Conserved Functions of Orthohepadnavirus X Proteins to Inhibit Type-I Interferon Signaling.

Orthohepadnavirus causes chronic hepatitis in a broad range of mammals, including primates, cats, woodchucks, and bats. Hepatitis B virus (HBV) X protein inhibits type-I interferon (IFN) signaling, thereby promoting HBV escape from the human innate immune system and establishing persistent infection. However, whether X proteins of Orthohepadnavirus viruses in other species display a similar inhibitory activity remains unknown. Here, we investigated the anti-IFN activity of 17 Orthohepadnavirus X proteins derived from various hosts. We observed conserved activity of Orthohepadnavirus X proteins in inhibiting TIR-domain-containing adaptor protein inducing IFN-ß (TRIF)-mediated IFN-ß signaling pathway through TRIF degradation. X proteins from domestic cat hepadnavirus (DCH), a novel member of Orthohepadnavirus, inhibited mitochondrial antiviral signaling protein (MAVS)-mediated IFNß signaling pathway comparable with HBV X. These results indicate that inhibition of IFN signaling is conserved in Orthohepadnavirus X proteins.

High genetic diversity of the himalayan marmot relative to plague outbreaks in the Qinghai-Tibet Plateau, China.

Plague, as an ancient zoonotic disease caused by Yersinia pestis, has brought great disasters. The natural plague focus of Marmota himalayana in the Qinghai-Tibet Plateau is the largest, which has been constantly active and the leading source of human plague in China for decades. Understanding the population genetics of M. himalayana and relating that information to the biogeographic distribution of Yersinia pestis and plague outbreaks are greatly beneficial for the knowledge of plague spillover and arecrucial for pandemic prevention. In the present research, we assessed the population genetics of M. himalayana. We carried out a comparative study of plague outbreaks and the population genetics of M. himalayana on the Qinghai-Tibet Plateau. We found that M. himalayana populations are divided into two main clusters located in the south and north of the Qinghai-Tibet Plateau. Fourteen DFR genomovars of Y. pestis were found and exhibited a significant region-specific distribution. Additionally, the increased genetic diversity of plague hosts is positively associated with human plague outbreaks. This insight gained can improve our understanding of biodiversity for pathogen spillover and provide municipally directed targets for One Health surveillance development, which will be an informative next step toward increased monitoring of M. himalayana dynamics.

Pasteurella multocida strains of a novel capsular serotype and lethal to Marmota himalayana on Qinghai-Tibet plateau in China.

Pasteurella multocida is a zoonotic pathogen causing serious diseases in humans and animals. Here, we report P. multocida from wildlife on China's Qinghai-Tibet plateau with a novel capsular serotype, forming a single branch on the core-genome phylogenetic tree: four strains isolated from dead Himalayan marmot (Marmota himalayana) and one genome assembled from metagenomic sequencing of a dead Woolly hare (Lepus oiostolus). Four of the strains were identified as subspecies multocida and one was septica. The mouse model showed that the challenge strain killed mice within 24 h at an infectious dose of less than 300 bacteria. The short disease course is comparable to septicemic plague: the host has died before more severe pathological changes could take place. Though pathological changes were relatively mild, cytokine storm was obvious with a significant rise of IL-12p70, IL-6, TNF-αand IL-10 (P < 0.05). Our findings suggested P. multocida is a lethal pathogen for wildlife on Qinghai-Tibet plateau, in addition to Yersinia pestis. Individuals residing within the M. himalayana plague focus are at risk for P. multocida infection, and public health warnings are necessitated.

Molecular characterization of Enterocytozoon bieneusi genotypes in wild Altai marmot (Marmota baibacina) in Xinjiang, China: host specificity and adaptation.

Enterocytozoon bieneusi is responsible for opportunistic infections leading to gastrointestinal diseases in humans and animals worldwide. A total of 334 fresh fecal samples were collected from wild Altai marmots (Marmota baibacina) in Xinjiang, China, and E. bieneusi was screened via PCR amplification of the internal transcribed spacer (ITS) region of the small submit ribosomal RNA (SSU rRNA). The results indicated that 22.8% (76/334) of the wild Altai marmot fecal samples were positive for E. bieneusi, and the highest positive rate was detected in Akqi (51.9%, 27/52), with a significant difference from other sampling sites (p < 0.01). Four known genotypes (BEB6, CHG3, GX2, and YAK1) and three novel genotypes (XJHT2 to XJHT4) were identified in the present study. Genotype XJHT3 was dominant and detected in 48 fecal samples. In the phylogenetic analysis, the novel genotypes XJHT2 and XJHT3 were clustered in Group 1 together with the known genotype YAK1, while genotypes CHG3 and BEB6 were clustered in Group 2. The novel genotype XJHT4 was clustered together with other rodent-derived genotypes and generated a novel Group 14. These data confirmed the host specificity and adaptation of E. bieneusi in rodents. These findings enrich our understanding of the prevalence and genetic diversity of E. bieneusi in wild Altai marmots in Xinjiang, China.

