1994 Vol.09(2)



Advances in Molecular Biology of Marekof Disease Virus

LUO Man-Lin, CA Bao-Xiang, CHEN Bu-Yan

1994, 9(2): 87

Preparation of Biotin HBV RNA Probe and Its Application in Serum Detection

CHEN Xiao-Xu, HOU Chu-Xia, LI Re-Lun

1994, 9(2): 95

epotits B virus (HBV)RNA probes were synthesized in vitro by transcription from recombinantsbetween HBV DNA and the transcriptfon vector SP(65).These probes can be labelled with biotin and behybridized spoifically with HBV DNA.Comparing with HBV DNA probe prepared by Nick-translation for detection of HBV DNA,HBVRNA probe are 10 times sensitive than that of HBV DNA.In addition,70 serum saniples of hepatitis B potients have been detected by hybridization withtwo probes.Pooitive proportion of HBV DNA corresponils to 31.42%, 28.57%(P0.25).The clinical application of HBV RNA probes was discusseil preluninarily.

Detection of HBsAgHBeAg and HBV DNA in Urinary cells of Patients with Hepatitis B an d HBsAg Carriers

TAN Wei-Guo, LI Fa-Qing, TUN Yu-Jiang, TUN De-Fu, GONG Xi-Beng-

1994, 9(2): 100

his paper reports the detection of HBsAg,HBeAg and HBV DNA in the urinary cells by IFARPHA ELISA and molecular hybridization techniques.10 HBsAg carriers and 89 patients with hepotitis B were exanlined.The results indicated that HBsAg,HBeAg and HBV DNA could be detected inthe urinary cells.It also susgests that the urine of carriers and patients with hepatitis B be infectious.

A New M ethod for the Study of HPV M olecular Epidemiology-Quamy Control Slot HybridizationCoslot

Zhang-Wei, JIN Shun-Jian, SUN Jian-Heng, WANG Xi-Xia, TUN Ai-Ru

1994, 9(2): 104

he infection of HPV is the most frequent among the viruses tropic to the epithelium of cervix.The different HPV types possess various infective location and the abilities of etiology,for example,HPV type 6B,type 11 were correlated with the cervical non-tumor lesions,otherwise,the HPV type16 and type 18 were clasely assoctated with cervical carcinoma,so that it is important to identify accu-rately HPV type in the moledular epidemiological researchment for the relationship between cervicalHPv infection and the development of cervical carcinoma.We reperted here a new DNA hybridization method and procedure,with which we can easily differ one HPV type from another.The advantages as follow compared with other hybridimtion method.1.It lreer,mes sllitable to detect small pieces of biopeies from cervix since the experiment procedure being simplified and specimens consuption being decreased;2.The sensitivity is increased by 1.68 foldcomparing with dot blot hybridization;3.It has a higher types specificity owing to medi

The Inhibitory Effects of Quinolones on Hepatitis B Virus in Vitro

FAN Chao, FU Xi-Xian, ZHANG Guo-Qiang, ZHANG Ai-Lin

1994, 9(2): 113

uinolones were studied for their ability to inhibit hepatitis B virus DNA replicatiun,HBsAg andHBeAg exporssion in a HBV-transfeeted eell line(2.2.15 cell).The survival cell rate was determinedby MTT assay.The result shows that ID50(the drug concentration that inhibits HBsAg or HBeAg secretion by 50%) were 110g/ml and 199g/ml with Pipemidic Acid,64g/ml and 111g/ml with Norfloxacin,93g/ml and 210g/ml with Ciprofloxacin,105g/ml and 217g/ml with Ofloxacin.CD_50(the drug concentration that reduces cell growth by 50%) were 219g/ml,90g/ml,181g/ml,160g/ml,reepectively.In the range of drug concentration selected,they partially or completelybleeked the production of HBV particles and its replicative interniediates,especially the seneration of suporcoiled viral DNA((sc DNA).

Detection and Significance of HCV RNA NSs Gene

GUO Lin-Sheng, TANG Zhen-E, HAO Lian-Jie

1994, 9(2): 119

rimer selection is important for the detection of HCV RNA with RT-nested PCR method Com.pered to primers from 5'-UTR gene,the primere from NS5 gene had also high sensitivity and specifityin the detection of HCV RNA.Sixty-nine of 131 cases of anti-HCV Ab positive and 8 of 22 cases ofanti-HCV Ab nesative sera were found to be NS_5 gene positive,the positive rate were 52. 68% and36.36%,resnytively.All anti-HCV Ab positive samplea were divided into several sroups,according totheir clinical beckground.Prevalence of NS_5 gene in HCC and CRF groupe were 16.67% and 34.38%,respectively,lowertian that of other groupe (50%-80%),Interestinsly,the patientswith active liver diseiises(such as HCC,CAH,LC,SH) were peitive in the lst PCR,then the patientswith porristent hepotitis and subclinical infection were peitive in the 2nd PCR.The high prevalence ofNS5 gene in groupe of BD,PD and IG warned us of screening BD strictly and using blood-preducts carefully.

