Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

Haplotype-resolved chromosome-scale genomes of the Asian and African Savannah Elephants.

The Proboscidea, which includes modern elephants, were once the largest terrestrial animals among extant species. They suffered mass extinction during the Ice Age. As a unique branch on the evolutionary tree, the Proboscidea are of great significance for the study of living animals. In this study, we generate chromosome-scale and haplotype-resolved genome assemblies for two extant Proboscidea species (Asian Elephant, Elephas maximus and African Savannah Elephant, Loxodonta africana) using Pacbio, Hi-C, and DNBSEQ technologies. The assembled genome sizes of the Asian and African Savannah Elephant are 3.38 Gb and 3.31 Gb, with scaffold N50 values of 130 Mb and 122 Mb, respectively. Using Hi-C technology ~97% of the scaffolds are anchored to 29 pseudochromosomes. Additionally, we identify ~9 Mb Y-linked sequences for each species. The high-quality genome assemblies in this study provide a valuable resource for future research on ecology, evolution, biology and conservation of Proboscidea species.

Hantaan virus inhibits type I interferon response by targeting RLR signaling pathways through TRIM25.

Hantaan virus (HTNV) is responsible for hemorrhagic fever with renal syndrome (HFRS), primarily due to its ability to inhibit host innate immune responses, such as type I interferon (IFN-I). In this study, we conducted a transcriptome analysis to identify host factors regulated by HTNV nucleocapsid protein (NP) and glycoprotein. Our findings demonstrate that NP and Gc proteins inhibit host IFN-I production by manipulating the retinoic acid-induced gene I (RIG-I)-like receptor (RLR) pathways. Further analysis reveals that HTNV NP and Gc proteins target upstream molecules of MAVS, such as RIG-I and MDA-5, with Gc exhibiting stronger inhibition of IFN-I responses than NP. Mechanistically, NP and Gc proteins interact with tripartite motif protein 25 (TRIM25) to competitively inhibit its interaction with RIG-I/MDA5, suppressing RLR signaling pathways. Our study unveils a cross-talk between HTNV NP/Gc proteins and host immune response, providing valuable insights into the pathogenic mechanism of HTNV.

EGR1 functions as a new host restriction factor for SARS-CoV-2 to inhibit virus replication through the E3 ubiquitin ligase MARCH8.

IMPORTANCE: Emerging vaccine-breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight an urgent need for novel antiviral therapies. Understanding the pathogenesis of coronaviruses is critical for developing antiviral drugs. Here, we demonstrate that the SARS-CoV-2 N protein suppresses interferon (IFN) responses by reducing early growth response gene-1 (EGR1) expression. The overexpression of EGR1 inhibits SARS-CoV-2 replication by promoting IFN-regulated antiviral protein expression, which interacts with and degrades SARS-CoV-2 N protein via the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. The MARCH8 mutants without ubiquitin ligase activity are no longer able to degrade SARS-CoV-2 N proteins, indicating that MARCH8 degrades SARS-CoV-2 N proteins dependent on its ubiquitin ligase activity. This study found a novel immune evasion mechanism of SARS-CoV-2 utilized by the N protein, which is helpful for understanding the pathogenesis of SARS-CoV-2 and guiding the design of new prevention strategies against the emerging coronaviruses.

Exploration of Parascaris species in three different Equus populations in China.