Arthrobacter zhaoxinii sp. nov. and Arthrobacter jinronghuae sp. nov., isolated from Marmota himalayana.

Four yellow-coloured strains (zg-Y815T/zg-Y108 and zg-Y859T/zg-Y826) were isolated from the intestinal contents of Marmota himalayana and assigned to the 'Arthrobacter citreus group'. The four strains grew optimally on brain heart infusion agar with 5 % defibrinated sheep blood plate at 30 °C, pH 7.0 and with 0.5 % NaCl (w/v). Comparative analysis of their 16S rRNA genes indicated that the two strain pairs belong to the genus Arthrobacter, showing the highest similarity to Arthrobacter yangruifuii 785T (99.52 %), which was further confirmed by the 16S rRNA gene and genome-based phylogenetic analysis. The comparative genomic analysis [digital DNA-DNA hybridization, (dDDH) and average nucleotide identity (ANI)] proved that the four strains are two different species (zg-Y815T/zg-Y108, 71.7 %/96.8 %; zg-Y859T/zg-Y826, 87.3 %/98.5 %) and differ from other known species within the genus Arthrobacter (zg-Y815T, 19.6-32.3 %/77.2-88.0 %; zg-Y859T, 19.5-29.3 %/77.4-86.3 %). Strain pairs zg-Y815T/zg-Y108 and zg-Y859T/zg-Y826 had the same major cellular fatty acids (iso-C16 : 0 and anteiso-C15 : 0), with MK-8(H2) as their dominant respiratory quinone (70.6 and 61.7 %, respectively). The leading polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol. The detected amino acids and cell-wall sugars of the two new species were identical (amino acids: alanine, glutamic acid, and lysine; sugars: rhamnose, galactose, mannose, glucose, and ribose). According to the phylogenetic, phenotypic, and chemotaxonomic analyses, we concluded that the four new strains represented two different novel species in the genus Arthrobacter, for which the names Arthrobacter zhaoxinii sp. nov. (zg-Y815T= GDMCC 1.3494T = JCM 35821T) and Arthrobacter jinronghuae sp. nov. (zg-Y859T = GDMCC 1.3493T = JCM 35822T) are proposed.

Taxonomic descriptions of Aeromicrobium duanguangcaii sp. nov., Aeromicrobium wangtongii sp. nov. and Aeromicrobium senzhongii sp. nov.

Six Gram-stain-positive, facultative anaerobic, nonmotile and rod-shaped strains, designated zg-Y50T, zg-Y1362, zg-Y1379T, zg-Y869, zg-629T and zg-Y636, were isolated from the intestinal contents of Marmota himalayana in Qinghai Province, PR China. Strains zg-Y50T, zg-Y1379T and zg-629T exhibited the highest 16S rRNA gene sequence similarities of 99.2, 98.9 and 98.8 % to Aeromicrobium choanae 9 H-4T, Aeromicrobium ginsengisoli JCM 14732T and Aeromicrobium flavum TYLN1T, respectively. Phylogenetic and phylogenomic analyses based on the 16S rRNA gene and genomic sequences, respectively, revealed that the six strains formed three distinct clades within the genus Aeromicrobium. The genome sizes of strains zg-Y50T, zg-Y1379T and zg-629T were 3.1-3.7 Mb, with DNA G+C contents of 69.6-70.4 mol%. Average nucleotide identity and digital DNA-DNA hybridization values between each novel strain and available members of the genus Aeromicrobium were all below species thresholds. All novel strains contained MK-9 (H4) as the major menaquinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the polar lipids. The predominant fatty acid of the six isolates was C18 : 1 ω9c. The cell-wall peptidoglycan contained ʟʟ-diaminopimelic acid as the diagnostic diamino acid. Based on the results from this polyphasic taxonomic study, three novel species in the genus Aeromicrobium are proposed, namely, Aeromicrobium duanguangcaii sp. nov. (zg-Y50T=GDMCC 1.2981T=KCTC 49764T), Aeromicrobium wangtongii sp. nov. (zg-Y1379T=GDMCC 1.2982T=KCTC 49765T) and Aeromicrobium senzhongii sp. nov. (zg-629T=CGMCC 1.17414T=JCM 33888T).

