Isolation and Phylogenetic Analysis of Halophilic Archaeon AJ6

Halophilic archaeon AJ6 was isolated and purified from the Altun Mountain National Nature Reserve of the Xinjiang Uygur Autonomous Region. Strain AJ6 is a Gram-negative rod whose size is 0.2–0.6 by 1.6–4.2 μm, wherein a few cells are globular. The optimum salt concentration for its growth is 20% NaCl and 0.6% Mg²⁺, and the optimum pH is 6.0–7.0. Morphological, physiological, and biochemical characteristics of strain AJ6 were observed. The 16S rRNA encoding gene (16S rDNA) sequence of strain AJ6 was amplified by PCR, and its nucleotide sequence was determined subsequently. “Clustalw” and “PHYLIP” software bags were used to analyze the 16S rDNA sequence; the homology was compared, and then the phylogenetic tree was established. The results indicate that strain AJ6 is a novel species of the genus Natrinema. The GenBank accession number of the 16S rDNA sequences of strain AJ6 is AY277584. Translated from Journal of Zhejiang University (Science Edition), 2005, 32(1) (in Chinese)     

表达鸡传染性支气管炎病毒JS/95/03株S1蛋白的重组杆状病毒构建

The recombinant baculovirus expressing S1 glycoprotein of nephropathogenic strain JS/95/03 of infectious bronchitis virus was generated by using the Bac-to-Bac baculovirus expression system.The BamH I-Sal Ⅰ fragment containing S1 gene from the recombinant plasmid pMDJS9503S1 was purified and cloned in frame into the baculovirus transposing vector pFASTBAC HTa under the polyhedrin gene promoter.The recombinant transposing plasmid pFASTJS9503S1 was screened and then transformed into Escherichia coli DH10BAC.The resulting recombinant bacmid rBacmidJS9503S1 was transfected into cells of the insect Spodoptera frugiperda(Sf9)and the recombinant baculoviruse rAcJS9503S1 was obtained.The lysates of cells infected with rAcJS9503S1 were analyzed by SDS-PAGE and the expressed product of S1 gene was detected by Western bloting and immunofluorescence assay(IFA).The results showed the recombinant baculovirus was fully capable of expressing S1 glycoprotein of JS/95/03.Maybe owing to the incomplete glycosylation in insect cells,the S1 gene product had a Mr of only 61000.In immunofluorescence test and Western blotting,the expressed product could react with polycolonal antibody against IBV M41 strain,indicating it possessed the antigenic properties specific for native S1 glycoprotein.     

Construction of prophylactic human papillomavirus type 16 L1 capsid protein vaccine delivered by live attenuated Shigella flexneri strain sh42

To express human papillomavirus （HPV） L 1 capsid protein in the recombinant strain of Shigella and study the potential of a live attenuated Shigella-based HPV prophylactic vaccine in preventing HPV infection, the icsA/virG fragment of Shigella-based prokaryotic expression plasmid pHS3199 was constructed. HPV type 16 L1 （HPV16L1） gene was inserted into plasmid pHS3199 to form the pHS3199-HPV16L1 construct, and pHS3199-HPV 16L1 was electroporated into a live attenuated Shigella strain sh42. Western blotting analysis showed that HPV 16L 1 could be expressed stably in the recombinant strain sh42-HPV 16L 1. Sereny test results were negative, which showed that the sh42-HPV16L1 lost virulence. However, the attenuated recombinant strain partially maintained the invasive property as indicated by the HeLa cell infection assay. Specific IgG, IgA antibody against HPV16L1 virus-like particles （VLPs） were detected in the sera, intestinal lavage and vaginal lavage from animals immunized by sh42-HPV 16L1. The number of antibodysecreting cells in the spleen and draining lymph nodes were increased significantly compared with the control group. Sera from immunized animals inhibited mufine hemagglutination induced by HPV 16L1 VLPs, which indicated that the candidate vaccine could stimulate an efficient immune response in guinea pig＇s mucosal sites. This may be an effective strategy for the development of an HPV prophylactic oral vaccine.     

Effect of amino acid mutation at position 127 in 3A of a rabbit-attenuated foot-and-mouth disease virus serotype Asia1 on viral replication and infection

An amino acid mutation(R127→I) in the 3A non-structural protein of an FMDV serotype Asia1 rabbit-attenuated ZB strain was previously found after attenuation of the virus. To explore the effects of this mutation on viral replication and infection, the amino acid residue isoleucine(I) was changed to arginine(R) in the infectious cDNA clone of the rabbit-attenuated ZB strain by sitedirected mutagenesis, and the R127-mutated virus was rescued. BHK monolayer cells and suckling mice were inoculated with the R127-mutated virus to test its growth property and pathogenicity, respectively. The effects of the R127 mutation on viral replication and virulence were analyzed. The data showed that there was a slight difference in plaque morphology between the R127-mutated and wild-type viruses. The growth rate of the mutated virus was lower in BHK-21 cells and its virulence in suckling mice was also attenuated. This study indicates that the R127 mutation in 3A may play an important role in FMDV replication in vitro and in pathogenicity in suckling mice.     

