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.

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.

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.

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.

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.

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.

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 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.

Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

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.

The complete mitochondrial genome of Amblyomma geoemydae (Ixodida: Ixodidae).

The complete mitochondrial genome of Amblyomma geoemydae is reported for the first time in this study. Its entire mitogenome is 14,780 bp in length, contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and two non-coding regions. The phylogenetic analysis by Maximum-likelihood method show that A. geoemydae and the others of genus Amblyomma are in the same clade, indicating that A. geoemydae belongs to the genus Amblyomma.