RABIES IN RODENTS AND LAGOMORPHS IN THE USA, 2011-20.

Rabies is an acute progressive encephalitis caused by infection with rabies viruses, with reservoirs among bats and mesocarnivores, but all mammals are susceptible. Despite its distribution and abundance, cases of rabies are much less common in rodents and lagomorphs. Familiarity with current rabies prevalence data is important for informed decisions on human postexposure prophylaxis after rodent and lagomorph bites. This study is an update of rabies cases reported in rodents and lagomorphs in the US from 2011 to 2020. Rabies reports were collected passively from laboratory testing agencies in the US and Puerto Rico from 2011 to 2020. Descriptive analysis was conducted to determine the percent positivity of rabies cases by species. A total of 401 cases of rabies in rodents and lagomorphs were reported from 2011 to 2020. Most reported cases were in groundhogs (Marmota monax), representing >90% of cases, and the trend closely aligned with rabies in raccoons (Procyon lotor). In any given year, the percent positivity of rabies in rodents and lagomorphs was <2.5%, and the trend of percent positivity from 2011 to 2020 was stable. Groundhog and North American beaver (Castor canadensis) percent positivity was significantly higher than the rest of the rodents and lagomorphs. Most rabies cases occurred during the months of May-September. Documented cases of rabies in rodents and lagomorphs are generally rare, but with variation between species. Groundhogs and North American beavers had rabies percent positivity similar to high-risk species, such as bats and raccoons, and constituted 97% of all rodent and lagomorph positive cases. Since 1993, the trend in rabies cases in groundhogs has significantly declined. These results can be used to help inform public health officials on rodent and lagomorph prevention and control efforts, as well as rabies postexposure prophylaxis.

Comparison of hematologic and serum biochemical variables in Brucella-infected and Brucella-free adult Himalayan marmots (Marmota himalayana).

BACKGROUND: The Himalayan marmot (Marmota himalayana) plays a reservoir role in the epidemiology of brucellosis. However, the changes in blood biochemical parameters are still unclear in Brucella-seropositive marmots. OBJECTIVES: The present study was designed to explore the hematologic and biochemical variable changes in Brucella-seropositive marmots. METHODS: Blood samples were collected from the dorsalis pedis vein of Himalayan marmots (24 Brucella-seropositive marmots and 24 Brucella-free marmots). Ten hematologic and 10 serum biochemical variable examinations were performed and analyzed. RESULTS: Our results showed that leukocyte, platelet, neutrophil, and lymphocyte counts significantly increased, while the level of carbon dioxide combining power decreased in Brucella-infected marmots. These findings indicate that Brucella triggers an immune response in Himalayan marmots. CONCLUSIONS: This study provides a preliminary investigation of the changes in blood biochemical analytes in Brucella-infected marmots. The interaction between Brucella infection and blood biochemical indices in Himalayan marmots should be further explored.

Aborted infection of human sodium taurocholate cotransporting polypeptide (hNTCP) expressing woodchuck hepatocytes with hepatitis B virus (HBV).

Due to the limited host range of HBV, research progress has been hindered by the absence of a suitable animal model. The natural history of woodchuck hepatitis virus (WHV) infection in woodchuck closely mirrors that of HBV infection in human, making this species a promising candidate for establishing both in vivo and in vitro HBV infection models. Therefore, this animal may be a valuable species to evaluate HBV vaccines and anti-HBV drugs. A significant milestone in HBV and hepatitis D virus (HDV) infection is the discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the functional receptor. In an effort to enhance susceptibility to HBV infection, we introduced hNTCP into the woodchuck hepatocytes by multiple approaches including transduction of vLentivirus-hNTCP in woodchuck hepatocytes, transfection of p-lentivirus-hNTCP-eGFP plasmids into these cells, as well as transduction of vAdenovirus-hNTCP-eGFP. Encouragingly, our findings demonstrated the successful introduction of hNTCP into woodchuck hepatocytes. However, it was observed that these hNTCP-expressing hepatocytes were only susceptible to HDV infection but not HBV. This suggests the presence of additional crucial factors mediating early-stage HBV infection that are subject to stringent species-specific restrictions.