Identification and characterization of Acidithiobacillus ferrooxidans with high activity and resistance isolated from ancient mine area

A highly active ferrous iron oxidation Acidithiobacillus ferrooxidans strain SY, was isolated from an ancient copper mining area in Daye, Hubei Province. Analysis of 16S rDNA sequence showed that the strain has high similarity to the sequence of A. ferrooxidans (DQ 062116.1). Physiological and biochemical determinations showed that the strain was a chemical energy autotrophically with the optimal growth pH at 2.0 and optimal growth temperature at 30 ℃. The MTC of the strain to resist Cu²⁺, Cd²⁺, Ni²⁺, and Zn²⁺ were respectively at 300, 350, 700, and 800 (mmol/L), demonstrated it has high resistance against multiple heavy metal ions. Bioleaching data of SY showed its bioleaching rate on native ore was as high as 84.28%, higher than bioleaching rate on the ore from other mining areas, showing it has very high superiority on bioleaching native ore. The strain SY has great potential in the application of native minerals bioleaching.     

新疆地区猪戊型肝炎血清流行病学调查

The purpose of the present study was to determine the prevalence of swine Hepatitis E virus (HEV) infection in Xinjiang. 813 swine serum samples collected from 1 to 12 months of age at 9 swine farms in Xinjiang region were tested by ELISA for the presence of IgG antibodies against HEV. The recombinant protein pUS 166 containing region 452-617aa of the ORF2 of HEV US strain was used as coating antigen. The result showed that anti -HEV IgG were detected in 265 of 405 pigs (65.43%) in one group and 238 of 408 pigs (58.33%) in another group, and that the seropositivity rate was not related to geographic district and breeds, but differed remarkably by age, being 40% among the 1- to 3-month-old piglets, but 77.33% among ones over 3-month-old. It suggested that swine HEV was widespread in different geographic regions of XinJiang.     

Effect of amino acid mutation at position 127 in 3A of a rabbitattenuated foot-and-mouth disease virus serotype Asia1 on viral replication and infection

An amino acid mutation(R127→I) in the 3A non-structural protein of an FMDV serotype Asia1 rabbit-attenuated ZB strain was previously found after attenuation of the virus. To explore the effects of this mutation on viral replication and infection, the amino acid residue isoleucine(I) was changed to arginine(R) in the infectious cDNA clone of the rabbit-attenuated ZB strain by sitedirected mutagenesis, and the R127-mutated virus was rescued. BHK monolayer cells and suckling mice were inoculated with the R127-mutated virus to test its growth property and pathogenicity, respectively. The effects of the R127 mutation on viral replication and virulence were analyzed. The data showed that there was a slight difference in plaque morphology between the R127-mutated and wild-type viruses. The growth rate of the mutated virus was lower in BHK-21 cells and its virulence in suckling mice was also attenuated. This study indicates that the R127 mutation in 3A may play an important role in FMDV replication in vitro and in pathogenicity in suckling mice.     

汉滩病毒M片段的核苷酸序列变异和系统发生树分析

The total RNA was extracted from two clinical blood samples of HFRS patients and the RNA was amplified by RT-PCRThe amplified DNA fragment of sample 613 involved nucleotides 1,471-1,873,and the sample 226 involved 540-1,244 nucleotides of M fragment of Hantaan virusThen the amplified PCR products were sequenced directlyThe sequencing results demonstrated that there was 84% identity between sample 613 and HV114 virus strain,but was 99% between sample 613 and HTN76-118 strainHowever,in sample 226,there was 95% sequence identity with HV114,82% with HTN76-118The results of phylogenetic tree analysis showed that HV613 was located in the same linage with HTN76-118,the HV226 was in the same linage with HV114 and A9 strains     

Study of the Antifungal Ability of Bacillus subtilis Strain PY-1 in Vitro and Identification of its Antifungal Substance （Iturin A）

A Bacillus strain,denoted as PY-1,was isolated from the vascular bundle of cotton.Biochemical,physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis.The PY-1strain showed strong ability against many common plant fungal pathogens in vitro.The antibiotics producedby this strain were stable in neutral and basic conditions,and not sensitive to high temperature.From theculture broth of PY-1 strain,five antifungal compounds were isolated by acidic precipitation,methanolextraction,gel filtration and reverse-phase HPLC.Advanced identification was performed by mass spec-trometry and nuclear magnetic resonance spectroscopy.These five antifungal compounds were proved to bethe isomers of iturin A:A2,A3,A4,A6 and A7.In fast atom bombardment mass spectrometry/mass spec-trometry collision-induced dissociation spectra,fragmentation ions from two prior linear acylium ions wereobserved,and the prior ion,Tyr-Asn-Gln-Pro-Asn-Ser-βAA-Asn-CO~ ,was first reported.     

