The characteristics of gut microbiota and commensal Enterobacteriaceae isolates in tree shrew (Tupaia belangeri).

BACKGROUND: Tree shrew is a novel laboratory animal with specific characters for human disease researches in recent years. However, little is known about its characteristics of gut microbial community and intestinal commensal bacteria. In this study, 16S rRNA sequencing method was used to illustrate the gut microbiota structure and commensal Enterobacteriaceae bacteria were isolated to demonstrate their features. RESULTS: The results showed Epsilonbacteraeota (30%), Proteobacteria (25%), Firmicutes (19%), Fusobacteria (13%), and Bacteroidetes (8%) were the most abundant phyla in the gut of tree shrew. Campylobacteria, Campylobacterales, Helicobacteraceae and Helicobacter were the predominant abundance for class, order, family and genus levels respectively. The alpha diversity analysis showed statistical significance (P < 0.05) for operational taxonomic units (OTUs), the richness estimates, and diversity indices for age groups of tree shrew. Beta diversity revealed the significant difference (P < 0.05) between age groups, which showed high abundance of Epsilonbacteraeota and Spirochaetes in infant group, Proteobacteria in young group, Fusobacteria in middle group, and Firmicutes in senile group. The diversity of microbial community was increased followed by the aging process of this animal. 16S rRNA gene functional prediction indicated that highly hot spots for infectious diseases, and neurodegenerative diseases in low age group of tree shrew (infant and young). The most isolated commensal Enterobacteriaceae bacteria from tree shrew were Proteus spp. (67%) and Escherichia coli (25%). Among these strains, the antibiotic resistant isolates were commonly found, and pulsed-field gel electrophoresis (PFGE) results of Proteus spp. indicated a high degree of similarity between isolates in the same age group, which was not observed for other bacteria. CONCLUSIONS: In general, this study made understandings of the gut community structure and diversity of tree shrew.

[Biological characteristics, relationship between antigen and serology of tupaial adenoviruses (type I and II)].

The strains of adenovirus were isolated from pharyngeal swabs, kidney cell cultures and stool of tupaias. They were identified by a series tests of biological characteristics and electron microscopy studies. 10 strains of tupaia adenoviruses (TAV) may be divided into two serum types: termed TAV-I and TAV-II. There was no cross-reactivity of neutralization antibody between TAV-I and TAV-II, except a slight cross-reactivity in the complement fixation test. TAV-I could hemagglutinate R.B.C. of rat, mouse, human 'O' type, and tupaia itself, but TAV-II, couldn't. The positive rate of TAV-I and TAV-II antibodies in blood was more than 50% in natural tupaia population, suggesting of TAV infecting latently in wild tupaia colony.

Isolation and characterization of a tupaia rhabdovirus.

Liver tumor biopsies of a 9-year-old moribund Tupaia (tree shrew) were explanted and cultured in vitro. The cell cultures degenerated spontaneously. A virus was isolated from the cell-free supernatant of these cultures and subsequent electron microscopy revealed rhabdovirus-like particles. Negative staining showed typical bullet-shaped particles 125-220 nm in length with a diameter of 68 nm studded with a dense layer of surface projections 9-11 nm in length. One end of the virion was flat, the other end was open; a distinct ribonucleoprotein (RNP) core was visible. The pitch of the RNP was 4.5 nm. Virus was assembled and matured by budding primarily into regions of dilated endoplasmic reticulum. The dimensions of the virion also were determined in ultrathin sections: the diameter and length of the virion were 52 and 125-255 nm, respectively, those of the RNP core were 39 and 120-240 nm. Only tupaia embryonic fibroblasts and kidney cells were susceptible to the rhabdovirus. The virus, when plaque-assayed on tupaia embryonic fibroblasts, grew to a titer of 1 X 10(6) PFU.

Analysis of the genomic termini of tupaia herpesvirus DNA by restriction mapping and nucleotide sequencing.

A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging.

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.

The nucleotide sequence of the early region of the Tupaia adenovirus DNA corresponding to the oncogenic region E1b of human adenovirus 7.

The nucleotide sequence of the early region E1b of the tree shrew (Tupaia) adenovirus (TAV) DNA has been determined. The sequenced region includes the genes for polypeptides of Mr 15 000, 44 000 and 13 400, which are analogous to the small and large E1b proteins and protein IX, respectively, of the three human adenovirus serotypes 5, 7, and 12. The hexanucleotide consensus signal AATAAA occurs only at the 3' terminus of the gene for protein IX suggesting that the E1 region of TAV encompasses one transcription unit. The amino acid sequences of the TAV polypeptides have a higher degree of homology to those of Ad7 and Ad5 than to those of Ad12.

Investigation of the virulence genes of herpes simplex virus 2 by experimental infection in vivo with defined intertypic recombinants of a virulent HSV-2 X an avirulent HSV-1.

