Different strategies of reducing non-viral nucleic acid of meta-virome in plasma: a comparative study / 中国输血杂志

【Objective】 To explore the influence of common methods of reducing non-viral nucleic acid on the abundance of plasma virus group. 【Methods】 Three kinds of library construction, five kinds of centrifugation conditions, two kinds of filters, four kinds of enzymes and four concentrations of chloroform were used to treat plasma samples added quantitatively 2.16 mL of pseudorabies virus(PRV) and 2.16 mL of porcine parvovirus(PPV). A total of 21.6 mL of plasma samples were processed, including 54 samples. Subsequently, nucleic acid was extracted, mitochondrial DNA(mtDNA) and two viruses were quantitated, the library of the next generation sequencing was constructed, Illumina NovaSeq 6000 was used for the next generation sequencing. The sequencing data were compared with Kraken Py 2.0 software, and the species annotation analysis was conducted. The corresponding species classification information of each segment was obtained to analyze the impact of different reducing non-viral nucleic acid methods on the relative abundance of microorganisms and two indicator viruses. 【Results】 After sequencing by Illumina NovaSeq 6000, 306.27 GB raw data and 193.17 GB clean data were obtained, with Q20>90%, Q30>85%, Error Rate of 0.03%, and average GC Content of 45.02%. The DNA library construction process significantly increased the proportion of microbial sequences and the PRV abundance [(91.8±0.5)%](P<0.05); RNA library construction and combined library construction can increase the abundance of Pestivirus, an RNA virus, and the PRV abundance was(17.7±3.3)% and(8.1±1.5)% respectively. The Ct value of mtDNA was increased and the proportion of human sequence decreased to less than(89.5±1)%, while the proportion of microbial sequence increased to (2.4±0.03)% after treatment of five centrifugation conditions(P<0.05); After centrifugation at 4℃, 100 g, 30 min, the PRV abundance was increased to (40.6±6)%, and centrifugation at 4℃, 4 000 g, 45 min reduced the PRV abundance to (4.1±0.01)%(P<0.05). Both of 0.22-μm filter and 0.45-μm filter increased the Ct value of mtDNA to above 25.56±0.13, decreased the proportion of human sequence to less than (86.1±0.6)%, increased the proportion of microbial sequence to (3.1±0.1)% and (3.4±0.2)%, and decreased the PRV abundance to (1.6±0.3)% and (4.1±0.7)%(P<0.05), while there was no statistical difference in the effect on PPV concentration and abundance. DNase Ⅰ and Benzonase increased the Ct value of PPV to 25.65±0.06 and 25.36±0.45, decreased the proportion of human sequence to (81.7±5.6)% and (72.8±6.7)%, and increased the proportion of microbial sequence and PRV abundance to (11.0±4.1)% and (16.1±4.7)%, (55.8±2.3)% and (39.0±8.9)%, respectively(P<0 05); After treatment with RNase A, the Ct value of PRV increased to 25.20±0.11, and the human sequence proportion decreased to (85.4±5.6)%(P<0 05); Lysozyme had no effect on removing non-viral nucleic acid. The chloroform of 1%, 5%, 10% and 20% increased Ct value of PRV and mtDNA to no less than 27.17±0.21 and 25.68±0.04; Only 10% chloroform increased the proportion of microbial sequences to (3.1±1.2)%(P<0.05); The abundance of PRV with 1% and 5% chloroform treatment was increased to (48.7±13.3)% and (42.1±5.5)%(P<0.05), while 10% and 20% chloroform reduced PRV abundance to (1.0±0.5)% and (3.4±2.8)%(P<0.05). There was no statistical difference in the effect of chloroform with four contents on PPV abundance. 【Conclusion】 Centrifugation at 4℃, 5 000 g, 10 min is suitable for increasing the overall abundance of virus, and centrifugation at 4℃, 100 g, 30 min is suitable for increasing the content of virus similar to PRV. 0.45-μm filter, DNase Ⅰ, Benzonase and low concentration chloroform can effectively reduce the proportion of non-viral nucleic acid sequence in plasma to increase the abundance of the indicated virus group. Thus, the enrichment effect of plasma meta-virome is closely related to the nature of the virus, and the appropriate virus enrichment method should be selected according to the research purpose to establish the corresponding enrichment strategy.