Characterization and comparative analysis of the Escherichia marmotae M-12 isolate from bank vole (Myodes glareolus).

The Escherichia marmotae is a bacterium of the Enterobacterales order, which was first isolated from the Himalayan marmot (Marmota himalayana). Recently E. marmotae has been shown to cause severe infections in humans. Wild animals were suggested to be a natural reservoir of this bacterium. The present study describes the first case of E. marmotae isolation from an apparently healthy wild bank vole (Myodes glareolus). Phenotype, as well as genotype-based techniques, were applied to characterize E. marmotae M-12 isolate. E. marmotae M-12 had the capsule-positive phenotype, high adhesion to human erythrocytes and HEp-2 cells as well as a low invasion into HEp-2 cells. E. marmotae M-12 was avirulent in mice. The phylogenomic analyses of E. marmotae showed dispersed phylogenetic structure among isolates of different origins. Virulome analysis of M-12 isolate revealed the presence of the following factors: siderophores, heme uptake systems, capsule synthesis, curli and type I fimbriae, flagella proteins, OmpA porin, etc. Comparative virulome analysis among available E. marmotae genomes revealed the presence of capsule K1 genes mostly in pathogenic isolates and OmpA porin presence among all strains. We assume that the K1 capsule and OmpA porin play a key role in the virulence of E. marmotae. Pathogenesis of the latter might be similar to extraintestinal pathogenic E. coli.

Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov., isolated from the respiratory tracts of marmots.

Four Gram-stain-negative, oxidase-positive, non-motile, cocci-shaped bacteria strains (ZJ106T, ZJ104, ZJ785T and ZJ930) were isolated from marmot respiratory tracts. Phylogenetic analyses based on 16S rRNA genes, 53 ribosomal protein sequences and 441 core genes supported that all four strains belonged to the genus Neisseria with close relatives Neisseria weixii 10022T and Neisseria iguanae ATCC 51483T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the species-level thresholds (95-96 % for ANI, and 70 % for dDDH). The major fatty acids of all four strains were C16 : 1 ω7c /C16 : 1 ω6c, C16 : 0 and C18 : 1 ω9c. Major polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. MK-8 was the major menaquinone. Based on Virulence Factor Database analysis, the four strains were found to contain NspA and PorB H-factor binding proteins that promote evasion of host immunity. Strains ZJ106T and ZJ104 contained structures similar to the capsule synthesis manipulator of Neisseria meningitidis. Based on phenotypic and phylogenetic evidence, we propose that strains ZJ106T and ZJ785T represent two novel species of the genus Neisseria, respectively, with the names Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov. The type strains are ZJ106T (=GDMCC 1.3111T=JCM 35323T) and ZJ785T (=GDMCC 1.1998T=KCTC 82336T).

Mapping small mammal optimal habitats using satellite-derived proxy variables and species distribution models.

Small mammal species play an important role influencing vegetation primary productivity and plant species composition, seed dispersal, soil structure, and as predator and/or prey species. Species which experience population dynamics cycles can, at high population phases, heavily impact agricultural sectors and promote rodent-borne disease transmission. To better understand the drivers behind small mammal distributions and abundances, and how these differ for individual species, it is necessary to characterise landscape variables important for the life cycles of the species in question. In this study, a suite of Earth observation derived metrics quantifying landscape characteristics and dynamics, and in-situ small mammal trapline and transect survey data, are used to generate random forest species distribution models for nine small mammal species for study sites in Narati, China and Sary Mogul, Kyrgyzstan. These species distribution models identify the important landscape proxy variables driving species abundance and distributions, in turn identifying the optimal conditions for each species. The observed relationships differed between species, with the number of landscape proxy variables identified as important for each species ranging from 3 for Microtus gregalis at Sary Mogul, to 26 for Ellobius tancrei at Narati. Results indicate that grasslands were predicted to hold higher abundances of Microtus obscurus, E. tancrei and Marmota baibacina, forest areas hold higher abundances of Myodes centralis and Sorex asper, with mixed forest-grassland boundary areas and areas close to watercourses predicted to hold higher abundances of Apodemus uralensis and Sicista tianshanica. Localised variability in vegetation and wetness conditions, as well as presence of certain habitat types, are also shown to influence these small mammal species abundances. Predictive application of the Random Forest (RF) models identified spatial hot-spots of high abundance, with model validation producing R2 values between 0.670 for M. gregalis transect data at Sary Mogul to 0.939 for E. tancrei transect data at Narati. This enhances previous work whereby optimal habitat was defined simply as presence of a given land cover type, and instead defines optimal habitat via a combination of important landscape dynamic variables, moving from a human-defined to species-defined perspective of optimal habitat. The species distribution models demonstrate differing distributions and abundances of host species across the study areas, utilising the strengths of Earth observation data to improve our understanding of landscape and ecological linkages to small mammal distributions and abundances.

