Neutralizing antibodies against adenovirus type 2 in normal and HIV-1-infected subjects: Implications for use of Ad2 vectors in vaccines.

Pre-existing neutralizing antibodies (NAbs) directed against vaccine vectors have attracted considerable research attention. Therefore, our aim was to establish a high-throughput economical neutralization assay to investigate the epidemiology of adenovirus type 2 (Ad2)-specific immunity in China and developed countries, including in a Chinese Human immunodeficiency virus (HIV)-1-infected population, and to guide the application of Ad2-vectored vaccines. We established a FluoroSpot-based anti-Ad2-virus neutralization assay using a recombinant replication-deficient Ad2 that expresses enhanced green fluorescent protein and standardized the critical parameters, including the choice of cell line, cell concentration, viral infective dose, and incubation time. The sera of 561 healthy individuals from China and developed countries and from 230 HIV-1-infected Chinese individuals were screened with this assay for Nabs against Ad2. The prevalence of anti-Ad2 NAbs was high in both China (92.2%) and developed countries (86.9%). Of the Ad2-seropositive individuals, 64.6% in China and 77.4% in developed countries had high NAb titers (> 810). The frequency of anti-Ad2 NAbs was higher in Anhui (97.5%) than in Beijing (88.7%). Their prevalence differed significantly according to age in Beijing, but not in Anhui Province, but by sex in neither province. Ad2 seroprevalence was as high among HIV-1-infected individuals (88.7%) as among healthy individuals (92.2%) in China. In conclusion, a simple, intuitive, high-throughput, economical fluorescence-based neutralization assay was developed to determine anti-Ad2 NAbs titers. Ad2 exposure was high in both healthy and HIV-1-infected populations in China, so vectors based on Ad2 may be inappropriate for human vaccines.

Regulation and quality evaluation system for HIV diagnostics in China.

A sophisticated regulatory framework has been constructed for Human immunodeficiency virus (HIV) diagnostics in China, which have developed over the past 30 years. China National Institutes for Food and Drug Control acts as the legal institution in this regulatory framework, launching important activities to ensure the quality of HIV diagnostics. These include the analysis of the main problems faced in developing domestic HIV diagnostics, by investigating the quality of HIV diagnostics and their development; exploring the key factors affecting the quality of HIV diagnostics, to determine the criteria for screening national reference samples; the development of new technologies and methods for preparing reference samples; and the establishment of nine types of national reference panels and nine national standards to evaluate the quality of HIV diagnostics. Based on these researches, a quality evaluation system was established, including nine types of national reference panels, nine national standards for HIV diagnostics, and five sample banks (HIV-positive sample bank, HIV-negative sample bank, common international genotype sample bank, seroconversion series sample bank, HIV virus bank) to evaluate the quality of HIV diagnostics in China. The regulatory framework and the quality evaluation system are pivotal in ensuring the quality of the HIV diagnostics licensed in China.

Hepatitis E genotype 4 virus from feces of monkeys infected experimentally can be cultured in PLC/PRF/5 cells and upregulate host interferon-inducible genes.

The understanding of the interaction between hepatitis E virus (HEV) and its host cells has been impeded greatly by the absence of a cell culture system. In this study, an efficient cultivation method was developed in PLC/PRF/5 cells for HEV genotype 4 from the feces of monkeys infected experimentally. Compared to minimal essential medium (MEM), mixed Dulbecco's Modified Eagle's Medium (DMEM)/M199 improved the infection efficiency of HEV in PLC/PRF/5 cells. The incubation time and temperature were set at 6 hr and 40°C, respectively. Compared to a 100% ELISA positive ratio (EPR) of 1 × 10(6) copies/ml HEV inoculated flasks, the ELISA positive ratio was 100%, 75%, 37.5%, and 100% for flasks inoculated with HEV incubated for 30 min under the conditions of pH 3.0, pH 11.0, 56°C and delipidation treatment, respectively. Gene expression profiles of HEV inoculated and control PLC/PRF/5 cells were assayed using a microarray. Four interferon-inducible genes, IFI27, IFI6, Mx1, and CMPK2, were up-regulated during HEV-infection. Furthermore, the replication of HEV was inhibited at 3-14 days after treatment with 500 IU/ml IFN-α2b.

Bisulfite and sulfite as derivatives of sulfur dioxide alters biomechanical behaviors of airway smooth muscle cells in culture.