For determination of the gene loci for virulence and tropism of herpes simplex virus 2 (HSV-2) intertypic recombinants of HSV-1 strain HFEM and HSV-2 strain 3345 were screened for their pathogenicity in the tree shrew. HSV-1 strain HFEM is not pathogenic in the tree shrew and can be recovered as a latent virus only from the spleen of the animals; but HSV-2 strain 3345, the other parental virus of the recombinants, was found to be virulent for the tree shrew and colonized the ganglia of latently infected animals. No clinical pictures were observed when the animals were inoculated with HSV recombinants RS1(3), RB2(3), RB2(8), RB2(9), RB2(10), RB5(2), RB7(3), and RB7(7). It was found, however, that HSV recombinant RB5(0) is pathogenic for the tree show. Studies of the state of viral latency in the animals infected with these recombinants revealed that only HSV recombinant RB5(0) was able to persist as latent virus in the spleen of latently infected animals. The genome of recombinant HSV-RB5(0) contains DNA sequences of HSV-2 strain 3345 with two interruptions at 0.22-0.33 and 0.54-0.59 viral map units. In these regions of HSV-RB5(0), DNA sequences of HSV-strain HFEM at 0.26-0.33 and about 0.57-0.58 viral map units, respectively, had been recombined. The results obtained in this study indicate that the recombinant HSV-RB5(0) acquired the virulent phenotype (v+) from parental virus HSV-2 strain 3345 but lost the phenotype for colonizing the ganglia (g+). Furthermore, it acquired the phenotype of parental virus HSV-1 strain HFEM for persisting in a latent state only in the spleen (s+ and g-) of the latently infected animals.

Tupaia (tree shrew) adenovirus DNA: sequence of the left-hand fragment corresponding to the transforming early region of human adenoviruses.

The nucleotide sequence of the left-hand region of the adenovirus DNA of the phylogenetically interesting tree shrew, (Tupaia belangeri) has been determined. Transcription signals, initiation codons, splice sites and termination codons were assigned on the basis of its relatedness to the EIA region of human adenoviruses 5, 7 and 12. A consensus sequence for encapsidation of adenoviral DNA that is based on the established packaging region of adenovirus 16 is proposed. The sequenced region includes the gene for a 18 000 dalton polypeptide that corresponds to the EIA proteins of the transforming region of the human serotypes. The deduced amino acid sequence of the 18 000 dalton TAV polypeptide contains a highly conserved central domain that is homologous to the middle of the 30 000 dalton polypeptides of human adenovirus indicating the significance of the central amino acid residues for the biological function of the EIA proteins.

Protein kinase and specific phosphate acceptor proteins associated with tupaia herpesvirus.

A protein kinase activity was found to be associated with tree shrew (tupaia) herpesvirus. The protein kinase was characterized with respect to its requirements for enzymatic activity. A divalent cation such as Mg2+ or Mn2+ was necessary as well as ATP as the phosphate donor. Distinct tupaia herpesvirus polypeptides (molecular weights of 100,000 [100K], 82K, and 53K) were found to be phosphate acceptor proteins when 5 mM Mg2+ was used. At a higher Mg2+ concentration (20 mM), additional viral proteins (220K, 71K, 31K, and 20K) were phosphorylated. The viral phosphoproteins were analyzed by chemical and enzymatic hydrolyses. The predominant sites of phosphorylation were the beta-OH groups of the serine and threonine residues of these tupaia herpesvirus proteins. Kinase activity was not stimulated by cyclic nucleoside monophosphates. Endogenously added proteins did not enhance protein kinase activity. Protein kinase activity was inhibited by 5'-p-fluorosulfonylbenzoyladenosine.

Analysis of polypeptides of the tree shrew (Tupaia) herpesvirus by gel electrophoresis.

The virion polypeptide composition of three independently isolated tree shrew herpesviruses (THV) was analysed by SDS-polyacrylamide slab gel electrophoresis and by a two-dimensional technique using isoelectric focusing. Two of the virus isolates analysed were from malignant tumours; the other isolate (THV, strain 1) was from an apparently healthy animal. The polypeptide patterns of the three purified Tupaia herpesvirus isolates were remarkably similar, each consisting of at least 35 polypeptides ranging in mol. wt. from 12,000 to 230,000. Whilst the majority of analogous polypeptides of the three viruses were of indistinguishable electrophoretic mobility, some (e.g. polypeptides of 82K to 86K) showed small differences in apparent mol. wt. which were characteristic of the virus strain. Comparative SDS-polyacrylamide gel electrophoresis made it possible to distinguish the Tupaia herpesvirus isolates from each other. At least five glycoproteins were found in purified THV virions. The two-dimensional electropherograms revealed at least 47 discernible protein spots, some of which were specific for a given THV isolate and which were detectable even in lysates of THV-infected cells.

Tree shrew (Tupaia) herpesviruses.