BACKGROUND: The roundworms, Parascaris spp., are important nematode parasites of foals and were historically model organisms in the field of cell biology, leading to many important discoveries. According to karyotype, ascarids in Equus are commonly divided into Parascaris univalens (2n = 2) and Parascaris equorum (2n = 4). METHODS: Here, we performed morphological identification, karyotyping and sequencing of roundworms from three different hosts (horses, zebras and donkeys). Phylogenetic analysis was performed to study the divergence of these ascarids based on cytochrome c oxidase subunit I (COI) and internal transcribed spacer (ITS) sequences. RESULTS: Karyotyping, performed on eggs recovered from worms of three different Equus hosts in China, showed two different karyotypes (2n = 2 in P. univalens collected from horses and zebras; 2n = 6 in Parascaris sp. collected from donkeys). There are some differences in the terminal part of the spicula between P. univalens (concave) and Parascaris sp. (rounded). Additionally, it was found that the egg's chitinous layer was significantly thicker in Parascaris sp. (> 5 µm) than P. univalens (< 5 µm) (F(2537) = 1967, P < 0.01). Phylogenetic trees showed that the sequences of Parascaris from Equus hosts were divided into two distinct lineages based on sequences of the COI and ITS. CONCLUSIONS: Comparing the differences in roundworms collected from three different Equus hosts, this study describes a Parascaris species (Parascaris sp.) with six chromosomes in donkeys. It is worth noting that the thickness of the chitinous layer in the Parascaris egg may serve as a diagnostic indicator to distinguish the two roundworms (P. univalens and Parascaris sp.). The Parascaris sp. with six chromosomes in donkeys in the present study may be a species of P. trivalens described in 1934, but the possibility that it is a new Parascaris species cannot be ruled out. Both karyotyping and molecular analysis are necessary to solve the taxonomic problems in Parascaris species.

H10Nx avian influenza viruses detected in wild birds in China pose potential threat to mammals.

H10 subtype avian influenza viruses (AIVs) have been isolated from wild and domestic avian species worldwide and have occasionally crossed the species barrier to mammalian hosts. Fatal human cases of H10N8 infections and the recent detection of human H10N3 infections have drawn widespread public attention. In this study, 25 H10Nx viruses were isolated from wild waterfowl in China during a long-term surveillance of AIVs. We conducted phylogenetic and phylogeographic studies of the hemagglutinin (HA) genes of global H10 viruses to determine the spatiotemporal patterns of spread and the roles of different hosts in viral transmission. We found the pattern of AIV transmission from wild birds to poultry to humans, and Anatidae have acted as the seeding population in the spread of the virus. Phylogenetic incongruence indicated complex reassortment events and our isolates were divided into eight genotypes (G1-8). We also found that the HA genes of the G8 viruses belonged to the North American lineage, indicating that intercontinental gene flow has occurred. Their receptor-binding specificity showed that the G1/4/5/6/7/8 viruses bind to both human-type α2,6-linked sialic acid receptors and avian-type α2,3-linked sialic acid receptors. Mouse studies indicated that the H10Nx isolates replicated efficiently in the respiratory system without preadaptation, but showed low pathogenicity in mice. The H10Nx isolates showed no (G2/4/7) or low pathogenicity (G1/3/5/6/8) in chickens, and the G6 and G8 viruses could be transmitted to chickens through direct contact. The asymptomatic shedding of these wild-bird-origin H10Nx isolates in chickens and their good adaptation in mice should increase the ease of their transmission to humans, and they therefore pose a threat to public health. Our findings demonstrate a further understanding of wild bird-origin H10 viruses and provide information for the continuous surveillance of H10 subtype viruses.

Yezo Virus Infection in Tick-Bitten Patient and Ticks, Northeastern China.

We identified Yezo virus infection in a febrile patient who had a tick bite in northeastern China, where 0.5% of Ixodes persulcatus ticks were positive for viral RNA. Clinicians should be aware of this potential health threat and include this emerging virus in the differential diagnosis for tick-bitten patients in this region.

The first genome assembly of the amphibian nematode parasite (Aplectana chamaeleonis).

Cosmocercoid nematodes are common parasites of the digestive tract of amphibians. Genomic resources are important for understanding the evolution of a species and the molecular mechanisms of parasite adaptation. So far, no genome resource of Cosmocercoid has been reported. In 2020, a massive Cosmocercoid infection was found in the small intestine of a toad, causing severe intestinal blockage. We morphologically identified this parasite as A. chamaeleonis. Here, we report the first A. chamaeleonis genome with a genome size of 1.04 Gb. The repeat content of this A. chamaeleonis genome is 72.45%, and the total length is 751 Mb. This resource is fundamental for understanding the evolution of Cosmocercoid and provides the molecular basis for Cosmocercoid infection and control.

Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling.