Sulfur dioxide (SO2) is a common air pollutant that triggers asthmatic symptoms, but its toxicological mechanisms are not fully understood. Specifically, it is unclear how SO2 in vivo affects airway smooth muscle (ASM) cells of which the mechanics is known to ultimately mediate airway hyperresponsiveness (AHR) - a hallmark feature of asthma. To this end, we investigated the effects of bisulfite/sulfite (1:3 M/M in neutral fluid to simulate the in vivo derivatives of inhaled SO2 in the airways), on the viability, migration, stiffness and contractility of ASM cells cultured in vitro. The results showed that bisulfite/sulfite consistently increased viability, migration, F-actin intensity and stiffness of ASM cells in similar fashion as concentration increasing from 10(-4) to 10(-1) mmol/L. However, bisulfite/sulfite increased the ASM cell contractility induced by KCl only at the concentration between 10(-4) and 10(-3) mmol/L (p < 0.05), while having no consistent effect on that induced by histamine. At the concentration of 10(0) mmol/L, bisulfite/sulfite became acutely toxic to the ASM cells. Taken together, the data suggest that SO2 derivatives at low levels in vivo may directly increase the mass, stiffness and contractility of ASM cells, which may help understand the mechanism in which specific air pollutants contribute in vivo to the pathogenesis of asthma.

ADAM33 protein expression and the mechanics of airway smooth muscle cells are highly correlated in ovalbumin-sensitized rats.

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as an asthma susceptibility gene; however, the role of ADAM33 in the pathogenesis and progression of asthma remains to be elucidated. As ADAM33 is predominantly expressed in airway smooth muscle cells (ASMCs), it is feasible to investigate whether ADAM33 protein expression is correlated with ASMC mechanics that are ultimately responsible for airway hyperresponsiveness in asthma. To determine this, Sprague Dawley rats were sensitized with ovalbumin (OVA) for up to 12 weeks to simulate asthma symptoms. Subsequently, ASMCs were isolated from the rats and cultured in vitro. The protein expression of ADAM33 and cytoskeletal proteins (including F­actin and vinculin), cell stiffness and contractility, as well as traction force were measured. The results demonstrated that compared with the non­sensitized rats, the protein expression of ADAM33 in ASMCs from the OVA­sensitized rats increased in a time­dependent manner, reaching a maximum level at 4 weeks of sensitization and gradually subsiding as OVA sensitization continued (P<0.001). The cell stiffness, traction force and expression of vinculin and F­actin changed similarly, resulting in a positive correlation with ADAM33 protein expression (Pearson's correlation coefficient, 0.864, 0.716, 0.774 and 0.662, respectively; P=0.1­0.3). The in vivo results of OVA­induced ADAM33 protein expression and its association with the mechanics of ASMCs suggested that ADAM33 is a mediator of ASMC dysfunction in asthma, and may provide a rationale for the therapeutic targeting of ADAM33 in the treatment of asthma.

[A flexion loading system for knee biomechanics research].

It is important to design and build a kinetic loading system for flexing movement of knee joint to study knee biomechanics. The system reported here includes driving device, control device, and flexion angle determination imaging system. The driving device was constructed with a stepper motor and a mechanical transmission with a serried of clamps, shanks and so on, and the driving device was controlled by the control device with micro-control unit, a computer and the serial 232. While the knee joint was driven to move by the stepper motor, the flexion angle of the knee was determined using imaging-based techniques. The system achieved accurate loading and control of speed, extent and duration of knee flexion, as well as fast and non-contract determination of flexion angle during knee flexing movement. The system is simple to build, easy to operate, highly accurate and reliable and it provides an important tool for the study of knee biomechanics, and potentially provides a tool for helping patients of knee surgery during their post operation recovery training.

Chronic exposure to sulfur dioxide enhances airway hyperresponsiveness only in ovalbumin-sensitized rats.

