Preliminary investigation and analysis of nucleotide site variability of nine glycoproteins on varicella-zoster virus envelope, Jilin Province, China, 2010-March 2024.

Varicella is endemic worldwide. In China, varicella has not yet been included in the list of legal infectious diseases, nor has a unified national surveillance program been established. And the live attenuated varicella vaccine has not been included in routine immunization. In this study, we analyzed for the first time the varicella epidemiology in Jilin Province in the past 20 years, and the nucleotide site, amino acid site and N-glycosylation site variation of glycoprotein in varicella-zoster virus (VZV) surface 9 in the past 15 years. The results showed that the reported incidence of varicella in Jilin Province in the last 20 years was fluctuating above and below 20/100,000, especially after the epidemic of the COVID-19, and fatal cases appeared in individual years. The genotypic branching of VZV was monitored as Clade 2 in the last 15 years. 9 glycogen nucleotide sites of VZV had different degrees of variability, and the variability had specificity. Therefore, it gives us the idea that in order to reduce the incidence of varicella and herpes zoster, a provincial or even national surveillance program should be introduced as early as possible, and the dynamic monitoring of the variability of the nucleotide sites of VZV should be strengthened at the same time as the vaccine immunization strategy is introduced.

Genome cloning and genetic evolution analysis of eight duck-sourced novel goose parvovirus strains in China in 2023.

Introduction: There are three major categories of waterfowl parvoviruses, namely goose parvovirus (GPV), Muscovy duck parvovirus, and novel goose parvovirus (NGPV). NGPV can infect both Cherry Valley ducks and mule ducks, resulting in short beaks and dwarfism syndrome, and the incidence of short beaks and dwarfism syndrome rises annually, posing a significant threat to the waterfowl breeding and the animal husbandry. Therefore, clarifying the biological characteristics and genetic evolution of NGPV is very important for the prevention and control of NGPV. Methods: Ducks with short beaks and dwarfism syndrome from Shandong and Henan Province were investigated by dissection and the tissue samples were collected for study. The NGPV genome was amplified by PCR, and the genome was analyzed for genetic evolution. Results: Eight strains of NGPV were isolated, which were designated as HZ0512, HZ0527, HZ0714, HZ0723, HZ0726, HZ0811, HZ0815, and HN0403. The nucleotide homology among these strains ranged from 99.9% to 100%. The eight strains, along with other NGPVs, belong to GPV. The eight strains showed a 92.5%-98.9% nucleotide homology with the classical GPV, while a 96.0%-99.9% homology with NGPV.Therefore, it can be deduced that there have been no major mutations of NGPV in Shandong and Henan provinces in recent years. Discussion: This study lays a theoretical foundation for further studying the genetic evolution and pathogenicity of NGPV, thereby facilitating the prevention and control of NGPV.

A case of mild partial androgen insensitivity syndrome in a juvenile boy.

Androgen insensitivity syndrome (AIS) is a rare disorder with X-linked recessive inheritance in 46 XY patients. The clinical manifestations vary between patients, especially regarding external genitalia development. Herein, the case of AIS in a 13-year-old male, who was born with hypospadias and presented to the hospital with gynaecomastia that had developed from 8 years of age, is reported. No micropenis, cryptorchidism or bifid scrotum were found. Testis volume was 12 ml on both sides. His testosterone and luteinizing hormone levels were normal compared with sex- and age-adjusted reference range. His bone age was approximately 13 years according to Greulich-Pyle assessment. Sequence analysis of the androgen receptor (AR) gene revealed a mutation (c.2041A>G) in exon 4, a novel mutation site in the AR gene. Prediction analysis suggested this to be a disease-causing variant. A milder clinical presentation and normal hormone levels in cases of partial AIS might differ from the usually reported signs and symptoms. A diagnosis of AIS should not be ignored in teenage patients who present with gynaecomastia and hypospadias, but normal hormone levels.