The clinical epidemiologic characteristics of patients infected with hepatitis C virus post blood transfusion in Shanghai area / 中华传染病杂志

Objective To investigate the clinical epidemiologic characteristics of patients with hepatitis C virus(HCV)infection post blood transfusion.Methods The polymerase chain reaction (PCR)and enzyme linked immunosorbent assay(ELlSA)were used to detect HCV RNA and antiHCV,respectively.Analysis was performed for patients' age distribution,cause of primary diseases,exposure years,ingredient and amount of transfusion,incubation period and liver function damage.The statistical processing were performed with chi-square test,t-test and correlation analysis.Results HCV RNA levels were higher than 3.0 log10 copy/mL in 85.3%infected patients with a median of 5.99log10 copy/mL,among which 19.7%patients showed viral load 3.0 to 4.0 log10 copy/mL and 69.9%showed 5.0 to 6.0 log10 copy/mL.Eighty-one point six percent(40/49)of infected persons were confirmed as HCV RNA positive by HCV RNA qualitative analysis,while 99.7%(383/384)patientswere detected as anti-HCV positive by serological test.The sensitivity of serological test was higher than both HCV RNA quantitative and qualitative assays(F=57.138,P=0.000;F=63.149,P=0.000,respectively).HCV infection post blood transfusion was more common in people of 30 to 60years old.Most cases(84.4%)got the first time exposure during 1990 to 1994.More than 10%cases had primary disease as obstetrics, orthopedics or gastrointestinal tract hemorrhage. Eighty percent received whole blood product transfusion.The mean interval between transfusion and clinical diagnosis was (86.0±54.6 ) months. Eighty nine percent of infected patients had liver function damage, while most of them showed elevated alanine aminotransferase (ALT) with no more than 5 upper limits of normal (ULN). Conclusions Post transfusion HCV infection mainly happened in adulthood. Infected patients usually have liver function damage with elevated ALT with no more than 5 ULN and medium HCV RNA levels.

DNA Sequence Features of Transfusion Transmitted Virus in Neonatal Infection / 实用儿科临床杂志

Objective To analyze the mutations in nucleotide sequences of transfusion transmitted virus(TTV) in neonatal infection.Methods Neonatal serum TTV-DNA was detected by a nested PCR technique.Fifteen Chinese neonates with positive TTV-DNA were diagnosed as TTV infection.ORF1 sequences of TTV-DNA from these neonates were determined.Results Homology of Chinese TTV(C01-C15) and Japanese TTV(N22)isolated ranged from 87.1%-97.7% at nucleotide level,but there were point mutations in Chinese TTV,such as GG→TT in locus 112 and 113,TTATC→CCTAT in locus 236-240.Conclusions Chinese and Japanese TTV isolated had the same genotype.Some gene mutations may increase the TTV pathogen,and result in neonatal hepatitis syndrome or hyperbilirubinemia.

Gene determination of transfusion transmitted virus-like mini virus / 吉林大学学报(医学版)

Objective To detect the transfusion transmitted virus-like mini virus(TLMV) sequence gene in patients with chronic hepatitis and to study the TLMV infection in population.Methods Two sets of primers in the most conservative regions of Japan strains TLMV-CBD279 and CBD231 were designed to amplify TLMV templete extracted from sera of patients with chronic hepatitis B using PCR.PCR products were cloned into pGEMR-T vector and sequenced.Results The results showed that 72%of TLMV sequence isolated in China was identical to that in Japan,suggesting that there was TLMV infection in patients with chronic hepatitis B in China.The patients with hepatitis virus C and the healthy blood donor had the highest infectious rates of TLMV.Conclusion TLMV infection exists in patients with chronic hepatitis B in China.

