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Objective@#To investigate the genetic characteristics of enterovirus A group 71 type (EV-A71) and etiological features of hand, foot and mouth disease (HFMD) in Xining city in 2017.@*Methods@#The pharyngeal swab specimens were collected from HFMD patients, and detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). For EV-A71 positive samples, virus isolation was performed. Then RNA of EV-A71 strains was extracted, and then VP1 coding region was amplified by RT-PCR. The phylogenetic tree was constructed by comparing with other genotypes and sub-genotypes strains of EV-A71.@*Results@#Total 57 strains of EV-A71 were isolated in Xining city in 2017, which all belonged to genotype C4a, and could be divided into two different lineages by phylogenetic analysis. The epidemic strains of EV-A71 in Xining City was closely related to other provinces of China in 2017.@*Conclusions@#C4a was the dominant genotype of EV-A71 in Xining city in 2017, and no other genotype was detected. In addition, EV-A71 isolated from Xining city was co-evolved with EV-A71 in other provinces of China.
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Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases worldwide. In the present study, a new virulent strain of PRRS virus (PRRSV), GDsg, was isolated in Guangdong province, China, and caused high fever, high morbidity, and high mortality in sows and piglets. The genome of this new strain was 15,413 nucleotides (nt) long, and comparative analysis revealed that GDsg shared 82.4% to 94% identity with type 2 PRRSV strains, but only 61.5% identity with type 1 PRRSV Lelystad virus strain. Phylogenetic analysis indicated that type 2 PRRSV isolates include five subgenotypes (I, II, III, IV, and V), which are represented by NADC30, VR-2332, GM2, CH-1a, and HuN4, respectively. Moreover, GDsg belongs to a newly emerging type 2 PRRSV subgenotype III. More interestingly, the newly isolated GDsg strain has multiple discontinuous nt deletions, 131 (19 + 18 + 94) at position 1404–1540 and a 107 nt insertion in the NSP2 region. Most importantly, the GDsg strain was identified as a virus recombined between low pathogenic field strain QYYZ and vaccine strain JXA1-P80. In conclusion, a new independent subgenotype and recombinant PRRSV strain has emerged in China and could be a new threat to the swine industry of China.
Subject(s)
China , Fever , Genome , Mortality , Nucleotides , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Swine , Swine DiseasesABSTRACT
Objective@#To investigate the genetic characteristics of enterovirus A 71 (EV-A71) and etiological features of hand, foot and mouth disease (HFMD) in Qinghai province from 2016 to 2017.@*Methods@#Specimens were collected from HFMD patients in Qinghai province, and detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). For EV-A71 positive samples, virus was isolated and RNA was extracted, and then VP1 coding region was amplified by RT-PCR. The phylogenetic tree was constructed by comparing with other genotypes and sub-genotypes strains of EV-A71.@*Results@#It was shown that 114 strains of EV-A71 were isolated in Qinghai province from 2016 to 2017, which all belonged to genotype C4a, and could be divided to two different lineages by phylogenetic analysis. From 2016 to 2017, the epidemic strains of EV-A71 in the different transmission chains of Qinghai province was closely related to other provinces of China.@*Conclusions@#C4a was the dominant genotype of EV-A71 in Qinghai province from 2016 to 2017, and no other genotype was detected. In addition, EV-A71 isolated from Qinghai province co-evolved with EV-A71 in other provinces of China.
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Globally, about 50% of liver cancer originates as a result of long term infection with hepatitis B virus (HBV), and some genotypes and mutations have been associated with an increased severity of infection. The aim of this study was to evaluate the genetic diversity of HBV in patients from Venezuela, with chronic infection, cirrhosis and hepatocellular carcinoma (HCC) and to compare the occurrence of mutations in all patient groups. Samples from patients with different pathologies of the liver, associated with HBV infection, were collected. The HBV S region was analyzed for genotype determination and, when available, the whole genome sequence was examined for mutations analysis. Genotype F was the most common genotype (87%). While the HBV subgenotype F3 was the most frequent genotype in the whole group of samples (44%), the subgenotype F2 predominated in HCC patients (56%). Mutations were more common in HCC and cirrhosis cases (p=0.01). The A1762T mutation was significantly associated with the advanced stage of liver disease (p=0.008). Additionally, mutations were more common in early stages of liver disease in HBV subgenotype F2- infected patients, and a significant association between this subgenotype and the emergence of T1753C, A1762T, A1762T/G1764A (p=0.04) and C1773T (p=0.001) mutations in chronic patients was found, when compared to the HBV subgenotype F3. By comparing F2 with all other HBV subgenotypes, a positive association for the three basal core promoter (BCP) mutants (A1762T, A1762T/G1764A p=0.01, G1764A p=0.04) was found. These results suggest that the HBV subgenotype F2 might be associated to more severe forms of liver disease in comparison with the HBV subgenotype F3.
