Molecular Prevalence, Genetic Diversity, and Tissue Tropism of Bartonella Species in Small Mammals from Yunnan Province, China.

Bartonella is an intracellular parasitic zoonotic pathogen that can infect animals and cause a variety of human diseases. This study investigates Bartonella prevalence in small mammals in Yunnan Province, China, focusing on tissue tropism. A total of 333 small mammals were sampled from thirteen species, three orders, four families, and four genera in Heqing and Gongshan Counties. Conventional PCR and real-time quantitative PCR (qPCR) were utilized for detection and quantification, followed by bioinformatic analysis of obtained DNA sequences. Results show a 31.5% detection rate, varying across species. Notably, Apodemus chevrieri, Eothenomys eleusis, Niviventer fulvescens, Rattus tanezumi, Episoriculus leucops, Anourosorex squamipes, and Ochotona Thibetana exhibited infection rates of 44.4%, 27.7%, 100.0%, 6.3%, 60.0%, 23.5%, and 22.2%, respectively. Genetic analysis identified thirty, ten, and five strains based on ssrA, rpoB, and gltA genes, with nucleotide identities ranging from 92.1% to 100.0%. Bartonella strains were assigned to B. grahamii, B. rochalimae, B. sendai, B. koshimizu, B. phoceensis, B. taylorii, and a new species identified in Episoriculus leucops (GS136). Analysis of the different tissues naturally infected by Bartonella species revealed varied copy numbers across different tissues, with the highest load in spleen tissue. These findings underscore Bartonella's diverse species and host range in Yunnan Province, highlighting the presence of extensive tissue tropism in Bartonella species naturally infecting small mammalian tissues.

Bayesian network-based survival prediction model for patients having undergone post-transjugular intrahepatic portosystemic shunt for portal hypertension.

BACKGROUND: Portal hypertension (PHT), primarily induced by cirrhosis, manifests severe symptoms impacting patient survival. Although transjugular intrahepatic portosystemic shunt (TIPS) is a critical intervention for managing PHT, it carries risks like hepatic encephalopathy, thus affecting patient survival prognosis. To our knowledge, existing prognostic models for post-TIPS survival in patients with PHT fail to account for the interplay among and collective impact of various prognostic factors on outcomes. Consequently, the development of an innovative modeling approach is essential to address this limitation. AIM: To develop and validate a Bayesian network (BN)-based survival prediction model for patients with cirrhosis-induced PHT having undergone TIPS. METHODS: The clinical data of 393 patients with cirrhosis-induced PHT who underwent TIPS surgery at the Second Affiliated Hospital of Chongqing Medical University between January 2015 and May 2022 were retrospectively analyzed. Variables were selected using Cox and least absolute shrinkage and selection operator regression methods, and a BN-based model was established and evaluated to predict survival in patients having undergone TIPS surgery for PHT. RESULTS: Variable selection revealed the following as key factors impacting survival: age, ascites, hypertension, indications for TIPS, postoperative portal vein pressure (post-PVP), aspartate aminotransferase, alkaline phosphatase, total bilirubin, prealbumin, the Child-Pugh grade, and the model for end-stage liver disease (MELD) score. Based on the above-mentioned variables, a BN-based 2-year survival prognostic prediction model was constructed, which identified the following factors to be directly linked to the survival time: age, ascites, indications for TIPS, concurrent hypertension, post-PVP, the Child-Pugh grade, and the MELD score. The Bayesian information criterion was 3589.04, and 10-fold cross-validation indicated an average log-likelihood loss of 5.55 with a standard deviation of 0.16. The model's accuracy, precision, recall, and F1 score were 0.90, 0.92, 0.97, and 0.95 respectively, with the area under the receiver operating characteristic curve being 0.72. CONCLUSION: This study successfully developed a BN-based survival prediction model with good predictive capabilities. It offers valuable insights for treatment strategies and prognostic evaluations in patients having undergone TIPS surgery for PHT.

Molecular epidemiological study of Scrub Typhus in residence, farm and forest habitats from Yunnan Province, China.