Screening of Phyllanthus niruri Leaves Phytoconstituents for Antiviral and Antibacterial Activity by Molecular Docking Studies.

The genus Phyllanthus belongs to one of the largest plant families, the Phyllantaceae (L.). Phyllanthus niruri is an annual perennial herb that grows in tropical Asia, America, China, and the islands of the Indian Ocean. Numerous alkaloids, steroids, flavonoids, lignans, coumarins, polyphenols, and lipids are present in Phyllanthus. The effects of plants have been studied for a variety of purposes, including their antioxidant (Giribabu et al., Evid Based Complement Alternat Med, 2014), anti-inflammatory (Porto et al., Revista Brasileira de Pharmacognosy, 2013), antinociceptive (Sathisha et al., Indian Drugs, 2009), analgesic (Mostofa et al., BMC Complement Altern Med, 2017), antiulcer (Mali et al., Biomed Aging Pathol, 2011), antiarthritic (Obidike and Salawu, Planta Medica, 2010), antiplasmodial (Shilpa et al., Environ Dis, 2018), immunomodulatory (Manikkoth et al., Anticonvulsant activity of Phyllanthus amarus in experimental animal models), anticonvulsant (Wasnik et al., Int J Pharm Sci Rev Res, 2014), antidepressant (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antiviral (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antitumor (Sharma et al., Asian Pac J Cancer Prev, 2009), hyperlipidemia (Khanna et al., J Ethnopharmacol, 2002), and antifertility (Ezeonwu, Inquiries J, 2011). For additional docking investigations with distinct proteins, the leaf chemicals are assessed, that is, the crystal structure of serine protease hepsin in complex with inhibitor [PDB ID:5 CE1] for antiviral activity human topoisomerase II beta in complex with DNA and etoposide [PDB ID:3QX3] and crystal structure of E. coli GyraseB 24 kDa in complex with 4-(4-bromo-1H-pyrazol-1-yl)-6-[(ethylcarbamoyl)amino]-N-(pyridin-3-yl) pyridine-3-carboxamide [PDB ID: 6F86] for antibacterial activity and have been selected. To evaluate the in silico results and grading of virtual screening, or molecular docking, ritonavir antiviral activity and ampicillin for antibacterial activity were used as a benchmark.

COMPARISON OF KETAMINE-MIDAZOLAM AND KETAMINE-MIDAZOLAM-BUTORPHANOL PREMEDICATION PRIOR TO SEVOFLURANE ANESTHESIA IN WOODCHUCKS (MARMOTA MONAX).

Cardiovascular disease is a frequent cause of death in the critically endangered Vancouver Island marmots (Marmota vancouverensis). This warrants the use of anesthetic protocols with minimal cardiovascular adverse effects. In this study, 12 adult male woodchucks (Marmota monax) were used as models for Vancouver Island marmots. The objective was to compare the physiological effects of two premedication protocols during induction and maintenance of anesthesia with sevoflurane. The two premedications were ketamine 10 mg/kg and midazolam 0.5 mg/kg (KM) or ketamine 10 mg/kg, midazolam 0.5 mg/kg, and butorphanol 1.0 mg/kg (KMB), administered intramuscularly prior to mask induction. Each marmot underwent three anesthetic events and protocols were assigned using a blinded randomized crossover design. Heart rate, respiratory rate, oxygen saturation, and body temperature were recorded throughout, and blood gases were assessed following induction. Resistance to induction was scored and time to induction was recorded. Although mask induction with sevoflurane was successful in all events (mean induction time of 2.1 min), KMB premedication resulted in a faster induction (mean induction time reduced by 1.2 ± 0.3 min) and lower resistance scores. Both protocols resulted in significant cardiovascular and respiratory depression; however, animals that received KMB were more hypercapnic than KM by 8.8 ± 2.8 mm Hg (P = 0.03) (mean venous partial pressure of carbon dioxide [PvCO2] for all: 79.9 mm Hg). In conclusion, if shorter induction times are desired, KMB premedication is preferred. However, cardiorespiratory variables including blood pressure should be monitored, and endotracheal intubation is recommended to allow for ETCO2 monitoring and provision of intermittent positive pressure ventilation.

