evidence that pangolins are susceptible to HPIV3 and HRSV infection. Therefore, public awareness of the threat of pangolin-borne pathogens is essential to stop their human consumption and to prevent zoonotic viral transmission.

Virome in healthy pangolins reveals compatibility with multiple potentially zoonotic viruses.

Previous studies have identified multiple viruses in dead or severely diseased pangolins, but descriptions of the virome in healthy pangolins are lacking. This poses a greater risk of cross-species transmission due to poor preventive awareness and frequent interactions with breeders. In this study, we investigated the viral composition of 34 pangolins with no signs of disease at the time of sampling and characterized a large number of arthropod-associated viruses belonging to 11 families and vertebrate viruses belonging to eight families, including those with pathogenic potential in humans and animals. Several important vertebrate viruses were identified in the pangolins, including parvovirus, pestivirus, and picobirnavirus. The picobirnavirus was clustered with human and grey teal picobirnaviruses. Viruses with cross-species transmission ability were also identified, including circovirus, rotavirus, and astrovirus. Our study revealed that pangolins are frequently exposed to arthropod-associated viruses in the wild and can carry many vertebrate viruses under natural conditions. This study provides important insights into the virome of pangolins, underscoring the importance of monitoring potential pathogens in healthy pangolins to prevent outbreaks of infectious diseases in domesticated animals and humans.

Weaning period and growth patterns of captive Sunda pangolin (Manis javanica) cubs.

This study tracked and recorded the weight changes of 13 captive Sunda pangolin cubs from lactation to maturity to explored the appropriate weaning time and reveal the rules of its weight growth. SPSS 25.0 was used to build a cubic equation model to fit the body weight change rules of 4 individuals who nonvoluntarily ingested artificial feed (NIAF) at 127 days after birth and 5 individuals who voluntarily ingested artificial feed (VIAF) at 86-108 days after birth. The body weight of NIAF cubs aged 0-120 days and VIAF cubs aged 0-150 days were estimated according to the fitting model. An independent sample T-test was performed on the mean body weight of the two groups during the late lactation period. The results showed that at 105 days after birth, the body weight of the VIAF group was significantly higher than that of the NIAF group (P = 0.049), and the body weight of the VIAF group was extremely significantly higher than that of the NIAF group at 114 days (P = 0.008); The peak cumulative body weight of the NIAF cubs during lactation appeared around 130 days of age (n = 3); The mortality rate was 66.7% (n = 3) after about 150 days if the feed was continuously consumed nonvoluntarily. It was concluded that the milk secretion period of the mother is about 0-5 months after giving birth; the weaning period of the cubs should be 4-5 months after birth. If the cubs don't follow the mother to eat artificial feed for 3 months after birth, it can start be induced with artificial diet which adds termites, and the time point cannot be later than 130 days, otherwise it is not conducive to the survival of the cubs; When sexually mature, the body length and body weight of female cubs account for about 84% and 60% of the adult, respectively; the body maturity and body weight of female cubs tend to be stable about 15.3 months and 16.4 months, respectively. Finally, a special needle-shaped nipples and nursing patterns of female Sunda pangolins were also recorded in this study. These findings play an important role in guiding the nursing of captive Sunda pangolin cubs and other pangolin cubs. It is expected to improve the survival rate of the cubs by exploring the appropriate weaning time and the rules of weight growth. By scientifically planning the reproductive cycle of the female Sunda pangolins, our goal is to expand the population size and eventually release to the wild, meanwhile improving knowledge of this critically endangered species.

Natural infection of pangolins with human respiratory syncytial viruses.

Respiratory syncytial virus (RSV) is an enveloped non-segmented negative sense RNA virus that belongs to Orthopneumovirus genus of the Pneumoviridae family in the order Mononegavirales. The virus is the leading cause of severe respiratory disease in children under two years of age and is responsible for substantial disease burden in infants and elder people in both developed and developing countries1,2. RSV is only known to circulate among humans, though it was first isolated from chimpanzees3. The virus can experimentally infect mice, rats, cotton rats, ferrets, and hamsters, but does not naturally circulate in these animal populations4. We found that Malayan pangolins (Manis javanica) were naturally infected with RSVs that have 99.4-99.8% genomic identity with strains circulating in humans. Phylogenetic analyses revealed that five RSVs in pangolins were RSV-A ON1 and seven were RSV-B BA genotypes, both of which are currently prevalent in humans worldwide. These findings suggest that humans might transmit their viruses to endangered wildlife.

