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The COVID-19 epidemic that occurred at the end of 2019 spreads rapidly to all parts of the world, putting the global public health system to a severe test. With the continuation of the epidemic, SARS-CoV-2 variants are constantly emerging. In particular, the mutation of the spike protein can cause changes in the infectivity and antigenicity of the virus, resulting in an increase in the infectivity and a decline in the protective efficacy of existing vaccines, and even the replacement of epidemic strains. This is also one of the reasons why the epidemic has not been effectively controlled so far. Nowadays, the main circulating variants have changed their characteristics to a certain extent, and the neutralization sensitivity of some variants to neutralizing monoclonal antibodies, immune sera and convalescent sera has decreased to a certain extent compared with the original strains. The emergence of variants is not only related to the characteristics of the virus itself, but also to the changes of transmission host and the chronic infection in people with deficient immunity. The emerging variants should be closely monitored, and their functional characteristics should be systematically studied so as to provide data for vaccine research and development and the designation of immunization strategies.
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In 2009, hepatitis E virus was first detected in wild rats ( Rattus norvegicus) in Germany and was designated as rat hepatitis E virus (rat HEV). Since then, rat HEV has been detected in various murine rodents in many geographic regions. The potential of rat HEV to infect human has been ignored as the viral genomic nucleotide sequences of rat HEV and the HEV strains of human sources are only about 50%-56% identical. Recently, a few clinical hepatitis E cases with chronic or acute rat HEV infection have been reported and raised many concerns. Here, advances in studies of the prevalence of rat HEV in animals and the clinical hepatitis E cases caused by rat HEV were reviewed.
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Metagenomic next-generation sequencing (mNGS) could be used for pathogen detection from nearly all types of clinical samples. Especially, the unique diagnostic capability of pathogen mNGS detecting unknown causative agent of infectious diseases makes this method become an importation complement and irreplaceable component for conventional routine laboratory test. However, the complexity of the testing process, the rapid product update, and the insufficiency in quality control and evaluation methods that all make clinical transformation, industry development, and regulation of this technology full of challenge and uncertainty. This review briefly introduces the technical advantages and challenges, and describes the general workflow and quality control steps in details. Finally, it focuses on current considerations regarding quality evaluation methods and standards for pathogen mNGS.
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Humans , Communicable Diseases , High-Throughput Nucleotide Sequencing , Metagenome , Metagenomics , Quality ControlABSTRACT
Objective:To analyze the neutralization properties of different genotypes and mutants of severe fever with thrombocytopenia syndrome virus (SFTSV).Methods:Pseudoviruses of SFTSV of different genotypes and mutants were constructed using VSVΔG-Fluc*G backbone. Neutralization assays were established based on the pseudoviruses. DNA vaccines for different SFTSV genotypes were prepared. Serum samples were collected from guinea pigs immunized with the DNA vaccines. Neutralizing antibodies in serum samples from immunized guinea pigs and naturally infected patients were detected using neutralization assays and analyzed.Results:The pseudoviruses of five genotypes and 43 mutants were successfully constructed and the neutralization assays based the pseudoviruses were successfully established after optimizing the reaction parameters. The dilution multiple corresponding to the inhibition rate of neutralizing antibody to half of the pseudovirus infection was taken as the titer of neutralizing antibody by the reduction in pseudovirus reporter gene. The neutralization antibody titers in naturally infected patients and immunized guinea pigs were respectively in the ranges of 1∶100-1∶43 000 and 1∶100-1∶2 500 when detected with the reference HB29 pseudovirus. The neutralization antibody titers ranged from 1∶100-1∶2 500 after immunization with different genotypes of DNA vaccines. No significant statistical difference in neutralization antibody titer was observed among different genotypes or mutant strains.Conclusions:The neutralization properties of different genotypes and mutants showed no significant change, which would be very useful for developing vaccines.
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Antibody dependent enhancement (ADE) is a common phenomenon in virology. It is involved in the mechanisms of infections caused by Dengue virus (DV), severe acute respiratory syndrome coronavirus (SARS-CoV), influenza virus, HIV and other viruses and affects the research and development of vaccines against them. Because the pre-existing specific antibodies or antibodies at sub-neutralizing titer can enhance the infectivity of viruses, leading to disease aggravation, vaccination may promote infection instead of preventing it. This article focused on the impact of ADE on the research and development of vaccines and the assessment of ADE.