The number of people suffering from scrub typhus, which is not of concern, is increasing year by year, especially in Yunnan Province, China. From June 1, 2021 to August 15, 2022, a total of 505 mammalian samples were collected from farm, forest, and residential habitats with high incidence of scrub typhus in Yunnan, China, for nPCR (nested PCR) and qPCR (quantitative real-time PCR) detection of Orientia tsutsugamushi. A total of 4 orders of murine-like animals, Rodentia (87.52%, n = 442), Insectivora (10.29%, n = 52), Lagomorpha (1.79%, n = 9) and Scandentia (0.40%, n = 2) were trapped. Comparing the qPCR infection rates in the three habitats, it was no significant difference that the infection rate of residential habitat (44.44%) and that of the farm habitat (45.05%, P>0.05), which is much larger than that of the forest habitat (3.08%) (P<0.001). Three genotypes (Karp-like, Kato-like and TA763-like) of O. tsutsugamushi were found from Yunnan, China in this study.

Detection of Alpha- and Betacoronaviruses in Small Mammals in Western Yunnan Province, China.

The genetic diversity of coronaviruses (CoVs) is high, and their infection in animals has not yet been fully revealed. By RT-PCR detection of the partial RNA-dependent RNA polymerase (RdRp) gene of CoVs, we screened a total of 502 small mammals in the Dali and Nujiang prefectures of Western Yunnan Province, China. The number of overall CoV positives was 20, including ß-CoV (n = 13) and α-CoV (n = 7), with a 3.98% prevalence in rectal tissue samples. The identity of the partial RdRp genes obtained for 13 strains of ß-CoV was 83.42-99.23% at the nucleotide level, and it is worth noting that the two strains from Kachin red-backed voles showed high identity to BOV-36/IND/2015 from Indian bovines and DcCoV-HKU23 from dromedary camels (Camelus dromedarius) in Morocco; the nucleotide identity was between 97.86 and 98.33%. Similarly, the identity of the seven strains of α-CoV among the partial RdRp sequences was 94.00-99.18% at nucleotide levels. The viral load in different tissues was measured by quantitative RT-PCR (qRT-PCR). The average CoV viral load in small mammalian rectal tissue was 1.35 × 106 copies/g; differently, the mean CoV viral load in liver, heart, lung, spleen, and kidney tissue was from 0.97 × 103 to 3.95 × 103 copies/g, which revealed that CoV has extensive tropism in rectal tissue in small mammals (p < 0.0001). These results revealed the genetic diversity, epidemiology, and infective tropism of α-CoV and ß-CoV in small mammals from Dali and Nujiang, which deepens the comprehension of the retention and infection of coronavirus in natural hosts.

Ecological study of cave nectar bats reveals low risk of direct transmission of bat viruses to humans.

Bats are reservoirs of various viruses. The widely distributed cave nectar bat ( Eonycteris spelaea) is known to carry both filoviruses and coronaviruses. However, the potential transmission of theses bat viruses to humans is not fully understood. In this study, we tracked 16 E. spelaea bats in Mengla County, Yunnan Province, China, using miniaturized GPS devices to investigate their movements and potential contact with humans. Furthermore, to determine the prevalence of coronavirus and filovirus infections, we screened for the nucleic acids of the Menglà virus (MLAV) and two coronaviruses (GCCDC1-CoV and HKU9-CoV) in anal swab samples taken from bats and for antibodies against these viruses in human serum samples. None of the serum samples were found to contain antibodies against the bat viruses. The GPS tracking results showed that the bats did not fly during the daytime and rarely flew to residential areas. The foraging range of individual bats also varied, with a mean cumulative nightly flight distance of 25.50 km and flight speed of up to 57.4 km/h. Taken together, these results suggest that the risk of direct transmission of GCCDC1-CoV, HKU9-CoV, and MLAV from E. spelaea bats to humans is very low under natural conditions.

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

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

The phylogeny of the Anderson's White-bellied Rat (Niviventer andersoni) based on complete mitochondrial genomes.

The phylogenetic structure of the genus Niviventer has been studied based on several individual mitochondrial and nuclear genes, but the results seem to be inconsistent. In order to clarify the phylogeny of Niviventer, we sequenced the complete mitochondrial genome of white-bellied rat (Niviventer andersoni of the family Muridae) by next-generation sequencing. The 16,291 bp mitochondrial genome consists of 22 transfer RNA genes, 13 protein-coding genes (PCGs), two ribosomal RNA genes, and one noncoding control region (D-Loop). Phylogenetic analyses of the nucleotide sequences of all 13 PCGs, PCGs minus ND6, and the entire mitogenome sequence except for the D-loop revealed well-resolved topologies supporting that N. andersoni was clustered with N. excelsior forming a sister division with N. confucianus, which statistically rejected the hypothesis based on the tree of cytochrome b (cytb) gene that N. confucianus is sister to N. fulvescens. Our research provides the first annotated complete mitochondrial genome of N. andersoni, extending the understanding about taxonomy and mitogenomic evolution of the genus Niviventer.