Mundialmente, alrededor del 50% del cáncer de hígado se origina como consecuencia de la infección a largo plazo con el virus de la hepatitis B (VHB), y algunos genotipos y mutaciones han sido asociados con severidad incrementada de la infección. El objetivo de este estudio fue evaluar la diversidad genética del VHB en pacientes de Venezuela con infección crónica, cirrosis y carcinoma hepatocelular (CHC) y comparar la ocurrencia de mutaciones en los tres grupos de pacientes. Se reunieron muestras de pacientes con diferentes patologías de la enfermedad del hígado asociada a la infección por VHB. La región S del VHB fue analizada para la determinación del genotipo y cuando estuvo disponible, la secuencia del genoma completo fue examinada para análisis de mutaciones. El genotipo F de VHB fue el más frecuente (87%). Mientras que el F3 fue el subgenotipo más encontrado en el grupo completo de muestras (44%), el F2 fue predominante en pacientes con CHC (56%). Las mutaciones fueron más comunes en casos de pacientes con cirrosis y CHC (p=0,01). La mutación A1762T estuvo asociada significativamente con estado avanzado de la enfermedad del hígado (p=0,008). Adicionalmente, las mutaciones fueron más comunes en estados tempranos de la enfermedad del hígado en pacientes infectados con el subgenotipo F2, encontrándose una asociación significativa entre este subgenotipo y la ocurrencia de las mutaciones T1753C, A1762T, A1762T/ G1764A (p=0,04) y C1773T (p=0,001) en pacientes crónicos, en comparación con el subgenotipo F3. Por otro lado, al comparar F2 con los demás subgenotipos de VHB, se encontró una asociación positiva para las tres mutantes del promotor basal de la cápside (PBC) (A1762T, A1762T/G1764A p=0,01, G1764A p=0,04). Estos resultados sugieren que el subgenotipo F2 de VHB puede estar asociado a formas más severas de la enfermedad del hígado en comparación al subgenotipo F3.
Subject(s)
Humans , Genetic Variation , Hepatitis B virus/genetics , Carcinoma, Hepatocellular/virology , Liver Neoplasms/virology , Mutation , Venezuela , GenotypeABSTRACT
Objective To study the etiological agent of hand, foot and mouth disease ( HFMD) and the genetic characteristics of coxsackievirus A16 ( CVA16 ) strains isolated from clinical specimens of patients with HFMD in Liaocheng city in 2013.Methods Throat swab and stool specimens were collected from patients with HFMD in the disease surveillance hospitals in Liaocheng city from January to December 2013.Samples pos-itive for CVA16 strains were screened out for the isolation of virus strains with rhabdomyosarcoma ( RD) cells and Vero cells.The entire VP1 coding regions of 9 randomly selected CVA16 isolates were amplified and se-quenced.BioEdit and MEGA4 softwares were used for homology analysis.A phylogenetic tree among the 9 CVA16 isolates and 56 CVA16 representative strains of known genotypes and subgenotypes was constructed.Re-sults The results of PCR analysis showed that 747(77.73%) out of 961 specimens were positive for HFMD and among them, 74 samples (9.91%) were positive for EV71 strains, 130(17.40%) were CVA16 strains and 543(72.69%) were other enterovirus strains.The 9 CVA16 strains clustered into the B2b evolution branch of B genotype with the representative strains, sharing 97.7%to 100%homologies in nucleotide sequences and 99.3%to 100%in amino acid sequences.Conclusion Although EV71 and CVA16 strains were identified, other enteric viruses were the predominant pathogens causing HFMD in Liaocheng city in 2013.The CVA16 iso-lates belonged to B2b subgenotype.The pathogen spectrum of HFMD had already changed.It is necessary to strengthen the surveillance for EV71, CVA16 and other enteric viruses and understand their genetic characteriza-tions, which would be of great significance for the prevention and control of HFMD.