Sulfur dioxide (SO(2)) is a common air pollutant that triggers asthmatic symptoms, but its toxicological mechanisms are not fully understood. Specifically, it is unclear how airborne SO(2) affects airway hyperresponsiveness (AHR) - a hallmark feature of asthma. To this end, we investigated the effects of chronic exposure to SO(2) on AHR, airway inflammation, tissue remodeling, cell stiffness (G') and contractility of the airway smooth muscle cell (ASMC). Newborn Sprague-Dawley (SD) rats sensitized to ovalbumin (OVA) was used as the model to mimic asthmatic symptoms. The experimental results show that exposure to SO(2): (1) significantly increased Penh (an indicator of AHR) in the OVA-sensitized rats (p<0.01) but not in the normal rats (p>0.05), which correlated with the increase of airway smooth muscle mass; (2) increased IL-4 production in BALF of both the normal (p<0.05) and OVA-sensitized rats (p<0.001), but decreased IFN-γ in BALF of only the normal rats, and in serum only increased IL-4 production of the OVA-sensitized rats (p<0.001); (3) increased ASMC stiffness (G') and contractility only in the OVA-sensitized rats (p<0.001, p<0.05, respectively). Taken together, these results demonstrate that SO(2) may be a universal airway inflammatory factor, but more importantly, specific to exacerbating AHR in asthmatics only. These findings uncover a potential mechanism of SO(2)-induced health effects and may provide a basis for therapeutic targets.

Optimization and proficiency testing of a pseudovirus-based assay for detection of HIV-1 neutralizing antibody in China.

Among the neutralizing antibody evaluation assays, the single-cycle pseudovirus infection assay is high-throughput and can provide rapid, sensitive and reproducible measurements after a single cycle of infection. Cell counts, pseudovirus inoculation levels, amount of diethylaminoethyl-dextran (DEAE-dextran), and the nonspecific effects of serum and plasma were tested to identify the optimal conditions for a neutralizing antibody assay based on pseudoviruses. Optimal conditions for cell counts, pseudovirus inoculation, and amount of DEAE-dextran were 1 × 10(4)cells/well, 200TCID(50)/well, and 15 µg/ml, respectively. Compared with serum samples, high-concentration anticoagulants reduced the relative light unit (RLU) value. The RLU value increased sharply initially but then decreased slowly with dilution of the plasma sample. Test kits containing 10 HIV-1 CRF07/08_BC pseudovirus strains and 10 plasma samples from individuals infected with HIV-1 CRF07/08_BC were assembled into two packages and distributed to nine laboratories with a standard operating procedure included. For the 10 laboratories that evaluated the test, 17 of 44 (37%) laboratory pairs were considered equivalent. A statistical qualification rule was developed based on the testing results from 5 experienced laboratories, where a laboratory qualified if at least 83% of values lied within the acceptable range.

A novel high-throughput vaccinia virus neutralization assay and preexisting immunity in populations from different geographic regions in China.

BACKGROUND: Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. METHODOLOGY/PRINCIPAL FINDINGS: A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. CONCLUSION: A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

Comparison between the automated Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 assay and its version 1 and Nuclisens HIV-1 EasyQ version 2.0 assays when measuring diverse HIV-1 genotypes in China.

BACKGROUND: Several commercially available HIV-1 viral load assays based on real-time detection technology and automated platforms are available. It is not clear how the diversity of HIV-1 genotypes impacts the ability to consistently detect HIV-1 viral loads. OBJECTIVES: To examine whether the diversity of HIV-1 genotypes impacts the ability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0), its version 1.0 (CAP/CTM v1.0) and the NucliSens EasyQ HIV-1 version 2.0 (EasyQ v2.0) assays to consistently determine the viral loads. STUDY DESIGN: The three assays were used to measure the viral load in 178 plasma samples with diverse genotypes from treatment-naive patients. RESULTS: CAP/CTM v2.0 showed significant correlation and high agreement with CAP/CTM v1.0 and EasyQ v2.0. CAP/CTM v2.0 showed excellent detection of clade B samples compared with CAP/CTM v1.0 and EasyQ v2.0. However, significant differences were observed when using CAP/CTM v2.0 to test clade BC and AE samples. The HIV-1 load measured by CAP/CTM v2.0 differed by >0.5logIU/ml in 59.52% and 72.62% of clade BC samples, and in 57.14% and 85.71% of clade AE samples, compared with CAP/CTM v1.0 and EasyQ v2.0, respectively. CAP/CTM v2.0 was more precise (13.18%) than EasyQ v2.0 (29.21%), and both assays showed good linearity (R≥0.9926). CONCLUSIONS: The three assays may not deliver consistent results for samples belonging to clades BC and AE. It is strongly suggested that the version of the HIV-1 viral load assay used initially is also used at follow-up.