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Human papillomavirus (HPV) infection is the most common sex-transmitted virus infection. Persistent infection of HPV could cause cancers in different parts of the body, such as cervix and anus. Up to now, three prophylactic HPV vaccines have been approved to prevent HPV infection and related diseases. It is well accepted that neutralizing antibodies are the mediator of protection. Analysis of immunogenicity has been playing a key role in HPV vaccine clinical trials, especially in the evaluation of durability of immune responses, immunobridging in young girls and boys, and reduced-dose schedules. There is still no unique method for the evaluation of immunogenicity of HPV vaccines in clinical trials. The data of immunogenicity from different clinical trials cannot be compared directly. It is urgently needed to establish a high throughput and protection-related unique assay as well as standard materials for its standardization. To accelerate the development and evaluation of prophylactic HPV vaccines, a standardized method should be well validated and transferred to different laboratories to make clinical immunogenicity data comparable.
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Cervical cancer is the second most common cancer in women worldwide. It is clear that persistent infection of high-risk human papillomaviruses (HR-HPVs) is the main cause for this disease. Among the several HPV types associated with carcinoma, HPV-16 is the most prevalent type and present in about 50% of tumor specimens. The major capsid protein (L1) of HPV can self-assemble into virus-like particles (VLPs) with immunogenicity similar to infectious virions. Neutralizing epitopes are the structural basis of the current prophylactic HPV vaccines. The efficacy of HPV vaccines is critically dependent upon the integrity of type-specific neutralizing epitopes. Recently, considerable headway has been made in studying the epitopes of HPV16 based on neutralizing antibodies. Notably, more and more HPV16 variants have appeared along with increasing immune pressure. To study the phenotypic variations in HPV16 L1 protein, 1 204 naturally occurring sequences were analyzed and a phylogenetic tree was then constructed including four clades. Moreover, after compared the aforementioned sequences with the 114K reference sequence, eight "hot mutation sites" , six "specialized mutation sites" and 20 "epitope-related mutation sites" were found. Generally, sera raised against VLPs can neutralize the corresponding HPV types, but not other types. However, it is not known whether intragenotypic variants of human papillomavirus type 16 (HPV-16) can be neutralized by sera vaccinated with a single variant VLPs. It is, therefore, imperative to understand the neutralizing epitopes and intragenotypic variants of HPV-16 for the production of prophylactic vaccines with high potency and broad coverage.
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Twenty-six novel tricyclic sophoridinic and matrinic derivatives containing a common chlorinated benzene fragment were designed, synthesized and evaluated for their anti-ebolavirus (EBOV) activities. Structure-activity relationship analysis indicated: (i) 12-dichlorobenzyl motif was beneficial for the activity; (ii) the chiral configuration at C5 atom might not affect the activity much. Among the target compounds, compound exhibited the most potent potency against EBOV with an IC value of 5.29 μmol/L and an SI value of over 37.8. Further anti-EBOV assay of identified its high effectiveness, and anti-MARV assay of suggested its inspiring broad-spectrum anti-filovirus activity. The results provided powerful information on further strategic optimization and development of this kind of compounds against filoviruses.
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Marburg virus (MARV) is a lethal virus that causes fatal hemorrhagic fever.It belongs to the Filoviridae family which also includes Ebola virus.MARV is similar to Ebola virus in structure and infection mechanism.Moreover,the diseases caused by them have similar clinical symptoms.However,researches on MARV are less than those on Ebola virus.In this review,we focus on the viral structure,especially the structure of MARV glycoprotein (GP) which determines its infectivity,functions of MARV GP as well as protective antibodies and vaccines against this protein.
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Zika virus (ZIKV) was first isolated in 1947. It is known to circulate mainly in tropical areas and can be transmitted through Aedes mosquitoes. In 2015, an outbreak of ZIKV began in Brazil and then spread rapidly to the Latin Americans and Caribbean. Accumulating evidence indicates that there are associations between ZIKV infection and human nervous disorders, such as the occurrence of microcephaly in neonates and Guillain-Barré syndrome in adults. The current ZIKV outbreak has been declared a public health emergency of international concern. Here, we review the virological features, transmission and epide-miological characteristics of ZIKV and the clinical featrues and prevention of ZIKV infection.