Five Tupaia herpesviruses have been isolated until now: four in our laboratory which were termed THV-2, 3, 4, and 5, whereas THV-1 has been isolated by Melnick and his colleagues. THV-2 was isolated from tumour cell culture of a high-grade malignant lymphoma of a Tupaia, THV-3 was released from a cell culture of another Tupaia lymphoma, THV-4 from a spleen tissue culture of a moribund animal with finely granulated liver cirrhosis, and THV-5 from cultured spleen cells of an apparently healthy tree shrew. THV-1 to 5 were efficiently propagated, plaque-purified and cloned on Tupaia embryonic fibroblasts. The five isolates of Tupaia herpesviruses are easily distinguished from each other by restriction enzyme analysis of their genomes. THV-1 to 4 are highly pathogenic (lethality 100%) for juvenile Tupaias by intravenous inoculation. In contrast, only 25% lethality was found by intraperitoneal administration. THV-1 to 4 can persist as a latent infection in spleens of Tupaias and rabbits, which allows the recovery of infectious virus from cultured spleens of both animals. THV-2 and 3 induced hyperplasia of the thymus of rabbits which developed malignant thymoma in a few cases. The biological properties and genomic size and structure indicate that THV cannot be considered to belong to one of the three existing subfamilies of herpesviruses.

Analysis of Tupaia herpesvirus proteins by one- and two-dimensional gel electrophoresis.

The polypeptide composition of four Tupaia herpesvirus strains--of which two were isolated from malignant tumors--was analysed by one- and two-dimensional gel analysis. It was found that purified virions of the tree shrew consist of a least 37 viral polypeptides when determined by SDS polyacrylamide gel electrophoresis. The number of viral polypeptides increased to 49 when analysed by the two-dimensional technique with the molecular weights ranging from 12.000 to 230.000. Variations in the viral polypeptide patterns made it possible to differentiate between the different virus isolated even by the one-dimensional method. The two-dimensional technique allows the identification of virus-specific spots among the polypeptides of virus-infected cells. Radiolabeling experiments identified at least five glycoproteins which seem to reside on the surface of the virus envelope. In addition, neutralisation and immunodiffusion tests were performed which revealed cross-reactivities among the tupaia herpesvirus strains. Rabbit anti-tupaia herpesvirus sera did not neutralize other animal or human herpesvirus.

Immunological relationship among adenoviruses of humans, simians, and nonprimates as determined by the neutralization test.

98 antisera prepared against 25 simian adenoviruses and 31 adenovirus types from various nonprimate mammals and birds were tested for their ability to neutralize 35 human adenovirus prototypes. Antisera to these human adenovirus types were similarly tested against 25 simian adenovirus prototypes. Although very few reactions to the human adenoviruses were found in antisera to nonprimate viruses, cross-reactions among the simian and human adenoviruses were more frequent. The majority of these reactions, however, were of low titer and of questionable significance. Two chimpanzee adenovirus strains--Pan 9 and SAV 21 (C 1)--were found with an immunological relationship to human adenoviruses: Pan 9 strain showed a two-way cross relationship to human adenovirus 4, and SAV 21 (C 1) was neutralized by antisera prepared against human adenoviruses 14 and 16.

Physical map of tupaia adenovirus DNA by cleavage with restriction endonucleases and partial denaturation.

The double-stranded DNA of an adenovirus of tupaia (TAV) which has a mol. wt. of 21.5 X 10(6) was analysed as follows: the cleavage sites of restriction endonucleases BamHI, EcoRI, KpnI, SmaI, BstEII, ClaI and HpaI were determined by complete, partial and double digestion followed by gel electrophoretic separation of the resulting fragments. Terminal HpaI fragments were determined by hydrolysing the intact DNA with exonuclease III before restriction enzyme cleavage. Partial denaturation mapping of uncleaved DNA and EcoRI fragments revealed the cleavage sites of EcoRI as well as A + T-rich regions at both termini of the genome.

Experimental infection and the state of viral latency of adult tupaia with herpes simplex virus type 1 and 2 and infection of juvenile Tupaia with temperature-sensitive mutants of HSV Type 2.

The susceptibility of adult Tupaia Belangeri to infection with herpes simplex virus (HVS) was investigated. Adult animals were inoculated intraperitoneally with HSV type 1 or 2. With the exception of HSV-2, strain HG-52, 10(5)--10(6) PFU of all HSV strains caused lethal infection irrespective of the age of the animals. Infections HSV was recovered from the spinal cord of those animals which had survived infection with a low dose of virus. The DNA of the recovered viruses was compared to the DNA of the inoculated HSV. The viral genome of the recovered HSV was unchanged as judged by analysis of the fragment pattern of the viral DNA's using restriction endonucleases. Animals which had survived the first HSV infection were protected against a second infection even at highly lethal doses of HSV-1 or 2. Juvenile Tupaia survived infection with temperature-sensitive mutants of HSV-2, strain HG-52, which induced protection against a second infection with lethal doses of HSV-1 or 2.

Identification of polypeptides of the tree shrew (Tupaia) adenovirus.

The virion polypeptides of the Tupaia (tree shrew) adenovirus were analyzed by SDS-polyacrylamide slab gel electrophoresis and by isoelectric focusing. The viral proteins from either nonradioactive or 35S-methionine-labeled virions formed distinct patterns of at least 15 polypeptides which were different from those of known human and animal adenoviruses. Two-dimensional analyses revealed the presence of 18 discernible polypeptides.