Detection of Human Cytomegalovirus in Neonatal Hepatitis by Nested PCR

CHAN Xiang-Nian, CHEN Jin-Dong, YAN Meng, LU Xiao, QIU Ding-Gong

1994, 9(2): 125

ixty-one children patients with neonatal hepatitis were investigated for human cytomesalovirus(HCMV)by single PCR and nested double PCR.of the 61 patients,33(54.1%)were positive forHCMV by single PCR,whereas 47(77.0%)were positive by nested double PCR.This showed that nest-ed double PCR is more sensitive than single PCR,and also revealed that HCMV can be one of the important agents for neonatal hepotitis.In addition,its advantage of high sensitivity and specificty com-bined with more rapid detection suggests that this precedure can be a potential diagnostic and epidemio-logical tool.

PCR Amplification CloningProtein Gene and Location of Major Capsid Of Baculovirus

WANG Jia-Wang, HUANG Yong-Xiu, TAN Ye-Beng, LI Shou-Dong, JI Xi-Feng

1994, 9(2): 130

he major eapeid protein gene(vp39)of Autographa californica nuclear polyhedrosis virus(AcNPV)was amplified successfully by PCR technique and cloned into pBluescript SK(+).With pure AcNPVvp39 gene as a probe,the major capeid protein gene(vp39)of Leucania seperata nuclear polyhedrosisvirus has been leeated on PstI-F.BamHI-C,EcoRI-C,E,XhoI-D,I,EcoRV-H,X fragnients by Southern blot.Besides amplification of the predicted 1406bp fragment including intact major capsid proteingene of AcNPV using XhoI and HindⅢ-digested AcNPV DNA as a teniplate.a ca.400 bp fragmenthas been amplified.In this paper we disscused the PCR amplification of ca.400 bp fragment of bothspecificity and nonspocificity.

Studies on Longan W itchesp Broom Disease

CHEN Jing-Yao, KE Chong, XIE Xu-Dong, LI Kai-Ben

1994, 9(2): 138

he results of longan seedlings with witches'broom disease immersed in tlie solutions of tetracycline-HCl(500,1500ppm for four hrs)and penicillin-GK(5000 ppm for four hrs)showed that thesymptom of the diseased seedlings were unable to be inhibited,which indicated that the disease was notcaused by MLO and BLO.The bunchy filan1entous viruses were found in the sieve cells of the diseasedleaf under electron micrcacope,no virus particle were deteeted in healthy plant used as control.Thevirus particle simil lar to that existed in the diseased leaves can be trapped from the salivary glands of Tessaratoma papillosa Drury(adults)and Cornegenapsylla sinica Yang et Li(adults)by SPA-ISEMmethod.It is concluded that the virus particles are the causal agent of Iongan wi tches'broom disease.

Studies on the TN Strain of Cucumber Mosaic Virus Inducing Tomato Necrosis Disease

CHENG Ning-Hui, PU Jie-Qin, FANG Zhong-Da

1994, 9(2): 143

lethal disease of tomato eecurred severely in the vicinity of Nanjing from 1991 to 1992 wascaused by a viral pathogen.A virus was isolated from the diseased tomato collected from field and.called the isolate TN.It infected 8 families 26 species host plants out of 10 families 39 species inoculated in the host ranse test and induced necrosis in tomato, pepper and tobacco.Its stability in extractedsap was TIP 50℃,DEP 510-3-10-4,and LIV 24-48hrs.The virus transmitted by aphid in non-persistence and reacted positively with CMV antiserum in agar diffusion test.Purified virions of the isolate TN were obtained by differential centrifugation and its morphology was spherical about 28nm in diameter by electron microscopy.Analysis of SDS-PAGE revealed that virion capsid protein had a singlecomponent (monomer),which molecular weight was 27500D.Viral RNAs contained normal CMVRNA 1-4 and additional lower molecular weight RNA5 by polyacrylamide gel electrophoresis. Theseresults were concurrent with the previous reports that

Preparation of High Titer Antiserum to Swee!Potato Feathery Mottle Virus(SPFMV)

MENG Qing, ZHANG He-Ling, SONG Ba-Fu, LONG Rui-Jie, GENG Ji-Yang, YANG Yong-Jia-

1994, 9(2): 151

weet potato feathery mottle virus(SPFMV)was inoculated to I.selosa to propagare by graftingand was purified wirh 0.2mol/L pH7.2 PBK.Further purification of SPFMV was conducted by sucrose cushion and sucrose density gradient centrifugation.Purified SPFMV had a OD260/280 ratio of1.25.Rilbbit was immuned with purifled viruses to prepare antiSerum.Antisera produced to purifiedvirus had a titer of 1:1096 in microprecipitin and ring precipitin tests.SPFMV in leaves of sweet potato and I.selosa were detected respectively by indirect enzyme-linked immunosorbent assay(ELISA)onnitreeellulose membranes.