Prevalence and Genotypes of Transfusion-Transmitted Virus in Children with Hepatitis and Normal Control / 대한소아소화기영양학회지

PURPOSE: Transfusion transmitted virus (TTV) is a newly discovered virus and to date the contribution of TTV to liver disease remains unclear. Little is known about the frequency of TTV infection in children in Korea. The purpose of this study was to investigate the prevalence and genotypic distribution of TTV carried by healthy children and patients with hepatitis in Korea. METHODS: Eighty eight of healthy children and three groups of patients with hepatitis-14 patients with chronic hepatitis B, 12 patients with chronic hepatitis C and 25 patients with hepatitis of unknown etiology-were tested. TTV DNA was detected by semi-nested PCR using primer sets generated from N-22 region and from 5' noncoding region (NCR) of the viral genome. PCR products derived from 8 patients with hepatitis and from 11 healthy children were sequenced and a phylogenetic tree was constructed. RESULTS: TTV was found by PCR with N22 primer in 11.3% of healthy children, 28.5% of children with hepatitis B, 25% of children with hepatitis C, 24% of children with hepatitis of unknown etiology. TTV DNA was found by PCR with 5'NCR primer in 32.9% of healthy children, 71.4% of patients with chronic hepatitis B, in 50% of patients with hepatitis C and in 48% of patients with hepatitis of unknown etiology. TLMV DNA was found in 48.9% of healthy children, 21.4% of patients with hepatitis B, 16.6% of patients with hepatitis C, 40% of patients with hepatitis of unknown etiology. Among the sequenced isolates, 10(52%) belonged to genotype 1 (G1) and others belonged to genotype 2 (G2) or genotype 3 (G3). Among the G1 sequences, 7 were grouped as G1a. CONCLUSION: TTV infection was common in healthy children and in patients with hepatitis. But, the prevalence of TTV DNA by 5'NCR primer was relatively high in patients with hepatitis B and there may be some association between TTV and hepatitis B virus infection. G1 was the major genotype of the studied population.

Distribution of Transfusion-transmitted Virus Genomic Groups in Blood Products / 대한진단검사의학회지

BACKGROUND: Transfusion-transmitted virus (TTV) is a small DNA virus with single-stranded, closed circular, antisense genome infecting humans. The TTV has been classified into five major genomic groups 1-5. There have been a few studies on TTV prevalence in blood donors and blood products in Korea. However there have been no reports on the TTV genomic groups in Korea. The aim of this study was to gain information on TTV genomic groups in blood products in Korea. METHODS: A total of 50 plasma samples from blood products (25 units each of red blood cell and whole blood) were tested. The samples are obtained from the segments of the blood products. TTV DNA was detected using polymerase chain reaction (PCR) with two sets of universal primers (A set and B set), and TTV genomic groups were determined using PCR with group specific primer sets. RESULTS: TTV DNA was detected in 96% (48/50) of the blood products: the TTV genomic group 3 was found the most frequently (52%, 26/50), followed by group 4 (46%, 23/50), group 1 (20%, 10/50), group 5 (10%, 5/20), and group 2 (2%, 1/50). There were seven blood products (14%) infected with TTVs but their genomic groups were not identified with group specific primer sets. Among the blood products, 44% (22/50) were infected with a unique TTV genomic group; 38% (19/50) were coinfected with TTV from 2 (28%, 14/50) or 3 (10%, 5/50) genomic groups. CONCLUSIONS: Blood products are frequently infected with TTV and all five known genomic groups are detected in Korea.