Comparative evaluation of the ViroSeq™ HIV-1 genotyping system and an in-house method for analysis of HIV-1 drug-resistance mutations in China.

BACKGROUND AND OBJECTIVE: With the introduction of the ViroSeq™ HIV-1 Genotyping System (ViroSeq™ assay) into China, it is important to evaluate the impact of the diversity of HIV-1 genotypes found in China on the performance of the ViroSeq™ assay compared with an in-house method. MATERIALS AND METHODS: A total of 318 plasma samples, collected from 206 HIV-1-infected patients receiving antiretroviral therapy and 112 treatment-naïve HIV-1-infected patients, were used for evaluating the concordance of genotypes, genotypic resistance mutations, and phenotypic resistance between the ViroSeq™ assay and an in-house method for analyzing HIV-1 drug resistance in China. RESULTS: A concordance of genotypes between the ViroSeq™ assay and the in-house method was observed for the 313 samples (98.4%), using the Stanford University HIV Drug Resistance Database (Version 6.0.5). The overall concordances of drug-resistance-related mutations (DRRMs) in the HIV-1 protease (PR) and reverse transcriptase (RT) coding sequences within the HIV-1 pol gene, scored by the ViroSeq™ assay and the in-house method, were 99.5% and 98.1%, respectively. Discrepancies between the two methods were found in 38 samples assayed for protease inhibitor (PI) DRRMs, 36 samples assayed for nucleoside reverse transcriptase inhibitor (NRTI) DRRMs, and 72 samples assayed for non-nucleoside reverse transcriptase inhibitor (NNRTI) DRRMs, and 100%, 88.9%, and 87.5% of the samples with discrepancies for PI, NRTI, and NNRTI DRRMs, respectively, were genotyped as subtype B. One NNRTI mutation (the RT mutation Y318F) was reported only by the ViroSeq™ assay, and this discrepancy resulted from the difference in the pol gene lengths generated by the two systems. Furthermore, the overall concordance of phenotypic resistance was 94.7% (301/318) between the two methods. CONCLUSION: The ViroSeq™ assay will be a useful tool for monitoring clinical drug resistance and for better management of HIV-1 patients receiving antiretroviral therapy in China.

The serological prevalence and genetic diversity of hepatitis E virus in farmed rabbits in China.

We identified and characterized a novel virus, designated rabbit hepatitis E virus (HEV), in rex rabbits farmed in China. Rabbit HEV is genetically related to but distinct from other known mammalian HEVs and avian HEV and may represent a novel genotype. To evaluate the spread and genetic variation of rabbit HEV, a total of 1094 serum samples were collected from various breeds of rabbits across ten counties in China. All sera were screened for the presence of anti-HEV antibody, HEV antigen and viral RNA. A total of 169 samples (15.4%), from nine of the ten counties, were found to be positive for HEV antibody. The seroprevalence was highest in Wuhan, Hunan Province (53.4%, 55/103). Samples positive for HEV antigen were detected in seven counties and the overall prevalence was 3.7% (41/1094). HEV RNA was detected in 22 samples and all but one of these samples was found to be positive for HEV antigen. Sequence analysis of the 304 bp amplicons within open reading frame 2 showed that all HEV isolates in this study clustered with known rabbit HEV strains, in a branch separate from genotypes 1 to 4. The rabbit HEV strains were genetically heterogeneous and divided into divergent groups. Strains from the same geographic region tended to cluster together. These results indicate that rabbit HEVs with considerable genetic diversity are prevalent in farmed rabbits in China. The potential zoonotic risk of rabbit HEV needs to be investigated and evaluated further.

Performance of NucliSens HIV-1 EasyQ Version 2.0 compared with six commercially available quantitative nucleic acid assays for detection of HIV-1 in China.