Primary drug resistance among HIV-1 patients in Hubei Province in 2020-2022 / 公共卫生与预防医学

Objective To investigate the prevalence of primary drug resistance among HIV-1 patients in Hubei Province from 2020 to 2022, and to provide corresponding basis and data support for HIV antiviral therapy (ART) in Hubei Province. Methods During 2020-2022, plasma samples of HIV-1 infected patients before ART were collected., Patients’ demographic data and baseline laboratory test data were also collected. HIV-1 pol region was amplified by in-house method for sub-type typing and drug-resistant mutation site analysis. Results The pol gene sequence was successfully amplified in 242 of 285 cases, with a success rate of 84.9%. CRF07_BC was the predominant HIV-1 sub-type, accounting for 47.11% (114/242), followed by CRF01_AE, accounting for 25.21% (61/242), sub-type B, accounting for 14.16% (35/242), and CRF55_01B, accounting for 4.13% (10/242). The primary resistance rate was 6.20% (15/242). The mutation site of nucleoside reverse transcriptase inhibitors (NRTIs) was mainly M184V, and the mutation sites of non-nucleoside reverse transcriptase inhibitors (NNRTIs) were mainly E138A/G/EG and V179E. These different mutation sites led to different degrees of drug resistance to 12 drugs. The incidence of drug resistance mutation of CRF55_01B sub-type was significantly higher than that of other sub-types. Conclusion The primary drug resistance rate of HIV-1 infected patients is at a slightly high level in Hubei Province, and close monitoring of primary drug resistance and mutation sites should be strengthened before ART, especially for CRF55_01B sub-type.

Resistance to deltamethrin and its association with mutation sites in the sodium iron channel domain III gene in Rhipicephalus microplus in Huaihua City of Hunan Province / 中国血吸虫病防治杂志

Objective To investigate the level of deltamethrin resistance and mutation sites in the sodium iron channel gene in Rhipicephalus microplus in Huaihua City, Hunan Province, and to examine the correlation between deltamethrin resistance and mutation sites in the sodium iron channel gene in Rh. microplus. Methods Rh. microplus was sampled from multiple yellow cattle farms in Huaihua City, Hunan Province from June to September 2022, and the level of resistance to deltamethrin was determined in ticks using the adult immersion test. The sodium iron channel domain III gene was amplified in deltamethrin-resistant and wild-type Rh. microplus using PCR assay. Following sequencing and sequence alignment, mutation sites were detected in bases. The sodium iron channel domain III gene in Rh. microplus was translated, and the signal peptide, transmembrane domain, and phosphorylation and glycosylation sites were detected in amino acid sequences. The tertiary structures of the sodium iron channel domain III protein of deltamethrin-resistant and wild-type Rh. microplus were deduced and compared, and the association be tween mutation sites in bases and resistance to deltamethrin was examined in Rh. microplus according the level of deltamethrin resistance, sequence alignment and protein tertiary structure. Results The median (LC50) and 95% lethal concentrations (LC95) of deltamethrin were 121.39 mg/L and 952.61 mg/L against Rh. microplus, with a resistance factor of 9.24 and level II resistance. The sequence of the sodium ion channel domain III gene was 1 010 bp in size, and mutation sites were detected in two neighboring bases in the sequence of the sodium ion channel domain III gene in deltamethrin-resistant Rh. microplus. Although no signal peptides were found in the sodium iron channel domain III protein of deltamethrin-resistant or wild-type Rh. microplus, 6 trans-membrane domains, 42 phosphorylation sites and 8 glycosylation sites were identified, with a significant difference in the tertiary structure of the sodium iron channel domain III protein between deltamethrin-resistant and wild-type Rh. microplus. Conclusions Level II resistance to deltamethrin is detected in Rh. microplus in Huaihua City, Hunan Province, and two mutation sites that correlate with the emergence of deltamethrin resistance are identified in the sequence of the sodium iron channel domain III gene in deltamethrin-resistant Rh. microplus.