黑色素抑制流感病毒诱导宿主细胞凋亡

The apoptosis induced by influenza virus in cultured MDCK cells was reported and the selective inhibitory effect of melamin on the apoptosis induced by influenza virus was investigated. The results showed that the DNA ladder could be first detected at 6 h post-infection (p.i.), accompanied by nuclear condensation and nuclear fragmentation could be easily detected at 12 h p.i. In addition, the apoptosis-induced activity of influenza virus A1/Jingfang 86-1 strain was more potent than that of B/Hufang 93-1 strain (P＜0.05). In the range of 20-125 μg/mL, melanin was found to significantly (P＜0.001) inhibit apoptosis induced by 64 hemagglutination unit influenza virus infection with a inhibitory rate comparable to that obtained by virazole and showed no cytotoxicity. The inital results suggested that the mechanism of melanin against the apoptosis induced by influenza virus was related to the blockage of viruses' adsorbtion to the host cells.     

A rhizobia strain isolated from root nodule of gymnosperm Podocarpus macrophyllus

Symbiotic nitrogen fixation of rhizobia and leguminous plants is considered as the most important biologic nitrogen fixation system on earth. Symbiotic nodulation of gymnosperm Podocarpus macro-phyllus and rhizobia has never been reported. In this study, 11 endophytic bacteria strains were isolated from root nodules of P. macrophyllus and its variation P. macrophyllus var. maki. The plant infection tests on these strains indicated that the isolated strains could be nodulated on P. macrophyllus plants, and weak nitrogenase activity of nodules was found in acetylene reduction method. According to the physiological and biochemical characteristics of the 11 strains, GXLO 02 was selected as the representative strain. 16S rDNA full-length sequence analysis of GXLO 02 confirmed that the representative strain GXLO 02 belongs to Rhizobium sp.     

The flhDC gene affects motility and biofilm formation in Yersinia pseudotuberculosis

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mu- tated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIII?flhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regula- tory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

Identification and Culture Condition Study of MarineActinomycete HT-8 with Nematicidal Activity against Pine Wood Nematode

Pine wilt disease (PWD) is a devastating disease to pine trees, which was caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, and causes major losses in coniferous forests in many countries and regions in the world. In order to screen actinomycete strains with high nematicidal activity against PWN, marine actinomycetes were isolated from the marine environment and their nematicidal activity were tested.The marine actinomycetes were isolated from the samples collected from the subtidal zones near Qingdao coast using the dilution and streak plate method and their culture supernatant were assayed in vitro for nematicidal activity against PWN using immersion test. The strain with high nematicidal activity was identified on the basis of morphology, cultural characteristics and 16S rDNA sequence analysis, and its optimal culture conditions for the production of nematicidal substances were investigated through the single-factor experiments.A total of 28 marine actinomycete strains were isolated from the samples. One of these strains, designated as strain HT-8 and isolated from sea sand, exhibited stronger nematicidal activity with a 88.30% corrected mortality of PWN treated with the culture supernatant for 30 h. The strain HT-8 was identified as Streptomyces termitum on the basis of morphology, cultural characteristics, 16S rDNA sequence and phylogenetic analysis. The results of the single factor experiment demonstrated that the optimal cultivation conditions of HT-8 for the production of nematicidal substances were inoculum age was 48 h, inoculum concentration was 6%, concentration of seawater was 100%, initial pH was 7.5 and incubating at 25 ℃ for 7 days.This study provides a theoretical basis for the development of marine microorganism resources and the utilization of natural nematicidal substances.     

Identification of a denitrifying bacterium and verification of its anaerobic ammonium oxidation ability

A strain D3 of denitrifying bacterium was isolated from an anammox reactor,and identi-fied as Pseudomonas mendocina based on the morphological and physiological assay,Vitek test,Biolog test,(G C) mol% content,and 16S rDNA phylogenetic analysis.As a typical denitrifying bac-terium,strain D3 achieved the maximal nitrate reduction rate of 26.2 mg/(L·d) at the nitrate concen-tration of 88.5 mg N/L.The optimal pH and growth temperature were 7.84 and 34.9℃,respectively.Strain D3 was able to oxidize ammonia under anaerobic condition.The maximum nitrate and ammo-nium utilization rates were 6.37 mg/(L·d) and 3.34 mg/(L·d) ,respectively,and the consumption ratio of ammonia to nitrate was 1:1.91.Electron microscopic observation revealed peculiar cell inclusions in strain D3.Because of its relation to anammox activity,strain D3 was presumed to be anammoxosome.The present investigation proved that denitrifying bacteria have the anammox ability,and the results have engorged the range of anammox populations.     