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Background: Cryptosporidiosis is one of the most difficult protozoan infections to treat, with only two drugs i.e. nitazoxanide and paromomycin known for treatment with variable response in different patients. Human cryptosporidiosis is accounted mainly by C. hominis and C. parvum. These two species or their subtypes are known to differ in clinical manifestations, and may differ in their response to drugs. So, we planned the study to see the effect of nitazoxanide and paromomycin on different isolates of Cryptosporidium in vitro. Methods: MDCK cell lines were used for in vitro growth of parasite and cytotoxicity of drugs to MDCK cells was determined by MTT assay after 3, 12 and 24 hours of drug exposure. Efficacy of non-toxic drug concentrations (<25% cytotoxic) on 12 Cryptosporidium isolates (7 C. hominis and 5 C. parvum) was determined at three different life cycle stages (in vitro growth, invasion and oocyst) by quantitative RT-PCR. Unpaired t-test was used to calculate the difference response of Cryptosporidium isolates to nitazoxanide and paromomycin. Results and conclusions: Cytotoxicity of nitazoxanide and paromomycin increased in dose and time dependent manner. After 24 hours of drug exposure, >25% cytotoxic effect was seen with nitazoxanide and paromomycin at concentrations of more than, 25μg/ml and 6mg/ml, respectively. Nitazoxanide was more effective than paromomycin in decreasing in vitro growth, invasion inhibition and reducing oocyst viability of Cryptosporidium isolates. Drugs effect was higher on growth inhibition followed by invasion inhibition and least in decreasing oocyst viability. Different isolates had variable response to drugs; cumulatively C. parvum isolates were more susceptible at particular drug concentrations than C. hominis isolates.
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Objective: To investigate the role of HBV subgenotypes B2, C2 in the carcinogenesis, treatment and prognosis of hepatocellular carcinoma (HCC). Methods: HBV genotypes and subgenotypes were detected in 462 HCC patients and 234 chronic hepatitis B (CHB) patients by a multiplex PCR assay, and HCC patients infected with HBV B2 or C2 were followed up for a year after surgical resection, transarterial chemoembolization(TACE) or a combination of both. Results: The HCC patients infected with HBV C2 had a higher chance to receive surgical treatment than those with B2 (P=0.007). Age of 40 years or older (P=0.030), male gender (P= 0.000), and viral load (>10 000 copies/ml) (P=0.017) were the independent risk factors for the carcinoge-nesis of HCC by using multivariate logistic analysis; however, there was no significant difference in the carcinogenesis of HCC between CHB patients with HBV subgenotypes B2 and C2. Age of 50 years or younger (P=0.044), infection with HBV B2 (P=0.027), and non-surgical treatment (P=0.000) were the independent risk factors for the recurrence of HCC. Thick trabecular type was more prevalent in HCC patients infected with HBV B2, C2 and genotype mixture (85.7%, 71.2% and 75.0%, respectively), and the proportions of histopathological types were not significantly different between HCC patients infected with HBV B2, C2 and genotype mixture. HBV subgenotype C2 was found in all HCC patients with rare histopathological type and subgenotype B2 and mixture were no found. Conclusion: There is no significant difference in the carcinogenesis of HCC between CHB patients with HBV subgenotypes B2 and C2. The HCC patients infected with HBV B2 have a lower chance to receive surgical treatment and are more severe than those with C2. HBV B2 is also closely associated with recurrence of HCC.
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Objective To study the genetic characterization of coxsackievirus A16 (CVA16)strains isolated during an epidemic of hand, foot, and mouth disease (HFMD) in Ningxia Hui Municipality in 2008. Methods Clinical samples were collected from HFMD patients in Ningxia Hui Municipality and CVA16 strains were isolated by viral isolation methods. Reverse transcriptionpolymerase chain reactions (RT-PCR), specific for CVA16 were performed with these CVA16 strains.Entire VP1 coding region amplification and sequencing were then performed and finally phylogenetic tree was constructed among Ningxia CVA16 strains and CVA16 representative strains of known genotypes and subgenotypes. Results 70 Ningxia CVA16 strains were isolated from HFMD patients in Ningxia in 2008 and the homology of nucleotide and amino acid were 90.8%-100.0% and 98.9%-100.0%, respectively. Phylogenetic characteristics of the strains reconfirmed that they could be divided into two distinct genotypes-A and B. Genotype B could be further divided into the subgenotypes B1 and B2, while all the 70 Ningxia CVA16 strains belonged to the co-circulated clusters B1a and Blb within subgenotype B1, which belonged to 2 viral transmission chains.Conclusion Our results indicated that subgenotype B1 CVA16 strains continued to circulate over a wide geographic area of mainland China since the first reported episode in Shenzhen city in 1999. Like other CVA16 strains isolated elsewhere in China, both Bla and Blb evolution branches were co-circulating in Ningxia Hui Municipality. Based on the close phylgenetic and chronological relationship with CVA16 isolated in other countries and regions near China. Our data confimed that these strains co-evolved and co-circulated with those from neighboring countries and regions.