BACKGROUND: Vector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive. RESULTS: We showed that TBEV structural protein, pre-membrane (prM) protein, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeding the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKV and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKV prM significantly promoted the replication of TBEV and Sendai virus. CONCLUSION: Our findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development.

Seroprevalence and risk factor analysis of Toxoplasma gondii infection in Siberian tigers (Panthera tigris altaica) and giant pandas (Ailuropoda melanoleuca) in China.

Toxoplasmosis, caused by Toxoplasma gondii, is a worldwide zoonosis. The aim of the present study was to detect the seroprevalence of T. gondii infection and associated risk factors among Siberian tigers (Panthera tigris altaica) and giant pandas (Ailuropoda melanoleuca) in China. Blood samples from 112 Siberian tigers and 22 giant pandas were tested for immunoglobulin G (IgG) against T. gondii by enzyme-linked immunosorbent assay (ELISA). The seroprevalence of T. gondii infection was 7.14% among Siberian tigers and 9.09% among giant pandas. No risk factors were found to be significantly associated with seroprevalence (P > 0.05). This is the first study to evaluate T. gondii infection in Siberian tigers on a large scale in China, and it also updates the information regarding the positivity rate of T. gondii infection among giant pandas in China.

Extensive diversity of RNA viruses in ticks revealed by metagenomics in northeastern China.

BACKGROUND: Ticks act as important vectors of infectious agents, and several emerging tick-borne viruses have recently been identified to be associated with human diseases in northeastern China. However, little is known about the tick virome in northeastern China. METHODS: Ticks collected from April 2020 to July 2021 were pooled for metagenomic analysis to investigate the virome diversity in northeastern China. RESULTS: In total, 22 RNA viruses were identified, including four each in the Nairoviridae and Phenuiviridae families, three each in the Flaviviridae, Rhabdoviridae, and Solemoviridae families, two in the Chuviridae family, and one each in the Partitiviridae, Tombusviridae families and an unclassified virus. Of these, eight viruses were of novel species, belonging to the Nairoviridae (Ji'an nairovirus and Yichun nairovirus), Phenuiviridae (Mudanjiang phlebovirus), Rhabdoviridae (Tahe rhabdovirus 1-3), Chuviridae (Yichun mivirus), and Tombusviridae (Yichun tombus-like virus) families, and five members were established human pathogens, including Alongshan virus, tick-borne encephalitis virus, Songling virus, Beiji nairovirus, and Nuomin virus. I. persulcatus ticks had significant higher number of viral species than H. japonica, H. concinna, and D. silvarum ticks. Significant differences in tick viromes were observed among Daxing'an, Xiaoxing'an and Changbai mountains. CONCLUSIONS: These findings showed an extensive diversity of RNA viruses in ticks in northeastern China, revealing potential public health threats from the emerging tick-borne viruses. Further studies are needed to explain the natural circulation and pathogenicity of these viruses.

Characterization and subcellular localization of Alongshan virus proteins.

Alongshan virus (ALSV) in the Jingmenvirus group within the family Flaviviridae is a newly discovered tick-borne virus associated with human disease, whose genome includes four segments and encodes four structural proteins (VP1a, VP1b, VP2, VP3, and VP4) and two non-structural proteins (NSP1 and NSP2). Here, we characterized the subcellular distribution and potential function of ALSV proteins in host cells. We found that viral proteins exhibited diverse subcellular distribution in multiple tissue-deriving cells and induced various morphological changes in the endoplasmic reticulum (ER), and NSP2, VP1b, VP2, and VP4 were all co-localized in the ER. The nuclear transfer and co-localization of VP4 and calnexin (a marker protein of ER), which were independent of their interaction, were unique to HepG2 cells. Expression of NSP1 could significantly reduce mitochondria quantity by inducing mitophagy. These findings would contribute to better understanding of the pathogenesis of emerging segmented flaviviruses.

The genetic characteristics of Sarcoptes scabiei from Chinese serow (Capricornis milneedwardsii) and goral (Naemorhedus goral arnouxianus) compared with other mites from different hosts and geographic locations using ITS2 and cox1 sequences.