BACKGROUND AND OBJECTIVES: Six HIV-1 viral load assays have been widely used in China. These include the Cobas Amplicor HIV-1 Monitor Version 1.5 ('Amplicor'), Cobas AmpliPrep/Cobas TaqMan HIV-1 test Version 1.0 ('CAP/CTM'), Versant HIV-1 RNA Version 3.0 (branched DNA [bDNA]-based assay; 'Versant bDNA'), Abbott RealTime HIV-1 assay ('Abbott RealTime'), NucliSens HIV-1 QT (nucleic acid sequence-based amplification assay; 'NucliSens NASBA'), and NucliSens EasyQ HIV-1 Version 1.1 ('EasyQ V1.1'). Recently, an updated version of EasyQ V1.1, NucliSens EasyQ HIV-1 Version 2.0 ('EasyQ V2.0') was introduced into China. It is important to evaluate the impact of HIV-1 genotypes on the updated assay compared with the other commercial available assays in China. METHODS: A total of 175 plasma samples with different HIV-1 clades prevalent in China were collected from treatment-naïve patients. The viral loads of those samples were determined with the seven HIV-1 viral load assays, and the quantitative differences between them were evaluated. RESULTS: Overall, EasyQ V2.0 exhibited a significant correlation (R = 0.769-0.850, p ≤ 0.001) and high agreement (94.77-97.13%, using the Bland-Altman model) with the other six assays. Although no significant differences between EasyQ V2.0 and the other six assays were observed when quantifying clade B' samples, there were statistically significant differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays when quantifying clade BC samples, and between EasyQ V2.0 and the Versant bDNA and Abbott RealTime assays when quantifying clade AE samples. For clade BC samples, the quantitative differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples and exceeded 1 log(10) IU/mL in approximately 15% of samples. For clade AE samples, the quantitative differences between EasyQ V2.0 and the CAP/CTM, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples, and the differences between EasyQ V2.0 and CAP/CTM exceeded 1 log(10) IU/mL in approximately 15% of samples. CONCLUSION: Genotypes may affect the quantification of HIV-1 RNA, especially in clade BC samples with respect to EasyQ V2.0 and the Amplicor, Versant bDNA, or Abbott RealTime assays, and in clade AE samples with respect to EasyQ V2.0 and the Versant bDNA or Abbott RealTime assays. It is therefore strongly suggested that, where possible, the HIV-1 viral load in infected patients be quantified at follow-up by the same version of the same assay that was used initially.

The effect of human immunodeficiency virus type 1 (HIV-1) gp41 variability on antibody detection.

To determine whether genetic variability influences the ability to detect antibody, nine gp41 ectodomain recombinant proteins from human immunodeficiency virus type 1 (HIV-1) CRF07_BC, CRF01_AE and subtype B' were expressed in a bacterial expression system and purified. An indirect sandwich ELISA was developed with individual purified recombinant proteins. Plasma samples from 26 individuals infected with HIV-1 of different subtypes and four samples from the 1st international antibody reference panel were tested against each recombinant protein by ELISA. Heat-map and two-dimensional hierarchical clustering methods revealed that ELISA reactivity against antigens derived from the same subtypes clustered together. This suggests a similar reactivity pattern among infections of the same subtype, and thus the antigenicity of gp41 recombinant proteins may vary depending on the subtype, and subtype-related serotypes may exist among these antigens. Using association analysis methods, eight signature sites related to the subtype-specific reactivity patterns were identified. This study provides valuable information for the development of diagnostic assays with the ability to detect broadly cross-reactive antibodies induced by infection with different HIV-1 subtypes.

Performance of the automated COBAS AmpliPrep/COBAS TaqMan HIV-1 test on a genetically diverse panel of specimens from China: comparison to the COBAS amplicor HIV-1 monitor test, v1.5.

OBJECTIVES: To investigate the impact of genetically diverse HIV samples from China on the performance of the automated COBAS(R) AmpliPrep/COBAS TaqMan(R) HIV-1 Test (CAP/CTM). METHODS: 185 samples from untreated HIV-1-infected patients were used to assess the performance of the CAP/CTM Test and COBAS Amplicor HIV-1 Monitor Test, v1.5 (Cobas). RESULTS: A comparison of the qualitative results of both assays showed concordance for 1 negative and 184 positive samples (100%, kappa = 1.000). A significant correlation (R = 0.862, p < 0.001) and high agreement (95.53%) was observed for 179 samples with viral loads within the dynamic ranges of both assays. For samples of clades predominant in China, the fitted regression line differed significantly from the line of equality, although significant correlations (R = 0.694-0.899, p < 0.05) and high agreements (91.30-95.35%) were found for the two assays. The mean differences for samples from clades B' and BC were significant (p < 0.001) whilst no great difference (7.19-10.88%) between the quantitative values for both assays was found by plotting the regression line. CONCLUSION: The viral loads of different HIV genotypes in China measured by the CAP/CTM and Cobas assays were comparable.