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Objective To establish HIV-1 seroconversion panels with the samples collected by National Institutes for Food and Drug Control ( NIFDC) and to evaluate the window periods of HIV enzyme immunoassay ( EIA) diagnostic kits used for blood screening with them. Methods Serum specimens were collected from different plasma donation stations in China. All suspected HIV infection specimens were screened for HIV by using the nucleic acid amplification testing (NAT), Western blot confirmatory assay and P24 quantitative detection assay. The HIV env gene sequences were amplified by RT-PCR for further confirmation of HIV infection. The PCR products were sequenced and genotyped. The confirmed seroconver-sion panels were used to evaluate the early detection capabilities of the 4th and 3rd generation HIV EIA diag-nostic kits used in blood screening in china. Results A total of 8 sets of HIV seroconversion panels com-prised of 36 samples were confirmed in this study, including 6 sets of AE subtype, 1 set of B subtype and 1 set of unknown genotype. Those seroconversion panels were tested with HIV diagnostic kits produced by 19 different manufacturers. For the early detection of HIV infection, the 4th generation HIV diagnostic kits with a score of 9. 4 points were better than the 3rd generation HIV diagnostic kits whose score was 3. 6 points (P<0. 01, t=8. 547). Some of the domestic 4th generation HIV diagnostic kits were similar to the imported kits in the early detection of HIV infection. In terms of the diagnosis of HIV infection, the HIV-1 NAT was at least 2 weeks earlier than the HIV EIA diagnostic kits. The sensitivity of confirmatory assay was lower than that of the diagnostic kits. Four out of five 4th generation HIV diagnostic kits showed declined signal to cut off ( S/CO ) ratio , indicating the probability of false detection during the second window period . Conclusion Eight sets of NIFDC HIV-1 seroconversion panels were established in this study. With those panels we found that there were differences in the window period between different EIA diagnostic kits used for HIV blood screening.
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Objective To analyze the practicability of using ELISA kit for the detection of hepati-tis E virus antigen ( HEV-Ag) in plasma donations and Biomex HEV seroconversion panels. Methods The HEV-Ag positive samples were screened out from 36 340 donated blood plasma samples. Real-time fluores-cent PCR was performed for the detection of HEV RNA in HEV-Ag positive samples. The open reading frame 2 (ORF2) in HEV RNA was amplified by nested RT-PCR and the amplified products were confirmed by sequencing analysis. Phylogenetic tree was constructed for HEV genotyping. Five Biomex HEV serocon-version panels were used in this study for the detection of HEV-Ag, anti-HEV antibody and HEV RNA as well as the correlation analysis between HEV-Ag and HEV RNA. Results Twenty-six out of 36 340 plasma samples (0. 07%) were positive for HEV-Ag. Of the 26 samples, 25 samples were positive for HEV RNA as indicated by the results of nested RT-PCR and 23 positive samples were confirmed by sequencing analysis. The positive rate of HEV RNA in blood plasma donators was 1 ∶ 1 580 (0. 06%). There were 17 samples of genotype 1 (74%) and 6 samples of genotype 4 (26%) according to the phylogenetic tree analysis. All of the HEV-Ag positive samples were also positive for HEV RNA as indicated by the analysis of Biomex sero-conversion panels. HEV-Ag was consistent with the peak of the HEV RNA concentration. Conclusion A close relationship between HEV-Ag and HEV RNA was observed. HEV-Ag screening could be used as a measure to reduce the risk of HEV transmission by blood transfusion.
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Objective To establish a high throughput phenotypic test for the detection of drug re-sistance in human immunodeficiency virus(HIV)strains. Methods The gene encoding luciferase was in-activated through restriction enzyme digestion and ligation. LacZ gene was used to replace the genes encoding original protease and reverse transcriptase. pol genes were amplified from pSG3△env plasmid and cloned in-to a new backbone plasmid through infusion. The factors that might affect the results of the test were opti-mized. Results The parental backbone plasmid pNL4-3. Lac was constructed,of which the gene encoding luciferase was inactivated and bearing the LacZ gene instead of genes encoding protease and reverse tran-scriptase. Several influential factors including cell numbers(10 000 / well),virus inoculation(200 TCID50 /well)and the concentration of DEAE-dextran(15 μg/ ml)were optimized. The reproducibility of this test was confirmed by testing 12 anti-HIV drugs against 2 pseudovirus strains 8 times,presenting the coefficient of variations(CVs)from 4. 32% to 28. 46% . Six types of pseudovirus were constructed and tested against the 12 anti-HIV drugs,the results of which were compared with those by using the pSG3△env-based pseud-ovirus test. The results of the two tests presented good consistency. Conclusion The high throughput phe-notypic test based on pNL4-3. Lac plasmid,combining the advantages of pSG3△env and pNL4-3 systems, could be used to analyze the drug resistance patterns of HIV-1 infectors and screen new drugs for antiretrovi-ral therapy in a rapid and effective way.