The Fluorescence Staining of Grass Carp Hemorrhage Virus(GCHV)Polypeptides

WANG Wei, CHEN Yan, CA Yi-Quan, KE Li-Hua

1994, 9(2): 157

t had been reported that the GCHV(Gtass Carp Hemorrhage Virus)is a new member.in Reoviri-dae and has 11 polypeptides by our laboratory,We did further experiments on the viral polypeptides.The virions(GCHV-875)were purified from the medium of infection culture cells by CsCl density gradient centrifugation,dissolved in the buffer containing Tris-HCl 250mmol/L,glycerin 25%,SDS86mmol/L,2-ME 320mmol/L(pH6.9)and then heated at 100℃for 3 min.After that,by addingan equal volume of FITC solution(2mg/M)in 1 mol/L NaHCO3,the viral polypeptides were labelledwith FITC,and analysed by SDS-PAGE. All the viral structural polypeptides(11 bands),namelyVP130,VP120,VP113,VP107,VP75,VP65,VP60,VP52,VP42,VP39 and VP31,wete visible under. UV light.These polypeptides were retrieved by cutting down each band from the gel and demonstrated kept immunological property.The method can determine 500ng protein band in gel under UVlight.

The Use of Ampligen Alone and in Combination with Ganclclovlr and Coumermycin A 1 for the Treatmen1 0r Ducks Congenitally Infectcd with Duck H epatitis B Virus

NIU Jian-Zhang

1994, 9(2): 160

mpligen,a known immunomodulator and interferon inducer.was used alone and in conibination with other antiviral agents to treat ducks congenitally infected with duck hepatitis B virus.Theseantiviral agents included the conventional nucleotide analogue gunciclovir and the prokaryotic DNA gyrase B inhibitor coumermycin A1.When use alone,ampligen decreased the amount of serum and liverviral DNA,but had no effeet on circulating duck hepatitis B surface antigen (DHBsAg).In combination with sanciclovir,the antiviral effect appeared at least additive with a greater inhibition of vicalDNA replication within the liver.The conibination of ampligen with coutiiermycin A1 also resulted ininhibition of viral replication but to a lesser extent than ampligen alone.When all three agents wereused tapether,viral DNA replication was inhibited to the greatest extent but as with previous treatmentregimes,DHRsAg levels remained unchanged.At the end of the treatnient period for all resinies,analysis of viral DNA forms in the liver s

Isolation and Detection of Spherical Viruses from Penaells orientalis K

CHEN Dai-Hua, ZHANG Jian-Gong, DAN Zheng-Li, GAO Wei, GAO Hua-Xin, TUN Zhi-An

1994, 9(2): 170

uring the cultivition of Penaeus orientalis K,outbreak of death or slowing down of growth couldoccurred sometimes.tuge amount of dispersive spherical viruses with diameter around 80nm were observed in the midgut cells of those sick shrimps under electron microscope.Hoat cell organelles,such asmitochondria,became over swollen,and even broken.The virus was isolated from the hepotopancreasand digestive tube of the sick shrimp and the viriolls were icosahedron with diameter of 80nm on the av-erage.From infection experiment the corrected mortality was as high as 60%.The salne morphological spherical virus with the same size was also isolated from infected died shrimps digeetive glands andtubes.

The Correlation among Viral Axes,Triangulation Number and Capsomeres


1994, 9(2): 174

his paper talculates the length of the 5,3,and 2 fold rotational symmetry axes and the derivesforuiulas from them,they are and where a5,a3,and a2 are the length of the 5,3,and 2 fold axes reslxctively,1 is the distaisee between any two adjacent capsomeres,Tis trtangulation number.The formula of the that we hove reportedis derived ty the new method.These formulas connect T,a5,a3, a2,r and 1.

The Correlation among Viral Axes,Triangulation Number and Capsomeres

GAO Wen-Tong, GAO Jiao-Wan, FAN Fu-Zhong

1994, 9(2): 180

very best choice of several rnain affecfing factore on the crude purification of PRV was reporteddetailediy in this paper.The advanee purification method of PRV with the Cs SO4 continuous densitygradient supercentrifusation for about 3 hours with 30% sugar cushion was tested for the first time.Asimple procedure of one cycle of different speed centrifugution and one cycle of Cs2SO4 continuous density gradient supercentrifugation was designed at last.The virus preparation showed a typical ultravio let absorption curve for nucleoproteiris,The yield of virus was 2.56mg/100g of infected squashleaves.