Trafficked Malayan pangolins contain viral pathogens of humans.

Pangolins are the most trafficked wild animal in the world according to the World Wildlife Fund. The discovery of SARS-CoV-2-related coronaviruses in Malayan pangolins has piqued interest in the viromes of these wild, scaly-skinned mammals. We sequenced the viromes of 161 pangolins that were smuggled into China and assembled 28 vertebrate-associated viruses, 21 of which have not been previously reported in vertebrates. We named 16 members of Hunnivirus, Pestivirus and Copiparvovirus pangolin-associated viruses. We report that the L-protein has been lost from all hunniviruses identified in pangolins. Sequences of four human-associated viruses were detected in pangolin viromes, including respiratory syncytial virus, Orthopneumovirus, Rotavirus A and Mammalian orthoreovirus. The genomic sequences of five mammal-associated and three tick-associated viruses were also present. Notably, a coronavirus related to HKU4-CoV, which was originally found in bats, was identified. The presence of these viruses in smuggled pangolins identifies these mammals as a potential source of emergent pathogenic viruses.

Molecular Detection of Novel Borrelia Species, Candidatus Borrelia javanense, in Amblyomma javanense Ticks from Pangolins.

A novel Borrelia species, Candidatus Borrelia javanense, was found in ectoparasite ticks, Amblyomma javanense, from Manis javanica pangolins seized in anti-smuggling operations in southern China. Overall, 12 tick samples in 227 (overall prevalence 5.3%) were positive for Candidatus B. javanense, 9 (5.1%) in 176 males, and 3 (5.9%) in 51 females. The phylogenetic analysis, based on the 16S rRNA gene and the flagellin gene sequences of the Borrelia sp., exhibited strong evidence that Candidatus B. javanense did not belong to the Lyme disease Borrelia group and the relapsing fever Borrelia group but another lineage of Borrelia. The discovery of the novel Borrelia species suggests that A. javanense may be the transmit vector, and the M. javanica pangolins should be considered a possible origin reservoir in the natural circulation of these new pathogens. To our knowledge, this is the first identification of a novel Borrelia species agent in A. javanense from pangolins. Whether the novel agent is pathogenic to humans is unknown and needs further research.

Successful captive breeding of a Malayan pangolin population to the third filial generation.

Pangolins are threatened placental mammals distributed in Africa and Asia. Many efforts have been undertaken in the last century to maintain pangolins in captivity, but only a few of them succeeded in maintaining and keeping this species in a controlled environment. This study reports the first systematic breeding of the Critically Endangered Malayan pangolin (Manis javanica) in captivity. Our captive breeding approach successfully improved the reproductive rate for both wild and captive-born female pangolins. From 2016 to 2020, we had 33 wild pangolins and produced 49 captive-born offspring spanning three filial generations. The female offspring further bred 18 offspring, of which 14 (78%) were conceived during the first time of cohabitation with males, and four offspring were conceived during the second cohabitation event, suggesting that they may practice copulation-induced ovulation. We observed that captive-born female pangolins could reach sexual maturity at 7-9 months (n = 4), and male pangolins could mate and successfully fertilise females at nine months age (n = 1). We also observed a female pangolin conceiving on the eighth day after parturition (the fifth day after the death of its pup). Our captive pangolins had a female-biased sex ratio of 1:0.5 at birth, unlike other known captive-born mammals. Also, captive-born pangolins were generally more viable after successful weaning and had a similar gestation length (~185 days) to wild pangolins. Most importantly, we report the first self-sustaining captive population of Malayan pangolins, and this species has an efficient reproduction strategy. These advances provide more comprehensive information for people to understand pangolins, and have implications for conserving endangered Malayan pangolins and providing scientific guidance to the management of other pangolin species.

Detection of Novel Spotted Fever Group Rickettsiae (Rickettsiales: Rickettsiaceae) in Ticks (Acari: Ixodidae) in Southwestern China.