Occult HBV among anti-HBc + alone and not alone samples: mutation analysis of S Gene / 中国输血杂志

【Objective】 To investigate the status of occult hepatitis B virus infection(OBI) among blood donors in Quzhou, Zhejiang, and to analyze the mutation of S region in blood donors with anti-HBc+ alone and non anti-HBc+ alone. 【Methods】 The OBI samples were screened by ELISA and NAT; the HBV DNA was amplified and sequenced; 20 anti-HBc+ alone and 25 not anti-HBc+ alone samples were obtained. 【Results】 The detection rate of OBI in Quzhou was 0.10%(155/161 045), and the positive rate of anti-HBc was 74.19%(115/155). The detection rate of OBI increased with the age of blood donors(P<0.05), but was not related to gender. The positive rate of anti-HBc+ alone was 22.58%(35/155), and that of not anti-HBc alone was 51.61%(80/155). Among the 45 OBI sequencing samples, the proportion of B and C genotype was 73.33%(33/45) and 20.00%(9/45), respectively. The mutation sites of blood donors in the anti-HBc+ alone group were more than those in the not anti-HBc+ alone group, and the mutation rates of S114T and V168A on MHR were significantly different(P<0.05). 【Conclusion】 The genotype of OBI infection in Quzhou is mainly type B. The mutation sites of blood donors with anti-HBc+ alone are higher than those with not anti-HBc+ alone, which may be more suitable as one of the OBI screening indicators.

[Resistance to deltamethrin and its association with mutation sites in the sodium iron channel domain III gene in Rhipicephalus microplus in Huaihua City of Hunan Province].

OBJECTIVE: To investigate the level of deltamethrin resistance and mutation sites in the sodium iron channel gene in Rhipicephalus microplus in Huaihua City, Hunan Province, and to examine the correlation between deltamethrin resistance and mutation sites in the sodium iron channel gene in Rh. microplus. METHODS: Rh. microplus was sampled from multiple yellow cattle farms in Huaihua City, Hunan Province from June to September 2022, and the level of resistance to deltamethrin was determined in ticks using the adult immersion test. The sodium iron channel domain III gene was amplified in deltamethrin-resistant and wild-type Rh. microplus using PCR assay. Following sequencing and sequence alignment, mutation sites were detected in bases. The sodium iron channel domain III gene in Rh. microplus was translated, and the signal peptide, transmembrane domain, and phosphorylation and glycosylation sites were detected in amino acid sequences. The tertiary structures of the sodium iron channel domain III protein of deltamethrin-resistant and wild-type Rh. microplus were deduced and compared, and the association be tween mutation sites in bases and resistance to deltamethrin was examined in Rh. microplus according the level of deltamethrin resistance, sequence alignment and protein tertiary structure. RESULTS: The median (LC50) and 95% lethal concentrations (LC95) of deltamethrin were 121.39 mg/L and 952.61 mg/L against Rh. microplus, with a resistance factor of 9.24 and level II resistance. The sequence of the sodium ion channel domain III gene was 1 010 bp in size, and mutation sites were detected in two neighboring bases in the sequence of the sodium ion channel domain III gene in deltamethrin-resistant Rh. microplus. Although no signal peptides were found in the sodium iron channel domain III protein of deltamethrin-resistant or wild-type Rh. microplus, 6 trans-membrane domains, 42 phosphorylation sites and 8 glycosylation sites were identified, with a significant difference in the tertiary structure of the sodium iron channel domain III protein between deltamethrin-resistant and wild-type Rh. microplus. CONCLUSIONS: Level II resistance to deltamethrin is detected in Rh. microplus in Huaihua City, Hunan Province, and two mutation sites that correlate with the emergence of deltamethrin resistance are identified in the sequence of the sodium iron channel domain III gene in deltamethrin-resistant Rh. microplus.

Analysis of S gene characteristic sequences and changes in properties of protein expression in HBV ASCs with low-level HBsAg.

