RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/µL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance. IMPORTANCE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats.

BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.

Antibody Response to SARS-CoV-2 in the First Batch of COVID-19 Patients in China by a Self-Developed Rapid IgM-IgG Test.

It has been over two years since the COVID-19 pandemic began and it is still an unprecedented global challenge. Here, we aim to characterize the antibody profile from a large batch of early COVID-19 cases in China, from January - March 2020. More than 1,000 serum samples from participants in Hubei and Zhejiang province were collected. A series of serum samples were also collected along the disease course from 70 patients in Shanghai and Chongqing for longitudinal analysis. The serologic assay (ALLtest) we developed was confirmed to have high sensitivity (92.58% - 97.55%) and high specificity (92.14% - 96.28%) for the detection of SARS-CoV-2 nucleocapsid-specific antibodies. Confirmed cases found in the Hubei Provincial Center for Disease Control and Prevention (HBCDC), showed a significantly (p = 0.0018) higher positive rate from the ALLtest than RNA test. Then, we further identified the disease course, age, sex, and symptoms that were correlating factors with our ALLtest results. In summary, we confirmed the high reliability of our ALLtest and its important role in COVID-19 diagnosis. The correlating factors we identified will require special attention during future clinical application.

Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

The coronavirus disease 2019 (COVID-19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen.

A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.

TITLE: Un nouveau dispositif de bandelette à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) pour la détection rapide de Toxoplasma gondii dans le sang des chats et chiens errants. ABSTRACT: Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire qui provoque la toxoplasmose et menace la santé humaine et les animaux à sang chaud dans le monde entier. Des méthodes de diagnostic simples et applicables sont nécessaires de toute urgence pour guider le développement d'approches efficaces pour la prévention de la toxoplasmose. La plupart des outils de diagnostic moléculaire pour l'infection par T. gondii nécessitent des compétences techniques élevées, un équipement sophistiqué et un environnement de laboratoire contrôlé. Dans cette étude, nous avons développé un test par bandelettes à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) qui cible spécifiquement les 529 pb qui détectent une infection par T. gondii. Ce nouvel appareil portable est universel, rapide, convivial et garantit une sensibilité expérimentale ainsi qu'un faible risque de contamination par aérosol. Notre test LAMP-LFD a une limite de détection de 1 fg d'ADN de T. gondii et ne montre aucune réaction croisée avec d'autres pathogènes parasites, y compris Cryptosporidium parvum, Leishmania donovani et Plasmodium vivax. Nous avons validé le test en détectant T. gondii dans l'ADN extrait d'échantillons de sang prélevés sur 318 chats et chiens errants prélevés dans les villes de Deqing, Wenzhou, Yiwu, Lishui et Zhoushan dans la province du Zhejiang, dans l'est de la Chine. Le dispositif LAMP-LFD a détecté la prévalence de l'ADN de T. gondii chez respectivement 4,76 et 4,69% des chats et chiens errants. En conclusion, le test LAMP-LFD développé est efficace, minimise la contamination par les aérosols et convient donc à la détection de T. gondii dans les établissements médicaux simples et sur le terrain.

Predictive Analysis of the Neutralization Activity in Convalescent Plasmas From COVID-19 Recovered Patients in Zhejiang Province, China, January-March, 2020.

Background: Convalescent plasma (CP) transfusion is considered to be the priority therapeutic option for COVID-19 inpatients when no specific drugs are available for emerging infections. An alternative, simple, and sensitive method is urgently needed for clinical use to detect neutralization activity of the CP to avoid the use of inconvenient micro-neutralization assay. Method: This study aims to explore optimal index in predicting the COVID-19 CP neutralization activity (neutralizing antibody titers, NAb titers) in an indirect ELISA format. Fifty-seven COVID-19-recovered patients plasma samples were subjected to anti-SARS-CoV-2 RBD, S1, and N protein IgG antibody by indirect ELISA. Results: ELISA-RBD exhibited high specificity (96.2%) and ELISA-N had high sensitivity (100%); while ELISA-S1 had low sensitivity (86.0%) and specificity (73.1%). Furthermore, ELISA-RBD IgG titers and pseudovirus-based NAb titers correlated significantly, with R2 of 0.2564 (P < 0.0001). Conclusion: ELISA-RBD could be a substitute for the neutralization assay in resource-limited situations to screen potential plasma donors for further plasma infusion therapy.