Development and evaluation of a national reference panel of HIV-1 protease and reverse transcriptase drug-resistance mutations for HIV-1 genotypic resistance assays in China.

BACKGROUND: Transmission of antiretroviral drug-resistant strains of HIV-1 has a major impact on the success of HIV treatment regimens in most countries where antiretroviral therapy (ART) is available. It is now recommended that HIV-1 drug resistance be monitored in order to recommend appropriate therapy for infected ART-naïve individuals, or when choosing a new regimen for those patients not responding to ART. Commercial assays for the analysis of HIV-1 genotypic resistance have been approved by the US FDA. In China, several laboratories and research enterprises have been developing related assays, but at present, no systematic standardization or quality control for genotypic resistance assays has been established in China. A national reference panel is needed to evaluate and control the quality of HIV-1 genotype resistance assays in China. METHODS: A panel of five HIV-1 stocks (G1, G2, G3, G4, and G5) with well-characterized drug resistance was used to evaluate the resistance-mutation inclusivity of HIV-1 clades prevalent in China. Six samples (GS1-GS6) were used to evaluate the limit of detection (LOD) for the viral load levels, and five samples (GSS1-GSS3, GSS5, and GSS6) were used to evaluate the LOD for the percentages of mutant species within the range of detection. The samples were evaluated by five separate laboratories using one or two methods each, generating seven datasets in all. RESULTS: In samples G1-G5, which were used to evaluate inclusivity of HIV-1 clades, 92.86-100%, 16.67-83.33% and 4.17-8.33% of the most, intermediately, and least common resistance mutations, respectively, were reported by the seven datasets. For the LOD samples, four of the seven datasets reported correct resistance mutations in samples with minimal viral loads of >2 x 10(3) copies/mL, as well as in samples with >40% of mutants and viral loads of about 1 x 10(4) copies/mL; the other three tests did not amplify the target region or identify the mutants. CONCLUSIONS: The results were quite variable between different tests. The panel of HIV-1 genotypic resistance could be used as a control for resistance testing in China.

Genotypic and phenotypic characterization of HIV-1 CRF01_AE env molecular clones from infections in China.

BACKGROUND: Although a great number of HIV-1 pseudoviruses have been generated for neutralization assays, circulating recombinant forms, such as CRF01_AE, are not included. Given the increasing prevalence of CRF01_AE, the establishment of a pool of CRF01_AE env isolates for the evaluation of potential HIV vaccines is needed. MATERIALS AND METHODS: Full-length env genes were cloned from HIV-1 CRF01_AE-infected plasma samples collected in China and used to establish Env-pseudotyped viruses. The neutralization phenotypes of the pseudoviruses were characterized by testing against broadly neutralizing monoclonal antibodies, coreceptor antagonists, and 42 plasma samples that include 3 main prevalent HIV-1 subtypes in China. RESULTS: Thirty-four genetically distinct CRF01_AE env genes were cloned and used to generate pseudotyped viruses. Of the 34 pseudoviruses, 32 used CCR5 as a coreceptor and 2 used CXCR4. The majority of pseudoviruses were resistant to the neutralizing antibodies IgG1b12 and 2G12 and susceptible to 2F5 and 4E10. There was significant variation of the neutralization susceptibility of pseudoviruses against 42 HIV-1-positive plasma samples. Based on their overall neutralization susceptibilities, the 34 CRF01_AE pseudoviruses were classified into 3 tiers: high, medium, and low. CONCLUSION: The CRF01_AE pseudovirus isolates should be included in the panel of pseudoviruses used to assess vaccine-elicited neutralizing antibodies.

A novel genotype of hepatitis E virus prevalent among farmed rabbits in China.