Objective: We detected the serum HBsAg immune complex (HBsAg-CIC) and sequenced the HBV S gene in these patients to reveal the association between sustained low-level expression of HBsAg and mutated S gene sequence characteristics, protein function changes, and HBsAg immune complex formation. Methods: A total of 204 samples were collected and divided into high-level (n = 60, HBsAg level >10 IU/ml) and low-level (n = 144, HBsAg level ≤ 10 IU/ml) HBsAg groups. The clinical and epidemiological data of the two groups were statistically compared. According to different serological patterns and genotypes, the HBsAg-CIC results of the high-level and low-level HBsAg groups were divided into different subgroups, and then the HBsAg-CIC positive rates among different subgroups were compared. We sequenced the S gene of HBV from the two groups and identified the relevant mutations in the MHR of the S gene. In addition, we compared the changes in HBsAg protein properties and functions after hot spot mutation in the MHR of the S gene. Results: Comparing the positive rates of HBsAg-CIC under different serological patterns and genotypes in the two groups, the HBsAg-CIC positive rate was higher in the low-level HBsAg group. Moreover, there was weak correlation between HBsAg-CIC and HBsAg or HBV DNA in both groups (r = 0.32, 0.27, 0.41, 0.48; P < 0.05). Sequencing of S gene in the two groups, showed that the hot-spot mutations were T126A, M133L/T/S, and F134L/T/I in MHR of S gene of genotype B, and hot-spot mutations were Q101R and I126S/T in MHR of S gene of genotype C. Additionally, the positive rate of MHR mutation in the S gene from HBsAg-CIC positive patients was higher in the low-level HBsAg group. Conclusion: The host immune process of clearing HBV seems to have multiple site mutations in MHR, which changes the physicochemical properties and functions of HBsAg and intensifies the formation of HBsAg-CIC, thus avoiding the effective recognition of HBsAg by the host and resulting in immune tolerance between the host and HBV, which may be one of the formation mechanisms of sustained low-level expression of HBsAg in the serum of HBV-infected persons.

Pathogenicity analysis and a novel case report of intronic mutations in CSF1R gene.

Colony-stimulating factor 1 receptor-associated leukoencephalopathy (CSF1R-related leukoencephalopathy) is a genetic disorder mutated in a single allele. It is characterized by an adult-onset along with predominantly cognitive impairment, accompanied by neuropsychiatric symptoms as well as motor symptoms such as Parkinsonism. In the current study, we confirmed a case of CSF1R-related leukoencephalopathy pedigree by genetic screening, and a new intron c. 1858 + 5 G > A mutation was detected in affected patients. After reviewing all previous reports of introns, we found that symptoms and clinical manifestations of the patients were typical and met the features of previous intron reports.

Identify pathogenic mutations of <i>CYP4V2</i> gene in Bietti crystalline corneoretinal dystrophy / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To identify pathogenic mutations of <i>CYP4V2</i> gene in two Chinese families with Bietti crystalline corneoretinal dystrophy(BCD)by Sanger sequencing. <p>METHODS: The relevant clinical examination of BCD patients were collected. Peripheral blood of patients and their family members was collected. Then DNA was extracted from peripheral blood, and Sanger sequencing was used to identify mutation sites.<p>RESULTS: Two probands of BCD from different families were collected. All the probands showed progressive decrease of visual acuity and typical crystal-like material deposition could be seen in the fundus. Sanger sequencing showed that proband 1 and her brother and sister all had homozygous mutation of c.802-8_810del17insGC in <i>CYP4V2</i> gene. On the other hand, proband 2 had a compound heterozygous mutation of c.219T>A(p.F73L)and c.802-8_810del17insGC in <i>CYP4V2</i> gene. <p>CONCLUSION: The most common mutation was c.802-8_810del17insGC in Chinese BCD patients. The homozygous c.802-8_810del17insGC mutation was the cause of BCD in the proband 1 family. On the other hand, proband 2 had c.802-8_810del17insGC heterozygous mutation and c. 219T>A(p.F73L)heterozygous missence mutation, all of which affected the normal coding of <i>CYP4V2</i> gene and led to disease.

A new method towards the detection of DNA mutation by Surface-Enhanced Raman Spectroscopy.