Seroprevalence of transfusion-transmissible infectious agents among volunteer blood donors between 2006 and 2012 in Zhejiang, China.

BACKGROUND: The high prevalence of hepatitis B and C viruses (HBV and HCV), paralleling the growing epidemic of human immunodeficiency virus (HIV) and Treponema pallidum (TP) infections in the general population, poses a great threat to blood safety in China. This study investigated the prevalence of serological markers for causative agents of transfusion-transmissible infections (TTI), i.e. HBV, HCV, HIV and TP, among volunteer blood donors in five cities/regions of Zhejiang Province, China. MATERIAL AND METHODS: We investigated whole blood and apheresis donations collected at the Blood Services in five cities/regions in Zhejiang Province between January 1, 2006 and December 31, 2012. Two rounds of serological testing were performed for HBsAg, anti-HCV, anti-HIV1/2 and anti-TP using different kits. The rates of serological positivity were calculated and further analysis was performed to examine the association between donors' characteristics and seroprevalence. RESULTS: Of the 1,615,120 donations, approximately 40% came from first-time donors and 60% from repeat donors. The overall seroprevalence rates of HBV, HCV, HIV and TP were 0.51%, 0.25%, 0.15% and 0.52%, respectively. The overall prevalences of HCV and HIV remained relatively steady, whereas the prevalence of TP increased sharply after 2010. However, the prevalence of TTI agents varied among volunteer blood donors in different cities/regions and demographic groups. DISCUSSION: We collected data on the seroprevalence of TTI agents among volunteer blood donors. Although the risk of TTI is low in China compared to that in some developing countries, sensitive screening methods and recruitment of regular donors are still very important for blood safety and availability.

Comparison of the KIR3DS1/Bw4 distribution in Chinese healthy and acute myeloid leukemia individuals.

Killer cell immunoglobulin like receptor (KIR) 3DS1 is one of the most important activating receptors and some studies revealed that KIR3DS1 combined with HLA ligand was not related to acute myeloid leukemia (AML), but rare data was reported in Chinese population. In this study, KIR3DS1 gene polymorphisms and HLA-Bw4 were investigated in 189 Chinese AML patients compared with 166 healthy individuals. The results showed that the distributions of KIR3DS1, Bw4, 3DS1/Bw4 and 3DS1/Bw4-80I were insignificantly different between AML and healthy individuals. This study suggests that the presence of 3DS1 and HLA-Bw4 ligands have no effect on AML disease.

Molecular basis and zygosity determination of D variants including identification of four novel alleles in Chinese individuals.

BACKGROUND: The frequency and molecular basis of the D variants have been reported in the Caucasian and African populations, but relatively little information was known in the Chinese population. Here, a study was investigated in Chinese persons with weak or discrepant D serologic typing. STUDY DESIGN AND METHODS: D variant was typed with a serologic method. The full coding regions of RHD of these variants were amplified with polymerase chain reaction and then directly sequenced. RHD zygosity test was performed using the hybrid Rhesus box technique and a multiplex ligation-dependent probe amplification (MLPA) assay was also used to analyze the variant alleles and RHD gene copy number. RESULTS: Twelve distinct RHD mutation alleles were found in 32 D variant individuals, with eight weak D and four partial D alleles. Weak D Type 15 and DVI Type 3 were the major weak D and partial D alleles in Zhejiang Han persons. Three novel weak D alleles (RHD weak D 95A, 779G, and 670G) and one new partial D allele (RHD130-132 del TCT) were identified. The results of RHD zygosity in three individuals disagreed between the RHD zygosity test and the MLPA assay. The most known variant alleles can be detected, but four novel alleles were missed using the RH-MLPA assay. CONCLUSION: The molecular basis and zygosity of D variants in Zhejiang Han persons were analyzed, and four novel RHD alleles were identified. These data extend the information of D variants and may help to improve the transfusion strategy of the D variants.