In total, 335 serum samples were collected from rabbits from two farms in Gansu province, China, and tested for anti-hepatitis E virus (HEV) antibody using EIA and for HEV RNA using nested RT- PCR with ORF2 primers. The overall prevalence of anti-HEV antibody and HEV RNA was 57.0% (191/335) and 7.5% (25/335), respectively. The positivity rate of HEV RNA in the anti-HEV antibody negative group (7.6% (11/144)) did not differ significantly from that in the positive group (7.3% (14/191)). The concordance between HEV RNA and anti-HEV antibody was 43.3% with no significant correlation (P < 0.05). All 25 amplicons from the ORF2 region were cloned and sequenced. On the basis of nucleotide sequence comparison, they had 84-99% identity to each other and 73-77%, 70-76%, 75-82%, 71-77%, and 53-65% with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively. Samples that were positive with the ORF2 primers were amplified using ORF1 region primers; 17 were positive and shared 71-78%, 73-76%, 74-82%, 72-78%, and 39-58% identity with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively, at the nucleotide level. Two representative full-length sequences were determined. These two sequences shared 85% identity with each other and had 74%, 73%, 78-79%, 74-75%, and 46-47% identity to full-length genotypes 1, 2, 3, 4, and avian HEV, respectively. Thus, the sequences isolated from the rabbits represent a novel genotype of HEV. This study provides novel information about HEV genotypes infecting rabbits as well as evidence of a new mammalian genotype of HEV.

Relationships among viral diagnostic markers and markers of liver function in acute hepatitis E.

BACKGROUND: Diagnosis of acute hepatitis E has been based in many clinics predominantly on detection of anti-HEV (hepatitis E virus) antibody. Now, new assays have been developed to detect other HEV markers. Our aim was to investigate the relationships among HEV diagnostic markers and liver function markers in acute hepatitis E. METHODS: Seventy serum samples were collected from non-A, non-B, non-C acute hepatitis patients and tested for HEV markers (HEV antigen and RNA and anti-HEV IgM) and markers of liver function [alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total iron binding capacity (TBA), gamma-glutamyl transferase (GGT), total bilirubin (TBIL), and direct bilirubin (DBIL)]. Partial open reading frame (ORF) 2 sequences from HEV RNA-positive samples were cloned and analyzed. RESULTS: The concordances between HEV antigen and HEV RNA and between HEV antigen and anti-HEV IgM were 77.1% and 72.9%, respectively, with significant correlations, while that between HEV RNA and anti-HEV IgM was 61.4% with no significant correlation. Eleven of 25 samples negative for anti-HEV IgM were positive for HEV antigen. The ALT, AST, ALP, TBA, GGT, TBIL, and DBIL levels did not differ significantly between the anti-HEV IgM-positive and -negative groups. However, the ALT, AST, ALP, TBA, and GGT levels were significantly higher in the HEV antigen-positive group than in the HEV antigennegative group. All of the HEV isolates cloned belonged to genotype 4. CONCLUSIONS: HEV antigen was highly correlated with HEV RNA and elevated ALT, AST, ALP, TBA, and GGT levels. Testing for HEV antigen in combination with anti-HEV IgM is useful for the diagnosis of HEV infection.

Comparative evaluation of the COBAS AmpliPrep/COBAS TaqMan HIV type 1 test (CAP/CTM) and VERSANT HIV type 1 RNA 3.0 assay (bDNA) for quantifying HIV type 1 viral loads in China.

In the present study, 277 clinical samples from untreated and treated HIV-1-infected patients with different clades were used to assess the agreement between the COBAS AmpliPrep/COBAS TaqMan HIV-1 test (CAP/CTM) and VERSANT HIV-1 RNA 3.0 Assay (bDNA). A qualitative comparison of the results of the two assays showed concordance for 255 positive and 15 negative samples (94.95%, kappa = 0.798). However, seven samples with viral loads close to the lower limit of detection for CAP/CTM were negative by bDNA. A significant correlation (r = 0.881, p < 0.001) was observed for 253 samples with viral loads within the dynamic ranges of the two assays, and Bland-Altman analysis showed good agreement (96.05%) between the two assays for these 253 samples [mean (+/-2 SD), 0.389(-0.385, 1.163)]. Furthermore, ART drugs had no impact on the performances of the two assays. For samples with different clades predominant in China, the fitted regression line differed significantly from the line of equality, although significant correlations (r = 0.850-0.891, p < 0.001) and good agreements (92.86-97.25%) were found for the two assays. The mean differences for clade B' and BC samples were significant (p < 0.01). Good precision for clade B' samples was achieved for the CAP/CTM (CV: 20.73%) and bDNA (CV: 12.19%) assays. Furthermore, for clades B', BC, and AE, both assays exhibited good linearities (r = 0.9773-0.9998). Thus, the CAP/CTM and bDNA assays could be useful for quantifying HIV-1 RNA in routine clinical samples and monitoring viral loads in treated and untreated HIV-infected patients in China.