Climate-driven marmot-plague dynamics in Mongolia and China.

The incidence of plague has rebounded in the Americas, Asia, and Africa alongside rapid globalization and climate change. Previous studies have shown local climate to have significant nonlinear effects on plague dynamics among rodent communities. We analyzed an 18-year database of plague, spanning 1998 to 2015, in the foci of Mongolia and China to trace the associations between marmot plague and climate factors. Our results suggested a density-dependent effect of precipitation and a geographic location-dependent effect of temperature on marmot plague. That is, a significantly positive relationship was evident between risk of plague and precipitation only when the marmot density exceeded a certain threshold. The geographical heterogeneity of the temperature effect and the contrasting slopes of influence for the Qinghai-Tibet Plateau (QTP) and other regions in the study (nQTP) were primarily related to diversity of climate and landscape types.

Fatty acid metabolization and insulin regulation prevent liver injury from lipid accumulation in Himalayan marmots.

Fat storage and weight gain are dominant traits for hibernating mammals. However, excessive fat accumulation may cause liver damage. Here, we explore the lipid accumulation and metabolic processes of the Himalayan marmot (Marmota himalayana), a hibernating rodent species. We find that the unsaturated fatty acid (UFA) content in food was consistent with a large increase in the body mass of Himalayan marmots. Metagenomic analysis shows that Firmicutes Bacterium CAG:110 plays a synergistic role by synthesizing UFAs, which is demonstrated by fecal transplantation experiments, indicating that the gut microbiome promotes fat storage in Himalayan marmots for hibernation. Microscopic examination results indicate that the risk of fatty liver appears at maximum weight; however, liver function is not affected. Upregulations of UFA catabolism and insulin-like growth factor binding protein genes provide an entry point for avoiding liver injury.

Cellulomonas xiejunii sp. nov., Cellulomonas chengniuliangii sp. nov. and Cellulomonas wangsupingiae sp. nov., three cellulolytic bacteria isolated from intestinal contents of Marmota himalayana.

Six facultative anaerobic, Gram-stain-positive, oxidase-negative, rod-shaped bacteria (strains zg-B89T, zg-B12, zg-Y338T, zg-Y138, zg-Y908T and zg-Y766), were isolated from the intestinal contents of Marmota himalayana in Qinghai Province, PR China. The 16S rRNA gene sequence analysis showed that zg-B89T showed highest similarity to Cellulomonas iranensis NBRC 101100T (99.5 %), zg-Y338T to Cellulomonas cellasea DSM 20118T (98.7 %), and zg-Y908T to Cellulomonas flavigena DSM 20109T (99.0 %). Phylogenetic and phylogenomic analysis based on 16S rRNA gene and 881 core genes revealed that these six strains formed three separate clades in the genus Cellulomonas. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between these three novel species and all members of the genus Cellulomonas were below species thresholds (95-96 % for ANI and 70 % for dDDH). The DNA G+C contents of zg-B89T, zg-Y338T and zg-Y908T were 73.6, 72.9 and 74.5 %, respectively. Strains zg-B89T and zg-Y908T had anteiso-C15 : 0, C16 : 0 and anteiso-C15 : 1 A, and zg-Y338T had anteiso-C15 : 0, C16 : 0 and iso-C16 : 0 as the main fatty acids. All novel type strains had MK-9 (H4) as the predominant respiratory quinone, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside as the major polar lipids, and rhamnose, ribose and glucose as the cell-wall sugars. The peptidoglycan amino acids of zg-B89T, zg-Y338T and zg-Y908T contained ornithine, alanine, glutamic acid and aspartic acid (except for zg-Y338T). Based on genotypic, phenotypic, phylogenetic and biochemical properties, the six uncharacterized strains represent three novel species in the genus Cellulomonas, for which the names Cellulomonas xiejunii sp. nov. (type strain zg-B89T=GDMCC 1.2821T=KCTC 49756T), Cellulomonas chengniuliangii sp. nov. (type strain zg-Y338T=GDMCC 1.2829T=KCTC 49754T) and Cellulomonas wangsupingiae sp. nov. (type strain zg-Y908T=GDMCC 1.2820T=KCTC 49755T) are proposed, respectively.