Variants of CD36 gene and their association with CD36 protein expression in platelets.

BACKGROUND: The relationship between CD36 expression level in platelets and polymorphism of the CD36 gene still needs to be explored. Here, we investigated polymorphisms of the CD36 gene and CD36 expression level in platelets in the Chinese Han population. MATERIALS AND METHODS: A total of 477 samples were sequenced for exons 2 to 14 of the CD36 gene using a polymerase chain reaction sequence-based typing method. In 192 of these individuals the expression levels of CD36 antigen were analysed by flow cytometry. The genotype-phenotype relationship in platelets was analysed. RESULTS: A total of 22 variants of the CD36 gene were identified, of which five variants (111 A>T, 681 C>A, 1172-1183 del12b, 1236 delT and 1395 A>C) were novel variations, and nine were also found in single nucleotide polymorphism database (dbSNP) but had not been confirmed in individuals with CD36 deficiency. Two variants (329-332 delAC and 1228-1239 del12bp) in the coding region are the most frequent mutations in the Chinese population. Type II CD36 deficiency was identified in seven of 192 individuals, giving a frequency of 3.6%. Individuals with CD36 variations or wild-type genotypes both showed CD36 antigen negative, low-level and high-level expression patterns in platelets. The frequency of the nt-132 A>C polymorphism in the 5'-UTR is relatively high in the Chinese population (0.3516): the expression of CD36 was lower in individuals with nt-132 A>C than in those with the wild-type genotype. DISCUSSION: The distribution of CD36 gene variants in the Chinese population is different from that previously reported. The levels of expression of CD36 antigen in platelets are not determined directly by the genotypes of the CD36 coding region. This suggests that the molecular basis of type II CD36 deficiency may be derived from combined effects of coding region and potential cis-regulatory elements in the 5'-UTR of the CD36 gene.

Genotyping of human neutrophil antigens by polymerase chain reaction sequence-based typing.

BACKGROUND: Genotyping for human neutrophil antigen (HNA) systems is required in the investigation of disorders involving alloimmunisation to HNA. We established a polymerase chain reaction sequence-based typing method for genotyping HNA and determined the genotype and allele frequencies of HNA in the Zhejiang Han population of China. MATERIALS AND METHODS: Four hundred, healthy unrelated Zhejiang Han individuals were recruited. Specific primers for HNA were designed and the polymerase chain reaction amplification conditions were optimised. Amplification amplicons were purified with enzyme digestion and then sequenced. RESULTS: The frequencies of the FCGR3B*01 and FCGR3B*02 alleles were 0.613 and 0.387; no FCGR3B*03 allele was found. The frequencies of the SLC44A2*1 and SLC44A2*2 alleles were 0.654 and 0.346, respectively, while the frequencies of the ITGAL*1 (HNA-5a) and ITGAL*2 (HNA-5b) alleles were 0.896 and 0.104. Only ITGAM*1 (HNA-4a) allele was found in this study. Six single nucleotide polymorphisms were confirmed on sequenced regions separate from HNA polymorphisms, including FCGR3B (IVS3+39G>A and IVS3+52G>A), CD177(172A>G), SLC44A2 (IVS5-44A>G and IVS7-15T>C) and ITGAM (IVS3+118T>C). DISCUSSION: The polymerase chain reaction sequence-based typing method for genotyping HNA is reliable. These data of HNA alleles frequencies could contribute to the analysis of alloimmunisation to HNA in China.

Allelic polymorphism, mRNA and antigen expression of KIR2DL1 in the Chinese Han population.