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BACKGROUND: Hepatitis B virus (HBV) detected in Korean patients almost belongs to genotype C, which is subdivided into subgenotype C1 (or Cs) and C2 (or Ce). It was recently reported that the risk of hepatocellular carcinoma is different between subgenotype C1 and C2. Thus, we studied the distribution of subgenotypes of HBV in Korean chronic hepatitis B (CHB) patients. METHODS: Specimens of 421 patients, who were diagnosed as CHB and underwent antiviral treatment, were used. After sequence analysis for HBV S gene, subgenotype was identified through phylogenetic analysis. Utilizing the same sequence data, the distribution of serotypes was also investigated. RESULTS: Among 421 patient specimens, genotype C was found in 419 (99.5%) and genotype B in 2 (0.5%). Among the genotype C strains, 417 strains were C2 subgenotype and 2 strains were mixed subgenotypes. However, C2 was evidently found even in the mixed sequences. Serotypes of 419 HBV with genotype C were classified as follows: adr, 385 (91.9%), adw, 22 (5.3%), ayr, 2 (0.4%) and mixed serotype, 10 (2.3%). Serotype of both HBV with genotype B was adw. CONCLUSIONS: It was found that HBV detected in Korean CHB patients under treatment almost all belong to the C2 (Ce) genotype.
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Humans , Antiviral Agents/therapeutic use , Blood Specimen Collection , Genotype , Hepatitis B virus/classification , Hepatitis B, Chronic/diagnosis , Korea , Phylogeny , Sequence Analysis, DNA , SerotypingABSTRACT
Objective To compare and evaluate two type-specific primers PCR genotyping methods of hepatitis B virus ( HBV) which were established by Naito et al ( Naito method) and our lab (new method). Methods The two genotyping methods were applied for detecting the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2 and the plasmids mixed with different proportion of subgenotypes B1 and C2. In addition, the genotypes of 113 serum samples of patients with chronic HBV infection from Shenzhen, Handan and Urumqi cities of China were identified by the two methods, respectively. The results were verified by PCR product based sequencing. Results The sensitivity of the two methods showed no difference when they were applied to detect the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2. While detecting the plasmids mixed with different proportion of subgenotypes B1 and C2, the sensitivity of the new method was superior than that of Naito method. Meanwhile, the specificity of the new method was obviously superior than that of Naito method. Both of the two methods were highly sensitive in identification of HBV genotypes of serum samples with a single genotype. However, the new method showed more sensitive in identification of the B/C mix strains than that of Naito method. The total coincidence rate between the two methods was 83. 2% (94/113), with the discrepancy of 16. 8% (19/113). Fifteen of the 19 inconsistent genotypes by the two methods were selected and their PCR products were sequenced directly. The sequencing results were identical with that of the new methods, but not with that of the Naito method. Conclusion The new method is more sensitive, and its specificity is superior to the Naito method. It could be used for clinical and epidemiological studies on HBV genotype and subgenotype in China.
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To investigate the distribution of Hepatitis B virus (HBV) genotypes among the population of Dai nationality in Xishuangbanna, Yurman Province HBV genotypes of the Serum samples were tested by PCR-RFLP. This is the first time to discover the B+E genotypes in China. This finding provides new information for understanding the distribution of HBV genotype in China and a provides a basis for establishing a Chinese gene bank.
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Hepatitis A virus (HAV) infection is a public health problem worldwide and the virus has been classified into six genotypes. In Brazil, the only genotype that has been found is genotype I, predominately from subgenotype IA. Here, the HAV genotypes were analyzed of 18 isolates circulating between 1996-2001 in Goiânia, state of Goiás, Brazil. Viral RNA was extracted from 18 serum samples and amplified (RT-PCR/nested-PCR), followed by the genomic sequencing of the VP1/2A junction region of the HAV genome. Sequences of 168 nucleotides were compared and analyzed using the BLAST N, Clustal X and PAUP v. 4.10b programs. All samples were classified as genotype I, with 10 belonging to subgenotype IA and eight to subgenotype IB. The subgenotype IA isolates showed greater diversity than the subgenotype IB isolates at the nucleotide level. Elevated identity values were found between isolates obtained in this study and those from other regions of the world, including Brazil, highlighting the high conservation among different isolates of this virus. However, changes in the HAV subgenotype circulation could also be observed during the evaluated period.