Differences in transcriptomic and metabolomic analyses of metabolites of shoots on tea plants of different ages and relevant regulatory network.

To investigate differences in fresh leaves of tea plants at different ages in gene expression, metabolism, and dried tea quality, and to provide references to a deep exploration on metabolite differential accumulation of fresh leaves of tea plants at different ages as well as the regulation mechanism, two groups of fresh leaves from tea plants at different ages (group JP: 20-, 200-, and 1,200-year tea plants; group YX: 50-, 100-, and 400-year tea plants) were chosen as materials, and their differences in gene expression, metabolites, and metabolic regulatory network were investigated by transcriptomics and metabolomics. A total of 12,706 differentially expressed genes (DEGs) were screened from the fresh tea leaves in the JP group, of which tea-20 vs. tea-200 had the largest number of DEGs, up to 9,041 (4,459 down-regulated genes, 4,582 up-regulated genes). A total of 644 common genes in the fresh leaves of three different ages of tea plants in the JP group were differentially expressed. A total of 8,971 DEGs were screened from the fresh leaf samples of tea plants in the YX group, of which the number of DEGs obtained in the tea-50 vs. tea-400 comparison combination was the largest with a total of 3,723 (1,722 up-regulated genes and 2,001 down-regulated genes). A total of 147 common genes were differentially expressed in the fresh leaves of three different tree ages in the YX group. The pathway enrichment analysis showed that most up-regulated DEGs and their related metabolic pathways were similar in the two groups, and that the metabolic pathways of common significant enrichment included flavonoid biosynthesis, phenylpropane biosynthesis, carbon metabolism, amino acid biosynthesis, and plant pathogen interaction. The metabolomics results showed that 72 and 117 different metabolites were screened from the JP and YX groups, respectively. Most of the different metabolites in the two groups were flavonoids, phenolic acids, amino acids, and their derivatives. Among them, the number of down-regulated flavonoids in older tea plants is generally higher than the number of up-regulated flavonoids. Moreover, according to the sensory evaluation results of dried tea of fresh leaves from tea plants of different ages, tea-1200 and tea-400 showed the highest sensory evaluation scores in their groups. With increase in plant age, the fragrance of the tea was more elegant, and it changed from a dense scent to a faint scent; the tea tasted sweet and its freshness increased, while the sense of astringency was weakened and the concentration declined. Therefore, the quality difference of tea of different tree ages is mainly related to secondary metabolic pathways such as the flavonoid biosynthesis pathway. With increase in tea age, a large number of gene expression in the flavonoid biosynthesis pathway is down-regulated, which reduces the content of bitter flavonoid substances in fresh leaves and makes tea soup more mellow.

A qualitative study of zoonotic risk factors among rural communities in southern China.

BACKGROUND: Strategies are urgently needed to mitigate the risk of zoonotic disease emergence in southern China, where pathogens with zoonotic potential are known to circulate in wild animal populations. However, the risk factors leading to emergence are poorly understood, which presents a challenge in developing appropriate mitigation strategies for local communities. METHODS: Residents in rural communities of Yunnan, Guangxi and Guangdong provinces were recruited and enrolled in this study. Data were collected through ethnographic interviews and field observations, and thematically coded and analysed to identify both risk and protective factors for zoonotic disease emergence at the individual, community and policy levels. RESULTS: Eighty-eight ethnographic interviews and 55 field observations were conducted at nine selected sites. Frequent human-animal interactions and low levels of environmental biosecurity in local communities were identified as risks for zoonotic disease emergence. Policies and programmes existing in the communities provide opportunities for zoonotic risk mitigation. CONCLUSIONS: This study explored the relationship among zoonotic risk and human behaviour, environment and policies in rural communities in southern China. It identifies key behavioural risk factors that can be targeted for development of tailored risk-mitigation strategies to reduce the threat of novel zoonoses.

[The including and recommendation of acupuncture in NGC and NICE].