KIR2DL1 is one important molecule of inhibitory receptors that recognizes a subset of HLA-C allelic products carrying Lys80. In this study, we have investigated the allelic polymorphism, mRNA and antigen expression level of KIR2DL1 in the Chinese Han population. KIR2DL1(∗)001,(∗)00302 and (∗)00401 alleles and seven genotypes including two copy 2DL1(∗)00302, one copy 2DL1(∗)00302, two copy 2DL1(∗)001, one copy 2DL1(∗)001, 2DL1(∗)00302/2DL1(∗)001, 2DL1(∗)001/2DL1(∗)00401, 2DL1(∗)00302/2DL1(∗)00401 were identified in the total 164 Chinese Han individuals. The frequency of NK cells expression KIR2DL1 was varied considerably. There was no disparity on the level of antibody-binding for different genotypes according to mean fluorescent intensity and there was almost similar frequency of antibody-binding NK cells except for group KIR2DL1(∗)00302 with one copy. The frequency of NK cells expression KIR2DL1 of the individuals in the group 2DL1(∗)00302 with one copy was lower than that in the group 2DL1(∗)00302 with two copies. The amount of transcript was variable among the individuals and the mean value of transcript abundance in 21 individuals with one copy was lower than that in 143 individuals with two copies. In conclusion, the frequency of NK cells expression KIR2DL1 and the mRNA transcript abundance were not associated with allelic polymorphism but with gene copy number.

A pilot study on screening blood donors with individual-donation nucleic acid testing in China.

BACKGROUND: Nucleic acid amplification testing (NAT) is not yet obligatory in China for blood donor screening and the risk of enzyme immunoassay (EIA)-negative, NAT-reactive donations in Chinese blood donors has rarely been reported. The aim of this study was to screen a population of Chinese blood donors using a triplex individual-donation (ID)-NAT assay and assess the safety benefits of implementing NAT. MATERIALS AND METHODS: Between 1st August, 2010 and 31st December, 2011 all donations at a Chinese blood centre were screened individually using the Procleix® Ultrio® assay, a multiplex NAT assay for the detection of hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA and human immunodeficiency virus-1 (HIV-1) RNA. All donations were also screened for HBsAg, anti-HIV and anti-HCV using two different EIA for each marker. Samples with discordant results between NAT and EIA were further tested with an alternative NAT assay (Cobas® TaqMan®). Potential yield cases (serologically negative/NAT-reactive donors) were further evaluated when possible. RESULTS: During the study period a total of 178,447 donations were screened by NAT and EIA, among which 169 HBV NAT yield cases (0.095%) were detected. No N AT yield cases were found for HIV-1 or HCV. For the HBV NAT yield cases, follow-up results showed that 11 (6.51%) were probable or confirmed HBV window period infections, 5 (2.96%) were chronic HBV carriers and 153 (90.53%) were probable or confirmed occult HBV infections. There was a statistically significant difference between the NAT-positive rates for first-time vs repeat donations (0.472% vs 0.146%, respectively; P<0.001). DISCUSSION: Our data demonstrate that the potential HBV yield rate was 1:1,056 for blood donations in the Zhejiang province of China. Implementation of NAT will provide a significant increment in safety relative to serological screening alone.

The relationship of platelet yield, donor's characteristic and apheresis instruments in China.

Platelet yield was associated with donor's characteristic and property of apheresis instruments. Here, we have analyzed the relationship of platelet yield, physiologic parameters of donors for different apheresis instruments in China. Data were consecutively retrieved from plateletapheresis donors during March 1, 2007 and March 1, 2012. Three different apheresis instruments MCS+, Amicus, Trima system were used for plateletapheresis and defined as group 1, 2 and 3 respectively. Totally 77,091 Plateletapheresis donations were performed in this study. 17 donations were finally aborted because of vasovagal reaction with syncope. 5861, 37,036, 34,177 donations were performed in group 1, 2 and 3 respectively. Hct and platelet values before donations were similar, but platelet yield and collection rate were showed significantly difference (p<0.05) among the three groups. The values of platelet and Hct in the males before donations were higher than those in the females, and the platelet yield and collection rate were showed significantly difference between the male group and female group (p<0.05). The overall reaction rate was 1.56%. Most donors were chosen the group 2 (51.6%) for next donation, followed by group 3 (33%) and group 1 (15.4%). We concluded that the platelet yield and collection rate in the male group were higher than those in the female group and the efficiency of plateletapheresis was associated with the kind of apheresis instruments and donor's characteristic. These data will help to work out suitable apheresis protocol based on the Chinese donor's characteristic.