Transfusion Transmitted Virus Hepatitis in Neonates and Curative Effects of Genciclovir / 实用儿科临床杂志

Objective To investigate the pathogenicity of transfusion transmitted virus (TTV) infection and assess the effect of genciclovir on TTV.Methods Serum TTV-DNA from 968 neonates was detected by a nested polymerase chain reaction technique and electropherosis. Alanine aminotrans ferase (ALT) and direct bilirubin (DB) were assayed in neonates with positive TTV-DNA.Genciclovir[10 mg/(kg?d)]was used to treat neonates with TTV-induced hepatitis.Results Among 968 neonates, 38 had positive TTV-DNA (4.0%). All neonates with positive TTV-DNA had normal serum levels of ALT and DB [(24.8?12.0) U/L and (17.6?6.8) ?mo l/L] 3 days after birth;But an elevated ALT and DB level [(95.5?16.4) U/L and (58.2?10.4) ?mol/L] occurred in 15 of them 2 weeks after birth,and were diagnosed as TTV-induced hepatitis.These patients had hypersomnia,jaundice and anorexia. Serum ALT and DB levels recovered to normal range one week after genciclovir therapy in 11 patients,so did the other 4 patients after 2 weeks therapy with genciclovir. Serum TTV-DNAs in all patients became negative 2 weeks after genciclovir therapy.Conclusion TTV infection exists in the neonates, and may be one of important causes of neonatal hepatitis.genciclovir might have a good anti-TTV effect.

The Prevalence of Transfusion-Transmitted Virus Infection in Children / 소아과

PURPOSE: Transfusion-transmitted virus(TTV) is an newly described nonenveloped human virus, with a circular, negative stranded DNA genome. Although a high prevalence of TTV infection in the normal population has been demonstrated, there is a still possibility of association with hepatitis according to the genotype of TTV. The aim of this study is to investigate the prevalence of TTV infection in Korean children. METHODS: Nested polymerase chain reaction(PCR) using priner sets generated from the noncoding region(NCR) of the viral genome was done in 105 children without liver disease, aged 0-15 years. We performed a second set of PCR using N22 primer in 88 children after the first set of PCR. RESULTS: The TTV DNA was detectable in 36(34%) of 105 children without hepatitis by 5'NCR primer. The prevalence of TTV varied with age:<1 y,16%(4/25); 1-3 y, 44%(15/31); 4-6 y, 31%(5/ 16); 7-9 y, 25%(3/12); 10-15 y, 14%(3/21). By using N22 primers, the prevalence of TTV DNA in children without hepatitis was 11.3%(11/88):<1 y 8%(2/25); 1-3 y, 13.7%(4/29); 4-6 y, 6.2%(1/16); 7-9 y, 33.3%(2/6); 10-14 y, 8.2%(1/12). CONCLUSION: Our result showed a high prevalence of TTV infection, varying with age, in Korean children. Further evaluation of genotypes of TTV in patients with hepatitis and normal children is needed.

Transfusion transmitted virus infection in the neonates / 中华传染病杂志

Objective To investigate the relationship between transfusion transmitted virus (TTV)infection and neonatal hyperbilirubinemia,the effect of TTV infection on the liver function, and analyse the feature of nucleotide sequences in TTV ORF1.Methods Serum TTV DNA,which were from 58 neonates with high direct bilirubin(DB,including 5 with hepatitis Syndrome),92 ones with high indirect bilirubin(IB),and 85 normal ones,was detected using a nested polymerase chain reaction technique(nPCR),electropherosis and sequence analyse,and serum alanine amniotransferase (ALT)was determined in all neonates.Results In DB neonates,TTV DNA were detected in 7 neo- nates(12.1%,including 3 neonates with hepatitis syndrome);in IB and normal ones,1 neonate had positive TTV DNA(1.1% and 1.25),respectively.Even if there were point mutations in Guangdong's TTV,the homology of Guangdong's TTV(GD1-9)and Japanese TTV(N22)ranged from 87.1%～97.8% at nucleotide level.Conclusion TTV infection may be one of important pathogenesis resulting in neonatal hyperbilirubinemia and liver damage in such patients.Guangdong's and Japanese TTV isolates had the same genotype,some gene mutations maybe increase the pathogenicity in TTV.