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Objective To sequence and analyze the full-length genome of one HEV strain,W2-1 isolated from a sporadic hepatitis E patient hospitalized in 1999 in Xinjiang,China.Methods Nested RT-PCR assays with 4 sets primers were used to amplify the entire genome.The PCR products were purified and sequenced.The full-length genome was acquired by assembling the fragmental sequences using the DNAstar 5.01 software.The genome of W2-1 was analyzed by comparing with the reference HEVs from GenBank.Results The complete genome of W2-1 is 7212 nt in length,including three open reading frames (ORF1-3) with 5079,1980 and 345 nt respectively,27 nt 5'UTR and 83 nt 3'UTR,and a 3' poly A tail.Phylogenetic analysis based on full-length genome showed that W2-1 belonged to genotype 1,subtype 1b.W2-1 had high homology with the HEV strains isolated in the large hepatitis E epidemic in Xinjiang in 1987-1989,sharing 97.2％-98.5％ nucleotide identity in the full length genome.W2-1 also showed high homology with 1b strains isolated in China after 2000,with 97.6％-99.2％ nucleotide identity.The specific amino acid sites in ORF1-3 proteins that distinct between genotype 1 HEV and the potential zoonotic strains did not change in W2-1.Conclusion W2-1 belongs to subtype 1b.The study indicates subtype 1b HEV has been circulating in China in a long period after hepatitis E outbreak in Xinjiang in 1986-1989.The amino acids of ORF1-3 of subtype 1b are conserved.
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Objective To study the in vivo expression and biodistribution of Ad5-Fluc (Adenovirus carrying firefly luciferase genes) in mice.Methods The recombinant Ad5-Fluc virus was constructed and infected to BALB/c or nude mice through three different routes.The protein expression level,tissue distribution and the characteristics of infection were analyzed by in vivo bioluminescence imaging technology.Results Compared to other two routes,the BALB/c mice infected through muscular route had the longest expression cycle (over 60 days) and the highest expression level,while the virus was transferred into the liver and spleen after infection.The nude mice had a significantly extended expression cycle than BALB/c mice.Moreover,the characteristic of liver tropism was eliminated after Ad5 F35 infection in mice,while maintained similar expression efficiency.Conclusion Due to the highest expression efficiency,the muscular route would be the optimal route for Ad5 vector based vaccination.In addition,Ad5F35 virus could become an ideal alternative vaccine vector for eliminating the liver tropism.
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Objective To evaluate the quality of four domestic and three imported fourth-generation HIV diagnostic reagents.Methods The specificity and sensitivity of these assays were analyzed when testing HIV negative samples and HIV-1 RNA positive samples.The relative seroconversion sensitivity index was analyzed when testing BBI seroconversion panels.Results The sensitivity of seven 4th-generation assays were 100％ (95％ CI:99.86％-100％ ),and one sample at the window period of HIV-1 infection were detected as positive.Of the seven assays,one imported assay exhibited the relative largeδ + value (1.0892),and the small δ+ value were found on the remaining six assays (0.0836-0.3003 ).For the samples negative for HIV antibody,varying degrees of false positives were observed on the seven assays ( specificity:97.80％ -99.60％,δ- value:-1.3803 to -0.4778).When testing the BBI seroconversion panels,the relative seroconversion sensitivity index of domestic assays were -0.500-0,however,which of imported assays were -0.600 and -0.700.Conclusion The seven reagents exhibited high sensitivity and specificity.The 4th generation HIV assays can be used as blood screening reagents to find the samples at window period of HIV-1 infection,thus indicating the certain meaning in reducing the transmission risk of HIV-1 for fourth-generation HIV diagnostic reagents.However,the better efficiency to detect HIV-1 early infection was observed on the imported assays than on the domestic assays.
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Objective To detect the protection induced by HPV-58 L2 11-200 AA in animal, and analyze the relationship between antibody or neutralizing antibody titers and the protection generated by the immunizmg agent. Methods The peptide of HPV-58 L2 11-200 AA was expressed in E. coli and the mice were immunized with the peptide after purification and adsorption with aluminum adjuvant. The protection provided by different immunizing doses was detected in the mouse model against the challenge of the pseud-ovirions of human papiilomavirus types 58. The total antibodies and neutralizing antibody titers of serum were tested with ELISA and neutralization assay against HPV-58 pseudovirus, respectively. The total antibodies or neutralizing antibody titers that can protect the mouse from infection were analyzed. Results The mice can be protected from the challenge with HPV pseudovirus when the immunizing dose was 8 μg. The neutralizing antibody can not be detected in the immune serum by neutralization assay against pseudovirus. The total anti-body level has a corresponding relationship with the protection showed in mouse model. The results of total antibodies detected by ELISA showed that when the titer of total antibodies was ≥25 000, luminescent signal can not be detected and the mice can be protected from pseudovirus infection. Conclusion HPV-58 L2 11-200 AA peptide can protect mice from pseudovirus infection. L2 peptide has a promising perspective to be a candidate vaccine and the level of total antibodies in the immune serum can be used as a surrogate for the evaluation of protection against HPV infection.