The diseases recommended to be treated by acupuncture and the recommendation in the National Guideline Clearinghouse (NGC) and the National Institute for Health and Care Excellence (NICE) were summarized, and the diseases recommended were analyzed. The guidelines which mentioned acupuncture in NICE and NGC were searched and classified according to the types of diseases and intensity of recommendations. As a result, 45 guidelines in NGC were included, involving 59 recommendations; 29 guidelines in the NICE, involving 29 recommendations. The diseases recommended by NICE and NGC were mainly pain-related diseases, and the diseases recommended by NGC were wider. However, both needed to further describe the specific operation methods of acupuncture.

Detection and genome characterization of two novel papillomaviruses and a novel polyomavirus in tree shrew (Tupaia belangeri chinensis) in China.

BACKGROUND: Papillomaviruses (PVs) and polyomaviruses (PyVs) infect diverse vertebrates including human and cause a broad spectrum of outcomes from asymptomatic infection to severe disease. There has been no PV and only one PyV detected in tree shrews, though the genomic properties of tree shrews are highly similar to those of the primates. METHODS: Swab and organ samples of tree shrews collected in the Yunnan Province of China, were tested by viral metagenomic analysis and random PCR to detect the presence of PVs and PyVs. By PCR amplification using specific primers, cloning, sequencing and assembling, genomes of two PVs and one PyV were identified in the samples. RESULTS: Two novel PVs and a novel PyV, named tree shrew papillomavirus 1 and 2 (TbelPV1 and TbelPV2) and polyomavirus 1 (TbelPyV1) were characterized in the Chinese tree shrew (Tupaia belangeri chinensis). The genomes of TbelPV1, TbelPV2, and TbelPyV1 are 7410 bp, 7526 bp, and 4982 bp in size, respectively. The TbelPV1 genome contains 7 putative open-reading frames (ORFs) coding for viral proteins E1, E2, E4, E6, E7, L1, and L2; the TbelPV2 genome contains 6 ORFs coding for viral proteins E1, E2, E6, E7, L1, and L2; and the TbelPyV1 genome codes for the typical small and large T antigens of PyV, as well as the VP1, VP2, and VP3 capsid proteins. Genomic comparison and phylogenetic analysis indicated that TbelPV1 and TbelPV2 represented 2 novel PV genera of Papillomaviridae, and TbelPyV1 represented a new species of genus Alphapolyomavirus. Our epidemiologic study indicated that TbelPV1 and TbelPV2 were both detected in oral swabs, while TbelPyV1 was detected in oral swabs and spleens. CONCLUSION: Two novel PVs (TbelPV1 and TbelPV2) and a novel PyV (TbelPyV) were discovered in tree shrews and their genomes were characterized. TbelPV1, TbelPV2, and TbelPyV1 have the highest similarity to Human papillomavirus type 63, Ursus maritimus papillomavirus 1, and Human polyomavirus 9, respectively. TbelPV1 and TbelPV2 only showed oral tropism, while TbelPyV1 showed oral and spleen tropism.

Author Correction: Characterization of a filovirus (Menglà virus) from Rousettus bats in China.

In the version of this Letter originally published, in the 'Phylogenetic analysis' section of the Methods, the authors mistakenly stated that the GenBank accession number for the Ravn virus genome sequence was FJ750958. The correct accession number is DQ447649 for Ravn virus, Kenya, 1987. Accordingly, the label 'RAVN2007' in Fig. 1b should have been 'RAVV1987'. This mistake does not change any conclusions in this study. This statement and figure have now been amended in all versions of the Letter, and the Supplementary Information file has been updated accordingly.

Characterization of a filovirus (Menglà virus) from Rousettus bats in China.

Filoviruses, especially Ebola virus (EBOV) and Marburg virus (MARV), are notoriously pathogenic and capable of causing severe haemorrhagic fever diseases in humans with high lethality1,2. The risk of future outbreaks is exacerbated by the discovery of other bat-borne filoviruses of wide genetic diversity globally3-5. Here we report the characterization of a phylogenetically distinct bat filovirus, named Menglà virus (MLAV). The coding-complete genome of MLAV shares 32-54% nucleotide sequence identity with known filoviruses. Phylogenetic analysis places this new virus between EBOV and MARV, suggesting the need for a new genus taxon. Importantly, despite the low amino acid sequence identity (22-39%) of the glycoprotein with other filoviruses, MLAV is capable of using the Niemann-Pick C1 (NPC1) as entry receptor. MLAV is also replication-competent with chimeric MLAV mini-genomes containing EBOV or MARV leader and trailer sequences, indicating that these viruses are evolutionally and functionally closely related. Finally, MLAV glycoprotein-typed pseudo-types transduced cell lines derived from humans, monkeys, dogs, hamsters and bats, implying a broad species cell tropism with a high risk of interspecies spillover transmission.