Gene quasispecies analysis of transfusion transmitted virus DNA in two patients with transfusion transmitted virus infection / 中国医师杂志

Objective To investigate gene variation and the relationship between gene variation and pathogenicity of transfusion transmitted virus(TTV).Methods The TTV DNA in the serum sample from a blood donor(BD) and a chronic non-A-G severe hepatitis(CSH) patient with TTV infection was amplified by using PCR.The purified PCR product was cloned and 10 clones from each case were sequenced.The sequences were compared among different clones and analyzed by Phylogentic tree.Results There were two different TTV strains in the BD and seven different TTV strains in the chronic non-A-G severe hepatitis patient.The TTV clones in the BD were of G1a subtype and those of the CSH were of G1a and G1b subtype.Conclusion Gene variant of TTV was much more complicated in the CSH patients than that in the BD ones.

THE INVESTIGATION OF DIFFERENT POPULATION＇S TRANSFUSION-TRANSMITTED VIRUS-DNA IN SHAANXI PROVINCE / 药物分析学报

Objective To investigate transfusion transmitted virus (TTV) infection among population of different groups in Shaanxi Province. Methods A nested polymerase chain reaction (PCR) with primers from ORF1 of TTV genome was established to detect TTV-DNA in serum of the patients. ResultsTTV-DAN was detected in the sera of 3 of 50 cases of general population(6％), 2 of 30 cases of vocational blood donors(6.7%),21 of 97 cases with Type B hepatitis (21. 6％), 9 of 35 cases of Type C hepatitis (25. 7％),and 23 of 40 cases with non-A～ non-G hepatitis (57.5 ％ ). ConclusionThere is TTV infection among general population in Shaanxi Province. TTV may be an impor- tant agent to cause non-A～non-G hepatitis. And the patients with HBV or HCV can have overlapping TTV infection.

Study on mother-to-child transmission route of transfusion transmitted virus infection / 中华传染病杂志

Objective To investigate the prevalence of TT virus infection in pregnant women in Shenzhen and the role of mother-to -child transmission route in the TTV infection. Methods TTV DNA were detected by nested PCR in sera, cord blood and breast milk samples from 400 mother-infant pairs. TTV DNA positive mothers were followed up for 6 months for monitoring TTV DNA. Thirty two TTV DNA PCR products amplified from blood and milk samples of 13 mother-infant fairs were cloned and sequenced. Results By means of PCR, TTV DNA were detected in 62 of mothers' serum samples, 4 of 400 cord blood samples, and 20 of 400 milk samples, respectively. Therefore, 6.5%(4/62) of infants are likely to be infected with TTV from their mothers during the fetal period. Direct sequencing of TTV DNA from 13 mother-child pairs showed identical isolates (nucleotide homology from 97.5% to 99%), Follow-up sera of 42children, whose mothers were TTV DNA positive, showed 16 cases turned out to be existence of TTV viremia at 6 months old. Conclusions Our data proved the mother-to-child transmission route of TTV infection.TTV can be transmitted transplacentally and postnatally by the way of breast feeding as well. The increase of TTV prevalence during the 6 months after birth, suggests the postnatal transmission route of TTV may be of more importance, and further study is needed to address whether preventive measure should be done.

Comparison of Transfusion Transmitted Virus Infection between Normal Children and Hepatitis B Virus Carrying Children / 实用儿科临床杂志

Objective To study the prevalence of transfusion transmitted virus (TTV) in normal children and hepatitis B virus (HBV) carrying children. Methods Anested polymerase chain reaction(nPCR) assay was established using two - set of primers designed from the first open read frame of TTV genome. Serum from the children who received physical examination was tested by nPCR assay for the presence of TTV- DNA. Results The total detectable rate of TTV - DNA in those 230 objects was 17.4% , in which the positive rate of TTV - DNA was 13.7% in normal children and was 25.7% in children carrying HBV. There was significant difference in the positive rate of TTV - DNA between normal children and the children carrying HBV. Conclusion There is TTV infection in normal children,and there is higher positive rate of TTV-DNA in children carrying HBV in Xinjiang.