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The serum samples and corresponding cervical swabs were collected fi'om 50 women with genital warts from Tianjin city,China.The neutralizing antibodies against HPV-16,-18,-58,-45,-6 and-11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV.The results revealed that 36%(18/50)of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560,of which 22%(11/50),12%(6/50),10%(5/50),4%(2/50),4%(2/50)and 2%(1/50)were against HPVs-6,-16,-18,-58,.45 and-11,respectively.Additionally,60%(30/50)of samples were HPV DNA-positive,in which the most common types detected were HPV-68(18%),HPV-16(14%),HPV-58(12%),HPV-33(8%)and HPV-6,HPV-11,HPV-18 and HPV-52(6% each).The concordance between HPV DNA and corresponding neutralizing antibodies was 56%(28/50)with a significant difference(P＜0.05).The full-length sequences of five HPV types(HPV-42,-52,-53,-58 and-68)were determined and exhibited 98%-100% identities with their reported genomes.The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.
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Objective To compare cellular immune responses in mice elicited by Chinese different AIDS candidate vaccines.Methods According to their different immunization procedures,BALB/c mice were immunized with 6 AIDS candidate vaccines,separately.Spleen cells were isolated for the detection of cellular immune response to HIV-specific peptides using enzyme-linked immunosorbent spot(ELISPOT)assay and intracellular cytokine staining(ICS)method.Results AIDS vaccines were evaluated by using potential T-cell epitopes(PTE)Gag,Env and Pol peptides pool and ELISPOT.The positive conversion rates for cellular immune response of 1#-6# vaccines fluctuated from 70% to 100%.The vaccine-induced cellular immune responses to specific peptides pool are different not only in magnitude but also in breadth.The Th1type cytokines,IFN-γand IL-2,were detected with ELISPOT in 1# and 2# vaccines.The productions of IFN-γand IL-2 induced by both of the two vaccines showed a moderate correlation(r1 =0.62,P1 ＜0.01 ;r2=0.79,P2 ＜ 0.01).The positive conversion rate of IFN-γ secreting cells of 1 # vaccine was 66.7%(10/15)mice detected with both ELISPOT and ICS.And the results tested by ELISPOT and ICS showed moderate correlation(r = 0.55,P ＜ 0.05).Conclusion The magnitude and breadth of cellular immune responses induced by different AIDS candidate vaccines are different.Being induced by different AIDS candidate vaccines,the IFN-γand other Th1 type cytokines detected by ELISPOT or ICS could be used to evaluate the cellular immune responses in mice.
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Objective To pan and characterize anti-HIV-1 Fab by the phage antibody library technology. Methods Total RNA were extracted from lymphocytes which were isolated from peripheral blood collected from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1. The genes of heavy chains Fd fragment and light chains of antibody were amplified by RT-PCR. The phagmids pComb3X cloned Fd and light chain genes were transformed into E. coli XL1-Blue by electroporation to construct phage Fab library. By three runs of "absorption-elution-neutralization-enrichment", the clones were induced by IPTG and characterized by ELISA. The positive clones were sequenced and analyzed the sequences. Subsequently, Fab antibodies of these positive clones were induced to expressed and purified, then the recombinant virus neutralization assay was performed. Results A phage Fab library was constructed with 8×106 members, and 11 positive clones were obtained by detecting IPTG-induced-expressing Fabs with ELISA. By analysis of the sequences, 10 light chain genes and 8 Fd genes were ensured to be obtained. Compared with the genes of anti-HIV-1 antibodies in HIV sequence database, the gene sequences we obtained were highly homologous to some patent genes of anti-HIV-1 gp120 antibodies in HIV sequence database( light chains with 60%-90% identity, Fd with 71%-85% identity); The CDRs of these positive clones were determined by comparing the positive clone genes with antibodies' genes in V base database, furthermore, CDRH3 of these positive clones has the length of 12-22 aa. Strand shift had little effect to improving affinity of our Fab clones. Fab antibodies were induced to express at the concentration of ＞ 10 mg/L. Three Fab antibodies neutralize HIV-1 virus to some extent. Conclusion The studies will provide the basis on further study on the anti-HIV-1 Fabs obtained successfully.