[A rare p phenotype caused by a 26-bp deletion in α 1,4-galactosyltransferase gene].

OBJECTIVE: To delineate serological features and genetic basis for a rare p phenotype of P1Pk blood group system found in a Chinese individual. METHODS: Serological assaying was carried out for a proband with unexpected antibody found in his serum using specific antibodies and panel cells. Coding regions and flanking introns of α 1,4-galactosyltransferase gene (A4GALT) associated with the p phenotype were screened with polymerase chain reaction and DNA sequencing. RESULTS: A rare p phenotype of the P1Pk blood group system has been identified with red blood cells from the proband, whose serum contained anti-Tja antibody which can agglutinate and hemolyze with other common red blood cells. Other members of the proband's family were all normal with P1 or P2 phenotype. DNA sequencing has identified in the proband a homozygous 26 bp deletion at position 972 to 997 of the A4GALT gene. The deletion has caused a shift of the reading frame, resulting in a variant polypeptide chain with additional 83 amino acid residues compared with the wild-type protein. Other family members were either heterozygous for above deletion or non-deleted. CONCLUSION: A 26 bp deletion at position 972 to 997 of the A4GALT gene has been identified in a Chinese individual with p phenotype.

Distribution of MICB diversity in the Zhejiang Han population: PCR sequence-based typing for exons 2-6 and identification of five novel MICB alleles.

The polymorphism of major histocompatibility complex class I chain-related gene B (MICB) and variations in MICB alleles in a variety of populations have been characterized using several genotyping approaches. In the present study, a novel polymerase chain reaction sequence-based typing (PCR-SBT) method was established for the genotyping of MICB exons 2-6, and the allelic frequency of MICB in the Zhejiang Han population was investigated. Among 400 unrelated healthy Han individuals from Zhejiang Province, China, a total of 20 MICB alleles were identified, of which MICB*005:02:01, MICB*002:01:01, and MICB*004:01:01 were the most predominant alleles, with frequencies of 0.57375, 0.1225, and 0.08375, respectively. Nine MICB alleles were detected on only one occasion, giving a frequency of 0.00125. Of the 118 distinct MICB ∼ HLA-B haplotypes identified, 42 showed significant linkage disequilibrium (P < 0.05). Haplotypes MICB*005:02:01 ∼ B*46:01, MICB*005:02:01 ∼ B*40:01, and MICB*008 ∼ B*58:01 were the most common haplotypes, with frequencies of 0.0978, 0.0761, and 0.0616, respectively. Five novel alleles, MICB*005:07, MICB*005:08, MICB*027, MICB*028, and MICB*029 were identified. Compared with the MICB*005:02:01 sequence, a G > A substitution was observed at nucleotide position 210 in MICB*005:07, and a 1,134 T > C substitution in MICB*005:08 and an 862 G > A substitution in MICB*027 were detected. In addition, it appears that MICB*028 probably arose from MICB*004:01:01 with an A to G substitution at position 1,147 in exon 6. MICB*029 had a G > T transversion at nucleotide position 730 in exon 4, compared with that of MICB*002:01:01. On the basis of the new PCR-SBT assay, these observed results demonstrated MICB allelic variations in the Zhejiang Han population.

Bridging HIV-1 cellular latency and clinical long-term non-progressor: an interactomic view.

Development of an effective HIV management is enticed by the fact that long-term non-progressors (LTNP) restrict viral replication spontaneously, but is hindered by HIV-1 latency. Given that the most overlapping characteristics found between HIV-1 LTNP and latency, detailed analysis of the difference would disclose the essentials of latency. In this study, microarray data from our previous study was combined with HIV-1 latency and LTNP data obtained from NCBI GEO database. Principal variance component analysis and hierarchical clustering verified the removal of batch effect across platform. The analysis revealed a total of 456 differential expressed genes with >2-fold change and B-statistic >0. Bayesian inference was used to reconstitute the transcriptional network of HIV-1 latency or LTNP, respectively. Gene regulation was reprogrammed under different disease condition. By network interference, KPNA2 and ATP5G3 were identified as the hubs in latency network which mediate nuclear export and RNA processing. These data offer comparative insights into HIV-1 latency, which will facilitate the understanding of the genetic basis of HIV-1 latency in vivo and serve as a clue for future treatment dealing with key targets in HIV-1 latency.