猪瘟病毒石门株NS2-3基因片段的序列测定及比较

A couple of CSFV specific primer (PF 5648 and PR 6604) was designed with the aid of computer primer designation software and synthesized based upon the relative conserved regions of published sequences of C strain. NS2-3 gene (p125 gene) fragment of CSFV Shimen strain was amplified successfully by RT-PCR from the anticoagulant blood of infected pig. The product length is 957 bp, located in the central of NS3, a putative NTPase and helicase domain. The obtained PCR product was cloned and then sequenced. The sequence showed that this fragment contained all of seven typical conserved segments of helicase superfamily, including two common NTP-binding motifs, namely, “A” site (GXGKT/S) and “B” site (3hy, 2x) D. Sequence homology analysis revealed that Shimen strain had the highest homology with Japanese strains (ALD and GPE^-), and slightly lower homology with other three CSFV strains (C, BresciaandAlfort). Shimen strain had also significant homology with two BVDV strains (NADL and SD-1). The deduced amino acid sequence homology of Shimen strain with five CSFV and two BVDV strains was all upper than 90%. It is further confirmed that this fragment is the most conserved in pestivirus amino acid sequence. It is consistent with its essential function in replication and translation of virus genome and in processing of polyprotein precursor.     

High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment （htt552） in primary rat astrocytes

Huntington＇s disease （HD） is caused by an expansion of polyglutamine tract in N-terminus of huntingtin （htt）. The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown. This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids （htt552） in rat primary cortical astrocytes. We have evaluated optimal conditions for the infection of astrocytes with adenoviral vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astrocytes expressed the transgene after infection. At 24 h postinfection, the highest rate of infection was 89 ± 3% for the wild-type （htt552-18Q） with a multiplicity of infection （m.o.i.） of 80, and the highest rate of infection was 91 ± 4% for the mutant type （htt552-100Q） with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin （htt552-100Q） produced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranuclear inclusions. The result of MTT （3-（4,5- Dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide） assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i, of 40 was optimal to our cell model. The present study demonstrates that adenoviral-mediated expression of mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.     

半嵌套式PCR技术区别检测犬细小病毒疫苗株和强毒株的研究

Two pairs of PCR primers were designed according to the sequances of the vaccine strain and virulent strain of CPV. Heminested PCR method was established. Result of the first PCR amplification showed the same amplified products of 574bp length, after the second PCR amplification, the virulent strain produced the length 364bp fragment, but the vaccine strain couldn' t produce that. The products of PCR were examined by electrophoresis and restriction enzyme digestion. The result showed the length of the fragment and enzyme sites were as the same as those designed. The PCR assay of CPV was proved to be specific and sensitive. It shows that this method may be used in discriminating the vaccine strain and virulent strain of CPV or monitoring the vaccinated canine in order to aviod disease and financial losing.     

Pathological Changes in Andrias davidianus Infected with Chinese Giant Salamander Ranavirus

Chinese giant salamander ranavirus(CGSRV) is an emerging pathogen in captive populations of the Chinese giant salamander(Andrias davidianus). We processed 140 morbid Chinese giant salamanders from seven captive breeding populations over five years, and describe the disease associated with CGSRV infection. The most common gross signs were significant swelling of the legs and coelomic cavity, erythema of the legs and ventrum in juveniles; cutaneous erosions and ulcerations in adults, particularly the limbs and the head; and marked petechial or ecchymotic hemorrhages of the internal organs, particularly the liver, spleen and kidney. Histological examination showed degeneration, necrosis, and inflammation in many organs, particularly in the organs where hemorrhage was observed. There was evidence of eosinophilic inclusion bodies in degenerated and necrotic cells. We identified virus particles and empty capsids without viral nucleoid in the inclusion bodies using electron microscopy. Virus particles were hexagonal or round shape, and appeared in paracrystalline arrays, aggregates, or singly. All enveloped viral particles were 140–160 nm. Polymerase chain reaction followed by sequencing verified that the virus particles were CGSRV. These results collectively support that CGSRV was the etiologic agent responsible for these mass die-offs of the Chinese giant salamander. The pathology described herein will be useful in diagnosing cases of ranaviral disease caused by CGSRV, and provide evidence that this pathogen is a significant threat to the Chinese giant salamander.