Chevrier's Field Mouse (Apodemus chevrieri) and Père David's Vole (Eothenomys melanogaster) in China Carry Orthohepeviruses that form Two Putative Novel Genotypes Within the Species Orthohepevirus C.

Hepatitis E virus (HEV) is the prototype of the family Hepeviridae and the causative agent of common acute viral hepatitis. Genetically diverse HEV-related viruses have been detected in a variety of mammals and some of them may have zoonotic potential. In this study, we tested 278 specimens collected from seven wild small mammal species in Yunnan province, China, for the presence and prevalence of orthohepevirus by broad-spectrum reverse transcription (RT)-PCR. HEV-related sequences were detected in two rodent species, including Chevrier's field mouse (Apodemus chevrieri, family Muridae) and Père David's vole (Eothenomys melanogaster, family Cricetidae), with the infection rates of 29.20% (59/202) and 7.27% (4/55), respectively. Further four representative full-length genomes were generated: two each from Chevrier's field mouse (named RdHEVAc14 and RdHEVAc86) and Père David's vole (RdHEVEm40 and RdHEVEm67). Phylogenetic analyses and pairwise distance comparisons of whole genome sequences and amino acid sequences of the gene coding regions showed that orthohepeviruses identified in Chinese Chevrier's field mouse and Père David's vole belonged to the species Orthohepevirus C but were highly divergent from the two assigned genotypes: HEV-C1 derived from rat and shrew, and HEV-C2 derived from ferret and possibly mink. Quantitative real-time RT-PCR demonstrated that these newly discovered orthohepeviruses had hepatic tropism. In summary, our work discovered two putative novel genotypes orthohepeviruses preliminarily named HEV-C3 and HEV-C4 within the species Orthohepevirus C, which expands our understanding of orthohepevirus infection in the order Rodentia and gives new insights into the origin, evolution, and host range of orthohepevirus.

Longitudinal Surveillance of Betacoronaviruses in Fruit Bats in Yunnan Province, China During 2009-2016.

Previous studies indicated that fruit bats carry two betacoronaviruses, BatCoV HKU9 and BatCoV GCCDC1. To investigate the epidemiology and genetic diversity of these coronaviruses, we conducted a longitudinal surveillance in fruit bats in Yunnan province, China during 2009-2016. A total of 59 (10.63%) bat samples were positive for the two betacorona-viruses, 46 (8.29%) for HKU9 and 13 (2.34%) for GCCDC1, or closely related viruses. We identified a novel HKU9 strain, tentatively designated as BatCoV HKU9-2202, by sequencing the full-length genome. The BatCoV HKU9-2202 shared 83% nucleotide identity with other BatCoV HKU9 stains based on whole genome sequences. The most divergent region is in the spike protein, which only shares 68% amino acid identity with BatCoV HKU9. Quantitative PCR revealed that the intestine was the primary infection organ of BatCoV HKU9 and GCCDC1, but some HKU9 was also detected in the heart, kidney, and lung tissues of bats. This study highlights the importance of virus surveillance in natural reservoirs and emphasizes the need for preparedness against the potential spill-over of these viruses to local residents living near bat caves.

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus.

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.

Detection of alpha- and betacoronaviruses in rodents from Yunnan, China.