It is generally believed that the self-folding of single-stranded DNA depends on the hydrophobic effect of its internal bases, but the folding of a single-stranded DNA in a solution was not disordered and would be affected by the stacking effect of adjacent bases. In this work, we developed a new method to explore the stacking between adjacent bases using Surface-Enhanced Raman Spectroscopy (SERS) for the first time. Acidic titanium ions were introduced into silver nanoparticles as an aggregating agent (Ag@ITNPs), and obtained a symmetrical spectrum by normalizing the peak to deoxyribose at 955 cm-1. Based on the influence of adjacent base stacking on the spectrum, we first identified the point mutation sites accurately by SERS. Also, the base content and the DNA frameshift mutations in ssDNA were precisely analyzed. This new method has a simple experimental process and can accurately capture the changes in the base ring breathing peak intensity caused by different adjacent bases, and thus will provide potential application value in the field of gene diagnosis.

Genetic Characterisation and Local Genotypes of Canine Parvovirus Strains Collected from Pet Dogs in Central and Eastern China During 2018-2019.

INTRODUCTION: Canine parvovirus type-2 (CPV-2) causes acute infectious diseases in puppies, which show high morbidity and mortality. Better effect of vaccination against these diseases could be achieved with deeper knowledge of CPV-2 genotype dissemination and mutation history. This study investigated CPV-2-positive samples collected recently over a wide region of China. MATERIAL AND METHODS: A total of 118 faecal samples from dogs identified as CPV-positive were collected from veterinary clinics in central and eastern China. Overall, 16 strains collected from Anhui, 29 from Henan, and 16 from Zhejiang Province were sequenced to determine the genotypic composition of CPV-2 and mutational complexity of CPV-VP2. RESULTS: The CPV-2a, CPV-2b, and CPV-2c genotypes were detected in Anhui and Henan Provinces, while CPV-2c alone was detected in Zhejiang Province. Sequence analysis of all strains showed 98.5%-99.8%, 98.3%-99.9%, and 98.7%-99.8% identity among the 16 Anhui, 29 Henan, and 16 Zhejiang strains, respectively. Strains collected from Anhui and Henan Provinces showed lower identity (97.0%), suggesting greater genetic divergence in central China. The mutation rates of Henan and Anhui strains were lower than that of Zhejiang strains. Major amino acid mutations occurred at sites 5, 370, 426, and 440. Epitope and entropy analyses implied these sites' likely conformance to the principles of mutation tendency, complexity, and diversity. CONCLUSION: The findings for the evolutionary structure of CPV-2 strains collected from three provinces in central and eastern China advance trend monitoring of the genetic variation in canine parvovirus and point to its implications in the development of novel vaccines.

Analysis of clinical characteristics and S gene sequences in chronic asymptomatic HBV carriers with low-level HBsAg.

BACKGROUND: During the natural hepatitis B virus (HBV) infection process, some infected subjects are characterized by a sustained low serum HBV surface antigen (HBsAg) expression level. Most members in this population are chronic asymptomatic HBV carriers (ASCs). To elucidate the mechanism underlying low-level HBsAg expression in ASCs, we sequenced the HBV S gene in these patients to reveal specific sequence characteristics. METHODS: Overall, 1308 cases of chronic ASCs were grouped according to their HBsAg serum expression levels (10 IU/mL). The clinical characteristics of the population were analysed in detail. The HBV S gene was sequenced from 276 ASC cases with low-level HBsAg expression. Additionally, 100 of 1032 ASC cases with high-level HBsAg expression were randomly selected for HBV S gene sequencing based on age matching according to the low-level HBsAg group. A comparative analysis was conducted with the HBV S gene sequences from ASCs with low HBsAg expression and the HBV reference S gene sequences from ASCs with high HBsAg expression. RESULTS: The population with low-level HBsAg expression displayed the following primary clinical characteristics: mostly chronic asymptomatic HBV carriers, older age (mean age 55.09 years), HBsAg/anti-HBe/anti-HBc (core) positivity as the main serological pattern (97.1%), low HBV DNA replication (1.32 ± 1.60 log10 IU/mL), a low HBV-DNA positive rate (45.65%) and primarily genotype B (82.54%) and serotype adw (84.13%). The comparative analysis of the HBV S gene sequences from ASCs with low-level HBsAg showed significant mutations (including co-mutations) on both sides of the main hydrophilic region (MHR). CONCLUSION: Significant mutations in multiple regions and at multiple sites (including co-mutations) on both sides of the MHR may be one cause of the low HBsAg expression level in this population.