Spotted fever group rickettsiae, mainly maintained and transmitted by ticks, are important etiological agents of (re)emerging zoonotic diseases worldwide. It is of great significance to investigate spotted fever group rickettsiae in ticks in different areas for the prevention and control of rickettsioses. In this study, a total of 305 ticks were collected from wild and domestic animals in Chongqing, Guizhou, Yunnan, and Guangxi provinces of southwestern China during 2017-2019 and examined for the presence of spotted fever group rickettsiae by PCR with primers targeting the partial gltA, ompA, rrs, and htrA genes. Results showed that two spotted fever group rickettsiae species, including the pathogenic Candidatus Rickettsia jingxinensis (Rickettsiales: Rickettsiaceae) and a potential novel species Rickettsia sp. sw (Rickettsiales: Rickettsiaceae), were identified. The Ca. R. jingxinensis sequences were recovered from Rhipicephalus microplus (Ixodida: Ixodidae) and Haemaphysalis longicornis (Ixodida: Ixodidae) ticks and phylogenetically clustered with previous Ca. R. jingxinensis, Ca. R. longicornii (Rickettsiales: Rickettsiaceae), and Rickettsia sp. XY118 (Rickettsiales: Rickettsiaceae) strains. Rickettsia sp. sw was detected in Amblyomma geoemydae (Ixodida: Ixodidae) and Rh. microplus. Interestingly, as far as we know, this was the first report of Rickettsia (Rickettsiales: Rickettsiaceae) in A. geoemydae. Phylogenetic analyses indicated that this potential novel species was closely related to R. aeschlimannii (Rickettsiales: Rickettsiaceae) with gltA and ompA genes and grouped in a cluster composed of R. montanensis (Rickettsiales: Rickettsiaceae), R. raoultii (Rickettsiales: Rickettsiaceae), R. aeschlimannii, R. massiliae (Rickettsiales: Rickettsiaceae), and R. rhipicephali (Rickettsiales: Rickettsiaceae) with htrA, while formed a separate clade with rrs. The pathogenicity of Rickettsia sp. sw should be further confirmed. These results expand the knowledge of the geographical distribution and vector distribution of spotted fever group rickettsiae in China and are useful for assessing the potential public health risk.

Molecular evidence of Candidatus Rickettsia longicornii and a novel Rickettsia strain from ticks in Southern China.

Ticks and tick-borne rickettsial diseases have been gaining greater attention in China over the past decade. However, most published studies to date have occurred in Northern China, with limited investigations occurring in China's southern provinces. As part of larger surveillance efforts, a cross-sectional survey was conducted in six sites at Guangdong, Guangxi and Yunnan investigating rickettsial infection in ticks. A total of 581 ticks were collected from hosts and screened via PCR, targeting rrs, gltA, ompB, sca4, and ompA gene fragments. Two of 12 Haemaphysalis formosensis ticks were infected with novel Rickettsia strain GD01, which was closest phylogenetically (97.3-98.9 % identity) to Rickettsia tamurae strain AT-1, but not within the same clade. Another detected strain (GD02) shared similar identity, 99-100 % across four gene targets, to recently detected Candidatus Rickettsia longicornii isolate ROK-HL727, with an overall prevalence of 12.5 % (71/569). The presence of such pathogens calls for increased public health attention and active surveillance in patients reporting recent tick bites.

Detection and molecular epidemiology of ferlaviruses in farmed snakes with respiratory disease in Guangxi Province, China.

We screened 104 snakes with respiratory disease, collected from 52 snake farms in Guangxi Province, China, for pathogens. Ferlaviruses were detected in 70 of 104 lung samples by reverse-transcription PCR; 34 of 52 of the snake farms were positive for ferlaviruses. No reovirus, adenovirus, sunshine virus, or nidovirus was detected in any of the snakes. We obtained 96 bacterial isolates from snake organs, of which the most commonly isolated species were Salmonella (18) and Proteus (16). Sequence analysis, based on 27 partial RNA-dependent RNA polymerase gene (L) sequences, revealed that ferlaviruses from Guangxi and the known GenBank strains clustered together and formed 3 genogroups. The nucleotide and deduced amino acid homologies of ferlaviruses were 84.3-100% and 95.0-100% within groups, respectively, and 77.0-81.6% and 90.4-95.2% between groups, respectively. Ferlaviruses from Guangxi had close genetic relationships with the known GenBank strains. Our results indicate that ferlaviruses are common in snakes with respiratory disease on the farms of Guangxi that we sampled, and that ferlavirus molecular epidemiology is both diverse and complex.