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Adolescent , Adult , Child , Child, Preschool , Female , Humans , Male , Young Adult , Hepatitis A virus/genetics , Hepatitis A/virology , RNA, Viral/genetics , Base Sequence , Brazil , Hepatitis A virus/isolation & purification , Molecular Sequence Data , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Young AdultABSTRACT
Objective To establish a hepatitis B virus (HBV) nested PCR (nPCR) for detection of genotypes A-D and subgenotypes B1,B2, C1 and C2. Methods The entire HBV nucleotide sequences of genotypes A-H retrieved from GenBank were compared and analyzed by DNAStar software. The PCR primers were designed by Primer Premier 5.0 software,and the nPCR for genotyping HBV/A-D as well as subgenotyping B1, B2,C1 and C2 were established. There were 3 steps in the process:step 1 for genotypes B, D and subgenotypes C1, C2 with the amplification of Mix A; step 2 for genotype A with the amplification of Mix B; step 3 for subgenotypes B1 and B2 with the amplification of Mix C in the second-ound PCR, based on the first-round amplification procedure. A total of 68 serum samples from patients with chronic HBV infection were detected by nPCR. 15 of 68 sera were selected randomly and their PCR products were directly sequenced to confirm the accuracy of the method. Results Among 68 serum samples of patients with chronic HBV infection detected by the nPCR, 23.53% (16/68) were infected with B2, 11.76% (8/68) with C1,48.53% (33/68) with C2,1.47% (1/68) with D,11.76% (8/68) with B2C2 mix strains,1.47% (1/68) with C2D mix strains and 1.47% (1/68) with B2/C1/D mix strains. The sequencing analysis of the 15 serum samples had the same results as detected by nPCR. Conclusion nPCR is a simple,rapid method and able to detect genotypes A-D and subgenotypes B1 ,B2 ,C1 and C2 subtypes of HBV with both high sensitivity and specificity.
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To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B+C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B+C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B+C) (P=0.077/P=0.663). In Dali, genotypes B, B+C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B+C) in patients.
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Objective To study the distribution of hepatitis B virus genotype and subgenotype in Handan and clarify the genotype-related difference in clinic.Methods PCR-RFLP was used to detect the HBV genotype and subgenotype in 108 HBV-infected patients from Handan Infectious Disease Hospital from January,2006 to April,2006.Analyze the correlation of HBV genotype and clinical characteristicly of patients.Results Among 108 HBV-infected patients,the proportion of genotype B and C is 6.5% and 93.5% respectively.All genotype B patients were subgenotype Ba.In genotype C group,88.1% was subgenotype C2;genotype distribution showed no significant difference between male and female.ALT and AST level were higher in genotype C group than those in genotype B group.Conclusion HBV genotype C is predominant in chronic hepatitis B patients in Handan city.HBV subgenotype C2 is the major subgenotype in Handan city.The positive rate of HBeAg and HBV DNA levels are higher in genotype C than those in genotype B group.Genotype C and genotype B have no difference in the severity of liver disease and HBV-DNA.associated with.Clinical feature of subgenotype C1 and subgenotype C2 is similar.
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This study was done in 2004. Subgenotypes of HIV were determined by DNA sequencing technique at 3 gene regions of HIV-l: envelope, protease and reverse transcriptase. Our results showed that the recombinant virus CRFOI AlE was still predominant (98%) in Ho Chi Minh city. A new recombinant form HIV-l of subtype B and CRFOIAIE was also detected. The presence of a new recombinant form of HIV-1 derived from two different subtypes warns that the HIV epidemic in this region is more and more complex with the immigration and co-infection with different genetic subtypes of HIV.
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HIV-1 , DNAABSTRACT
The detecting of HCV-RNA and genotyping of HCV in sera of 65 paid blood donors which are positive for anti-HCV were conducted,indicating that 60 of them appeared positive for HCV-RNA,and their genotype of HCV was classified as genotype- I . It was suggested that the genotype- I of HCV could be considered as the predominant infectious strain in this group of paid blood donors. It was found that the endonuclease digestion method of typing based on HCV 5' -noncoding region PCR was better compared with the direct method of typing based on HCV core region PCR in terms of its senstivity,economy and simplicity.