Sequence analysis of persistently low level expression of hepatitis B surface antigen S gene in patients with hepatitis B virus infection / 中华传染病杂志

Objective To reveal the characteristics of S gene sequence of hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-infected patients with low HBsAg level.Methods From February 2016 to December 2017, 1 308 serum samples of inactive HBsAg carriers were collected from the 903rd Hospital of PLA and Hangzhou Jianggan District People′s Hospital.The cases were divided into high-level group and low-level group according to the level of serum HBsAg (10 IU／mL) expression.The HBV S gene was sequenced in patients with low-level HBsAg expression.In addition, in patients with high-level HBsAg, 100 patients were randomly selected (stratified sampling) for HBV S gene sequencing based on the matching of age and serological pattern (hepatitis B e antigen [HBeAg] negative) of low-level HBsAg group.A comparative analysis was conducted between HBV S gene sequences from inactive HBsAg carrier in low HBsAg expression group and the HBV reference S gene sequences from inactive HBsAg carrier in high HBsAg expression group .The results of normal distribution data were expressed as Mean ±SD, and analyzed using t-test.The results of non-normal distribution data were expressed by M(QR), and analyzed using Mann-Whitney U test.Chi-square test or Fisher exact test was used to compare continuous variables and classification variables between the two groups .Results There were 276 serum samples from the low level group and 1 032 serum samples from the high level group , including 257 HBsAg／HBeAg／anti-HBc-positive cases, 753 HBsAg／anti-HBe／anti-HBc-positive cases, and 22 HBsAg／anti-HBc-positive cases.Successful HBV S gene sequencing was performed on 126 out of 276 patients in the low-level HBsAg group.According to the age inthe low-level HBsAg group, 100 samples with negative HBeAg in the high-level HBsAg group were randomly selected , among which 94 patients were genotyped and hemotyped.The results showed that there were statistically significant differences in HBV serological markers , HBV DNA level and HBV genotype distribution between the high level group (94 cases) and the low level group (126 cases) (all P＜0.05).The ASC-R-B and ASC-R-C genotypes reported in this study had high homology (99.6%-100.0%) with those reported in Shanghai , Chengdu, Wuhan, Yunnan and Beijing of China , and high homology (98.2%-99.6%) with those reported in Japan and Korea of NCBI genotype B and C reference sequences, but had low homology with patients far away from China (98.2% in Canada and 98.7% in Indonesia).In genotype B of the low level group , the amino acid mutation number of SHB protein was 71, and the hot spot mutation number was 19, both higher than those in the high level group (39 and 8, respectively). The difference was statistically significant (χ2 ＝12.303 and 4.766, respectively, both P＜0.05).Amino acid mutation sites in the low HBsAg group were mainly distributed on both sides of the major hydrophilic region (MHR) (amino acid residues 40 -49 and 198 -220).There were no significant differences in amino acid mutation number and hot spot mutation number between the two groups of C genotype (χ2 ＝0.383 and 0.409, respectively, both P＞0.05).For genotype B, 12 single point mutations and 4 dual co-mutations were found in low level group.Among them, one single point mutation (S210R) and 3 dual co-mutations (G44E／V+T45P／I, G44E／V+L49P／R and N40S+I208T) were not hot spot mutations , while 2 dual co-mutations and 2 single point mutations were found in high level group.The difference between two groups was statistical significant (χ2 ＝7.533,P ＝0.006).For genotype C, 5 single point mutations ( T5A, A45T, T47A／K, Q101R and I126S／T) were found in low level group and 1 single point mutation (N3S) in high level group.The difference in mutation frequency between two groups were statistical significant (χ2 ＝47.914,P＝0.000).Conclusions Significant mutations in multiple regions and at multiple sites ( including co-mutations) on both sides of the MHR may be one of the causes of low HBsAg expression level in this population .