Serological characteristic and molecular basis of A2 subgroup in the Chinese population.

BACKGROUND: A2 phenotype is a common subgroup of blood group A, but the serological characteristic and genetics basis of A2 phenotype currently was rare reported in the Chinese Han population. Here, a large scale study of the serology and genetics of A2 and A2B phenotypes was performed. METHODS/MATERIALS: 11263 Chinese individuals with group A and AB phenotypes were determined for A2 antigen with the standard serological method. The full coding region of the ABO gene was sequenced in the individuals with A2 and A2B phenotypes. Some samples including each ABO genotypes were chosen for determining the activity of glycosyltransferase A (GTA) in plasma. RESULTS: 134 individuals were assigned as A2 and A2B phenotypes in 11263 individuals. There was imbalance in A2 and A2B phenotypes and the proportion of A2B among AB samples was significantly higher than that of A2 in group A samples. All samples of the A2 and A2B phenotypes were classified into A2-related allele group, A1-related allele group and the other group based on kind of the ABO genotype. Four novel A2-related alleles (A217, A218, A219, A220) were identified. The individuals with same genotype showed different agglutination strength with anti-A1 and anti-H on their RBCs. The plasma from individuals with A2-related allele had almost no GTA activity, while plasma from individuals with A1-related allele had some GTA activity. CONCLUSION: A2 and A2B phenotypes could derive from different genotypes and the serological characteristic may be heterogeneity in the Chinese Han population.

A dispermic chimera was identified in a healthy man with mixed field agglutination reaction in ABO blood grouping and mosaic 46, XY/46, XX karyotype.

BACKGROUND: Chimerism is the presence of two or more genetically distinct cell populations in one organism. Here, we reported the identification of dispermic chimerism in a 25-year-old male. METHODS: Blood grouping was performed with standard gel centrifugation test cards. ABO and HLA-A,-B,-C,-DRB1 and -DQB1 loci genotyping was determined with PCR sequence-based typing. A quantitative analysis of dual red cells populations was measured by flow cytometer. The karyotype was analyzed by G-banded chromosomes. Short tandem repeat (STR) analysis was performed on blood, buccal mucosal and hair shafts samples. RESULTS: A mixed-field agglutination with anti-B antibody was observed with gel centrifugation tests, which showed a double populations of O and B groups RBCs. Two groups RBCs were also observed by flow cytometer with nearly 90% O group cells and 10% B group cells. The normal O01,O02,B101 alleles were identified in DNA sample of the proband. STR analysis revealed three alleles for D8S1179,D3S1358,TH01,D13S317,D16S539,D2S1338,D19S433,TPOX and D18S51 loci. HLA-DRB1 and -DQB1 loci had three alleles and a karyotypic mosaic was found with 60% 46, XY and 40% 46, XX karyotype in the proband. In all studies, the third allele was attributable to a dual paternal contribution. CONCLUSION: A individual with dispermic chimerism was identified, which would generate by fertilization of an oocyte and the corresponding second polar body by two different sperms.

[Study on a novel mutation of B glycosyltransferase gene related with an ABx variant].

OBJECTIVE: To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system. METHODS: Serological assays were used to characterize the erythrocyte phenotypes and salivary ABH secretors. All of the seven exons and flanking introns of ABO glycosyltransferase gene were amplified with polymerase chain reaction (PCR). And the products were sequenced bidirectionally following enzyme digestion. Exons 6 and 7 were also subcloned and analyzed for haplotypes of the ABO gene. RESULTS: Erythrocytes of the proband have expressed a strong A antigen and a weak B antigen, which was identified as a rare ABx variant in addition with other serological features. Nine heterozygous sites in exon 6 (297A/G) and exon 7 (467C/T, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 808T/A, 930G/A) of the coding region of the ABO gene were identified. Based on haplotype analysis, one allele was determined as common A102, whilst another was consistent with B101 except for an 808T>A mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B. CONCLUSION: The 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.