Scabies is a common parasitic disease in many mammalian species, caused by the infestation of Sarcoptes scabiei. There is no consistent conclusion on whether Sarcoptes mites from different hosts or geographic locations have apparent genetic divergence. In this study, we collected and morphologically identified S. scabiei from Chinese serow and goral, and we described the genetic diversity of S. scabiei and other mites based on phylogenetic analyses of the ITS2 and cox1 sequence fragments, including data available in GenBank. The mites isolated from Chinese serow and goral were S. scabiei, and they were morphologically similar. The phylogenetic trees and haplotype networks showed that S. scabiei from other locations worldwide did not cluster according to host divergence or geographical distribution. Additionally, the Fst values were - 0.224 to 0.136 and - 0.045 to 1 between S. scabiei from different hosts, including humans and domestic and wild animals, based on partial ITS and cox1 sequences. Worldwide S. scabiei samples formed three clusters (with H2, H5, and H12 at their centers) in the ITS and one cluster (with C9 at the center) in the cox1 haplotype phylogenetic network. The S. scabiei collected from Chinese serow and goral were morphologically similar and had the same genotype. A study on the genetic characteristics of S. scabiei from Chinese serow and goral together with other mites from different hosts and geographic locations around the world showed no obvious divergence. These findings indicated that scabies likely is a zoonotic disease and that the global prevalence of scabies is probably related to the worldwide trade of domestic animals.

The chromosome-scale genome of the raccoon dog: Insights into its evolutionary characteristics.

The raccoon dog (Nyctereutes procyonoides) is an invasive canid species native to East Asia with several distinct characteristics. Here, we report a chromosome-scale genome of the raccoon dog with high contiguity, completeness, and accuracy. The intact taste receptor genes, expanded gene families, and positively selected genes related to digestion, absorption, foraging, and detoxification likely support the omnivory of raccoon dogs. Several positively selected genes and raccoon dog-specific mutations in TDRD6 and ZP3 genes may explain their high reproductivity. Enriched GO terms in energy metabolism and positively selected immune genes were speculated to be closely related to the diverse immune system of raccoon dogs. In addition, we found that several expanded gene families and positively selected genes related to lipid metabolism and insulin resistance may contribute to winter sleep of the raccoon dog. This high-quality genome provides a valuable resource for understanding the evolutionary characteristics of this species.

Gene rearrangements in the mitochondrial genome of ten ascaris species and phylogenetic implications for Ascaridoidea and Heterakoidea families.

The Ascaridoidea family and Heterakoidea family are the most common and typical representative of large parasites. Although our understanding of these parasites' diversity has expanded by analyses of some mitochondrial genes, there is limited information on these species' evolutionary rates. Here we determined ten complete mitogenome sequences of five subfamilies of Ascaridoidea and one subfamily of Heterakoidea. The phylogenetic tree divided the Ascaridoidea into six monophyletic major clades, and the divergence time of Heterakoidea family and Ascaridoidea family can be placed during the early Carboniferous Period (300-360 Mya). The reconstruction of the ancestral state showed that the gene orders of all species in Ascaridoidea were conserved, and the Heterakoidea had obvious genome rearrangement. The conserved blocks between them were divided into five and the main types are tandem-duplication/random loss (TDRL). These results will help to better understand the gene rearrangements and evolutionary position of ascaris species.

Equus roundworms (Parascaris univalens) are undergoing rapid divergence while genes involved in metabolic as well as anthelminic resistance are under positive selection.