Analysis of gene mutation sites in patients with failed anti-HIV-1 treatment in Lincang City from 2011 to 2018 / 中华疾病控制杂志

Objective To understand the major genotype-resistant mutation sites and change trends of HIV/AIDS patients with failure of antiviral therapy (ART) in Lincang City, Yunnan Province. Methods The In-House method was used to amplify the Pol gene region in the plasma samples of HIV/AIDS patients with failure of ART in Lincang City from 2011 to 2018. The target sequence was spliced and submitted to the HIV resistance database to identify and analyze the HIV-1 subtypes and resistant mutation sites. Results The 950 strains of HIV/AIDS patients with antiviral failure were mainly CRF08_BC, accounting for 75.5% (717/950), and the total gene mutation rate was 67.1% (637/950), which was dominated by non-nucleoside reverse transcriptase inhibitors (NNRTIs), accounting for 62.4% (593/950); followed by nucleoside reverse transcriptase inhibitors (NRTIs), accounting for 34.7% (330/950); protease inhibitors (PIs) was 7.5% (71/950). A total of 15 NRTIs of resistance-related mutation sites were detected, mainly M184V (29.3%) which was detected mostly in AZT/D4T+3TC+NVP programs; including 17 kinds of NNRTIs, mainly was K103N/S (25.1%)，the most detected in AZT/TDF+3TC+EFV programs. There were 22 kinds of PIs，mainly secondary sites were L10F/V/I (2.2%) and L33F (2.1%). The top three NRTIs mutation sites in the area were changed from T69D/N/G，M184I/V and D67N/G/S to M184I/V, K70R/Q/E/T and T215Y/F/V/I/N/A/D. NNRTIs mutation sites were changed from V179D/T/E/F, E138A/K/G/R and Y181C/F/G/V to K103N/S, E138A/K/G/R and V179D/T/E/F. The mutation sites of the first three PIs did not change much. Conclusions The second-line regimen based on PIs is a better choice in free antiviral treatments. Mastering the drug resistance of different gene mutations is beneficial to the compatibility of first-line drugs, thus delaying the use of second-line drugs.

Genomic changes in an attenuated genotype I Japanese encephalitis virus and comparison with virulent parental strain.

Genotype I Japanese encephalitis virus (JEV) strain SCYA201201 was previously isolated from brain tissues of aborted piglets. In this study, we obtained an attenuated SCYA201201-0901 strain by serial passage of strain SCYA201201-1 in Syrian baby hamster kidney cells, combined with multiple plaque purifications and selection for virulence in mice. We investigated the genetic changes associated with attenuation by comparing the entire genomes of SCYA201201-0901 and SCYA201201-1. Sequence comparisons identified 14 common amino acid substitutions in the coding region, with two nucleotide point mutations in the 5'-untranslated region (UTR) and another three in the 3'-UTR, which differed between the attenuated and virulent strains. In addition, a total of 13 silent nucleotide mutations were found after attenuation. These substitutions, alone or in combination, may be responsible for the attenuated phenotype of the SCYA201201-0901 strain in mice. This information will contribute to our understanding of attenuation and of the molecular basis of virulence in genotype I strains such as SCYA201201-0901, as well as aiding the development of safer JEV vaccines.

Evolution of Vertebrate Ryanodine Receptors Family in Relation to Functional Divergence and Conservation.