BACKGROUND: Rodents represent the most diverse mammals on the planet and are important reservoirs of human pathogens. Coronaviruses infect various animals, but to date, relatively few coronaviruses have been identified in rodents worldwide. The evolution and ecology of coronaviruses in rodent have not been fully investigated. RESULTS: In this study, we collected 177 intestinal samples from thress species of rodents in Jianchuan County, Yunnan Province, China. Alphacoronavirus and betacoronavirus were detected in 23 rodent samples from three species, namely Apodemus chevrieri (21/98), Eothenomys fidelis (1/62), and Apodemus ilex (1/17). We further characterized the full-length genome of an alphacoronavirus from the A. chevrieri rat and named it as AcCoV-JC34. The AcCoV-JC34 genome was 27,649 nucleotides long and showed a structure similar to the HKU2 bat coronavirus. Comparing the normal transcription regulatory sequence (TRS), 3 variant TRS sequences upstream the spike (S), ORF3, and ORF8 genes were found in the genome of AcCoV-JC34. In the conserved replicase domains, AcCoV-JC34 was most closely related to Rattus norvegicus coronavirus LNRV but diverged from other alphacoronaviruses, indicating that AcCoV-JC34 and LNRV may represent a novel alphacoronavirus species. However, the S and nucleocapsid proteins showed low similarity to those of LRNV, with 66.5 and 77.4% identities, respectively. Phylogenetic analysis revealed that the S genes of AcCoV-JC34, LRNV, and HKU2 formed a distinct lineage with all known coronaviruses. CONCLUSIONS: Both alphacoronaviruses and betacoronaviruses were detected in Apodemus chevrieri in the Yunnan Province of China, indicating that Apodemus chevrieri is an important host for coronavirus. Several new features were identified in the genome of an Apodemus chevrieri coronavirus. The phylogenetic distance to other coronaviruses suggests a variable origin and evolutionary route of the S genes of AcCoV-JC34, LRNV, and HKU2. These results indicate that the diversity of rodent coronaviruses is much higher than previously expected. Further surveillance and functional studies of these coronaviruses will help to better understand the importance of rodent as host for coronaviruses.

Novel bat adenoviruses with low G+C content shed new light on the evolution of adenoviruses.

Bats have been reported to carry diverse adenoviruses. However, most bat adenoviruses have been identified on the basis of partial genome sequences, and knowledge on the evolution of bat adenoviruses remains limited. In this study, we isolated and characterized four novel adenoviruses from two distinct bat species, and their full-length genomes were sequenced. Sequence analysis revealed that these isolates represented three distinct species of the genus Mastadenovirus. However, all isolates had an exceptionally low G+C content and relatively short genomes compared with other known mastadenoviruses. We further analysed the relationships among the G+C content, 5'-C-phosphate-G-3' (CpG) representation and genome size in the family Adenoviridae. Our results revealed that the CpG representation in adenoviral genomes depends primarily on the level of methylation, and the genome size displayed significant positive correlations with both G+C content and CpG representation. Since ancestral adenoviruses are believed to have contained short genomes, those probably had a low G+C content, similar to the genomes of these bat strains. Our results suggest that bats are important natural reservoirs for adenoviruses and play important roles in the evolution of adenoviruses.

Detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in China.

BACKGROUND: In recent years, novel hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses have been discovered in various species of bat around the world, indicating that bats may act as natural reservoirs for these hepatitis viruses. In order to further assess the distribution of hepatitis viruses in bat populations in China, we tested the presence of these hepatitis viruses in our archived bat liver samples that originated from several bat species and various geographical regions in China. METHODS: A total of 78 bat liver samples (involving two families, five genera, and 17 species of bat) were examined using nested or heminested reverse transcription PCR (RT-PCR) with degenerate primers. Full-length genomic sequences of two virus strains were sequenced followed by phylogenetic analyses. RESULTS: Four samples were positive for hepadnavirus, only one was positive for hepevirus, and none of the samples were positive for hepatovirus or hepacivirus. The hepadnaviruses were discovered in the horseshoe bats, Rhinolophus sinicus and Rhinolophus affinis, and the hepevirus was found in the whiskered bat Myotis davidii. The full-length genomic sequences were determined for one of the two hepadnaviruses identified in R. sinicus (designated BtHBVRs3364) and the hepevirus (designated BtHEVMd2350). A sequence identity analysis indicated that BtHBVRs3364 had the highest degree of identity with a previously reported hepadnavirus from the roundleaf bat, Hipposideros pomona, from China, and BtHEVMd2350 had the highest degree of identity with a hepevirus found in the serotine bat, Eptesicus serotinus, from Germany, but it exhibited high levels of divergence at both the nucleotide and the amino acid levels. CONCLUSIONS: This is the first study to report that the Chinese horseshoe bat and the Chinese whiskered bat have been found to carry novel hepadnaviruses and a novel hepevirus, respectively. The discovery of BtHBVRs3364 further supports the significance of host switches evolution while opposing the co-evolutionary theory associated with hepadnaviruses. According to the latest criterion of the International Committee on Taxonomy of Viruses (ICTV), we hypothesize that BtHEVMd2350 represents an independent genotype within the species Orthohepevirus D of the family Hepeviridae.