BACKGROUND: The evolution of parasites is often directly affected by the host's environment. Studies on the evolution of the same parasites in different hosts are of great interest and are highly relevant to our understanding of divergence. METHODS: Here we performed whole-genome sequencing of Parascaris univalens from different Equus hosts (horses, zebras and donkeys). Phylogenetic and selection analyses were performed to study the divergence and adaptability of P. univalens. RESULTS: At the genetic level, multiple lines of evidence indicate that P. univalens is mainly separated into two clades (horse-derived and zebra & donkey-derived). This divergence began 300-1000 years ago, and we found that most of the key enzymes related to glycolysis were under strong positive selection in zebra & donkey-derived roundworms, whereas the lipid-related metabolic system was under positive selection in horse-derived roundworms, indicating that the adaptive evolution of metabolism has occurred over the past few centuries. In addition, we found that some drug-related genes showed a significantly higher degree of selection in diverse populations. CONCLUSIONS: This work reports the adaptive evolution and divergence trend of P. univalens in different hosts for the first time. Its results indicate that the divergence of P. univalens is a continuous, dynamic process. Furthermore, the continuous monitoring of the effects of differences in nutritional and drug histories on the rapid evolution of roundworms is conducive to further understanding host-parasite interactions.

Biological characteristics of two mesenchymal stem cell cultures isolated from the umbilical cord and adipose tissue of a neonatal common hippo (Hippopotamus amphibius).

Mesenchymal stem cells (MSCs) are multipotent adult stem cells and can be isolated from many tissues of the body. Due to their potentials to treat various diseases and be applied in animal breeding, MSCs have been isolated and identified regarding their biological properties. Common hippos (Hippopotamus amphibius) are a vulnerable species and yet the cryopreservation of their genetic materials is scare. In this study, we successfully established two MSC cultures (UC-MSCs and AT-MSCs) from the umbilical cord and adipose tissue of a neonatal common hippo and comparatively described their features. Both UC-MSCs and AT-MSCs showed fibroblastoid morphology and could be continuously passaged for over 17 passages without dramatic signs of senescence. The cell cultures had normal chromosome composition, say, 17 pairs of autosomes and 1 pair of X chromosomes. UC-MSCs and AT-MSCs displayed similar gene expression profiles. They were positive for CD34, CD45, CD73, CD90 and CD105 and negative for HLA-DR. They demonstrated stemness maintenance by expression of classical stem cell markers. UC-MSCs and AT-MSCs manifested different differentiation potentials into other cell lineages. In summary, these two cell cultures demonstrated the essential properties of mesenchymal stem cells and might play a role in the future research.

The global succinylation of SARS-CoV-2-infected host cells reveals drug targets.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.

Molecular Detection of Parvovirus in Captive Siberian Tigers and Lions in Northeastern China From 2019 to 2021.

The fact that wild felines are carriers of pernicious infectious viruses should be a major concern due to the potential cross-species transmission between the felines and human or domestic animals. However, studies on the virus in the captive wild felines, especially in tigers, are thin on the ground. In this study, we screened four infectious viruses, namely, feline parvovirus (FPV), feline coronavirus (FCoV), canine distemper virus (CDV), and influenza A virus (IAV), in the blood samples of 285 captive Siberian tigers (Panthera tigris altaica) and in the spleen samples of two deceased lions (Panthera leo), which were collected from 2019 to 2021 in three Siberian Tiger Parks from the northeast of China. Nucleic acids isolated from the blood samples collected from tigers and the spleen samples collected from two deceased lions were positive for FPV by PCR, and the positive rate was 4.6% (13/285) in tigers. Furthermore, the VP2 gene of FPV was amplified by nested PCR, and the sequences of the VP2 gene from these six FPV positive strains shared 98.3-99.9% homology with the reference. The key amino acid sites of VP2 protein were consistent with that of FPV reference strains. Phylogenetic analysis based on the VP2 gene showed that in this study, FPV-positive strains were grouped within the FPV clade and closely related to the Asian strains clade. The results of this study showed that FPV circulated in the captive Siberian tigers and lions in northeastern China and provided valuable information for the study of FPV epidemiology in wild felines. Therefore, we suggest that regular antibody monitoring and booster immunization for tigers should be performed.

Highly Pathogenic Avian Influenza A(H5N8) Clade 2.3.4.4b Viruses in Satellite-Tracked Wild Ducks, Ningxia, China, 2020.

During October 2020, we identified 13 highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b viruses from wild ducks in Ningxia, China. These viruses were genetically related to H5N8 viruses circulating mainly in poultry in Europe during early 2020. We also determined movements of H5N8 virus‒infected wild ducks and evidence for spreading of viruses.