Ryanodine receptors (RyRs), the large homotetrameric protein complexes, regulate the release of calcium from intracellular stores into the cytosol and play vital roles in the excitation-contraction coupling of cells. However, the evolutionary relationship of RyRs in vertebrates has yet to be elucidated. We identified 22 RyRs from Homo sapiens, Mus musculus, Rattus norvegicus, Gallus gallus, Anolis carolinensis, Rana catesbeiana, and Danio rerio. The phylogenetic relationship, motifs analysis and reconstruction of ancestral RyRs showed that the members of RyR family in vertebrates were grouped into three clades: the RyR1 clade, the RyR2 clade, and the RyR3 clade. Positive selection existed in RyR gene evolution, which is consistent in three site models, and gene ontology (GO) analysis showed that the evolution of RyR family in vertebrates promotes RyRs function differentiation. At last, we predicted 140 mutation sites which may be involved in diseases and 57 phosphorylation sites among RyR1 sequence in human, as well as 61 mutation sites and 70 phosphorylation sites in human RyR2 sequences. Most of these potential sites are arranged in clusters. Our work provides insight into the origin and evolutionary process of RyRs in vertebrates, facilitating their functional investigations in the future.

Tryptophan and aspartic acid residues present in the glycerophosphoryl diester phosphodiesterase (GDPD) domain of the Loxosceles laeta phospholipase D are essential for substrate recognition.

It is known that the family of phospholipases D (PLD) from spiders of the genus Loxosceles, hydrolyze the substrates sphingomyelin and lisophosphatidylcholine, by their catalytic acid-base action which involves two histidines. However, little is known about the amino acids that participate on substrate recognition. In this study we identified highly conserved amino acids of the glycerophosphoryl diester phosphodiesterase (GDPD) domain of recombinant LlPLD1, which interact with the substrate sphingomyelin. The mutation of W256 to serine and D259 to glycine decreased significantly the sphingomyelinase and hemolytic activity when compared to wild type LlPLD1. The interaction of LlPLD1 with sphingomyelin was also strongly reduced in both mutants LlPLD1-W256S and LlPLD1-D259G. The results show the importance of these residues in the interaction of the protein with its substrate sphingomyelin in cell membranes.

Quick Detecting Mutation Site of 315 Codon in katG Gene of INH resistant MTB by Stem-ring Molecular Probe in Liquid / 中华医院感染学杂志

OBJECTIVE To detect 315 codon of mutation site in katG of isoniazid(INH)-resistant Mycobacterium tyberculosis(MTB) by stem-ring molecular probe quickly and detect out the fluorescence sign of hybridization between amplified products of katG 315 codon and probe in liquid by fluorescence spectrophotometer.The results were confirmed by sequencing.METHODS The software,Beacon designer,was used to design the katG 315 codon stem-ring molecular probe and the amplification system,and the relationship between the way and sequencing of the amplification products were compared.RESULTS The difference between PCR products from standard strain and INH-resistant one was significant in detecting the fluorescent light by use of fluorescence spectrophotometer.We detected fluorescent light signal between the 16 INH resistant strains and 10 H37RV standard strains.The resistant rate to INH detected was about 44%,and the rate of coincidence was about 97.5%.CONCLUSIONS The stem-ring molecular probe technology show high sensitivety in detecting mutation site of nucleic acid.The rate of coincidence is good between fluorescence spectrophotometer way and sequencing.

Detecting Micro-luminescence of 330 Codon in emb gene of Mycobacterium tuberculosis by Molecular Beacon Hybridization / 中华医院感染学杂志

OBJECTIVE To design molecular beacon detecting embB330 codon of ethambutol-resistant Mycobacterium tuberculosis(MTB),meanwhile,and try to detect fluorescence of mutation site of embB330 codon in liquid by fluorescence microscope by compareing the mutation strains and standard strains.METHODS The software,Beacon designer,was used to design molecular beacon detecting embB330codon and detecting fluorescence signal from hybridization between the amplified product and probe by fluorescence microscope,and to confer to the sequencing results.RESULTS The difference between PCR products from standard strain and ethambutol-resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope.We detected fluorescent light signal between the 33 ethambutol-resistant strains and 10 H37RV standard strains.The rate of ethambutol-resistant strains was about 3%,and the rate of sequencing was about 3%.CONCLUSIONS The technology of molecular beacon effectually can detect mutation single base site of embB330codon.Fluorescence microscope owns characteristics such as high sensitiveness to detect the fluorescent light.