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Spike‐protein‐based pseudotyped viruses were used to evaluate vaccines during the COVID‐19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus‐like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild‐type severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS‐COV‐1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild‐type VLP pseudotyped virus over 100‐fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS‐CoV‐2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze–thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra‐ and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high‐throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS‐CoV‐2 based on VLP pseudotyped virus. [ABSTRACT FROM AUTHOR]

Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3‐△env backbone plasmid HpaI and truncating the C‐terminal 21 amino acids of the SARS‐CoV‐2 spike protein (S), high‐titer SARS‐CoV‐2‐Sdel21‐AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96‐well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS‐CoV‐2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development. [ABSTRACT FROM AUTHOR]

SARS‐CoV‐2 breakthrough infections in vaccinated individuals underscore the threat posed by continuous mutating variants, such as Omicron, to vaccine‐induced immunity. This necessitates the search for broad‐spectrum immunogens capable of countering infections from such variants. This study evaluates the immunogenicity relationship among SARS‐CoV‐2 variants, from D614G to XBB, through Guinea pig vaccination, covering D614G, Alpha, Beta, Gamma, Delta, BA.1, BA.2, BA.2.75, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB, employing three immunization strategies: three‐dose monovalent immunogens, three‐dose bivalent immunogens, and a two‐dose vaccination with D614G followed by a booster immunization with a variant strain immunogen. Three distinct immunogenicity clusters were identified: D614G, Alpha, Beta, Gamma, and Delta as cluster 1, BA.1, BA.2, and BA.2.75 as cluster 2, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB as cluster 3. Broad‐spectrum protection could be achieved through a combined immunization strategy using bivalent immunogens or D614G and XBB, or two initial D614G vaccinations followed by two XBB boosters. A comparison of neutralizing antibody levels induced by XBB boosting and equivalent dosing of D614G and XBB revealed that the XBB booster produced higher antibody levels. The study suggests that vaccine antigen selection should focus on the antigenic alterations among variants, eliminating the need for updating vaccine components for each variant. [ABSTRACT FROM AUTHOR]

At present, the horse or human rabies immunoglobulin (RIG) used for postexposure prevention of human rabies (PEP) has high cost and limited availability. It is strongly encouraged to replace RIG with equivalent or more effective and safer products. Mouse and human monoclonal antibodies have been shown to protect rodents from lethal rabies virus (RABV) attacks. In this study, we reported a human–mouse chimeric monoclonal antibody, 12‐2A12, which showed a strong neutralization potency and a wide breadth against multiple street viruses of RABV in vitro. The antibody binded the viral glycoprotein (G) with nanomolar affinity. The complex structure of 12‐2A12 bound to RABV G revealed that the antibody recognizes an epitope that partially overlaps with the recognition region for the nicotinic acetylcholine receptor (nAChR). The antibody therefore would interfere with the nAChR/G interaction to block the viral receptor binding. In addition, comparison of our complex structure with the G structure in the acidic state reveals a clear steric clash, highlighting that the antibody would further prevent the conformational changes of the viral glycoprotein that are essential for membrane fusion. In light of these functional and structural data, we believe that 12‐2A12 might be developed to be included in an antibody cocktail for potential use in human rabies PEP. [ABSTRACT FROM AUTHOR]

Rabies is a viral disease that is nearly 100% fatal once clinical signs and symptoms develop. Post‐exposure prophylaxis can efficiently prevent rabies, and antibody (Ab) induction by vaccination or passive immunization of human rabies immunoglobulin (HRIG) or monoclonal antibodies (mAbs) play an integral role in prevention against rabies. In addition to their capacity to neutralize viruses, antibodies exert their antiviral effects by antibody‐dependent cellular cytotoxicity (ADCC), which plays an important role in antiviral immunity and clearance of viral infections. For antibodies against rabies virus (RABV), evaluation of ADCC activity was neglected. Here, we developed a robust cell‐based reporter gene assay (RGA) for the determination of the ADCC activity of anti‐RABV antibodies using CVS‐N2c‐293 cells, which stably express the glycoprotein (G) of RABV strain CVS‐N2c as target cells, and Jurkat cells, which stably express FcγRⅢa and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT‐luc/FcγRⅢa cells). The experimental parameters were carefully optimized, and the established ADCC assay was systematically validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 guideline. We also evaluated the ADCC activity of anti‐RABV antibodies, including mAbs, HRIG, and vaccine induced antisera, and found that all test antibodies exhibited ADCC activity with varied strengths. The established RGA provides a novel method for evaluating the ADCC of anti‐RABV antibodies. [ABSTRACT FROM AUTHOR]

An important goal of the Hepatitis E virus (HEV) vaccine is to prevent adverse pregnancy outcomes caused by different HEV genotypes during pregnancy, but studies directly evaluating maternal vaccination for HEV are lacking. Here we report maternal vaccination using HEV 239 vaccine in a pregnant rabbit model. Two dose of accelerated vaccination schedule (0, 7 days) induced high titers of anti‐HEV protective antibodies in a short period of time in pregnant rabbits, which could protect the pregnant rabbits from HEV infection and adverse pregnancy outcomes. In addition, the immunized rabbits transfer maternal antibodies to pups through the placenta and breast milk, which protect neonates against HEV infection. Our results suggest that, besides vaccinating nonpregnant individuals, HEV 239 vaccine may also be discreetly considered for maternal vaccination. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the virus causing the ongoing global coronavirus disease 2019 (COVID‐19) pandemic, is believed to be transmitted primarily through respiratory droplets and aerosols. However, reports are increasing regarding the contamination of environmental surfaces, shared objects, and cold‐chain foods with SARS‐CoV‐2 RNA and the possibility of environmental fomite transmission of the virus raises much concern and debate. This study summarizes the current knowledge regarding potential mechanisms of environmental transmission of SARS‐CoV‐2, including the prevalence of surface contamination in various settings, the viability and stability of the virus on surfaces or fomites, as well as environmental factors affecting virus viability and survival such as temperature and relative humidity. Instances of fomite transmission, including cold‐chain food transmission, and the importance of fomite transmission in epidemics, are discussed. The knowledge gaps regarding fomite transmission of SARS‐CoV‐2 are also briefly analyzed. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor receptor 2 (VEGFR2)/KDR plays a critical role in tumor growth, diffusion, and invasion. The amino acid sequence homology of KDR between mouse and human in the VEGF ligand‐binding domain was low, thus the WT mice could not be used to evaluate Abs against human KDR, and the lack of a suitable mouse model hindered both basic research and drug developments. Using the CRISPR/Cas9 technique, we successfully inserted different fragments of the human KDR coding sequence into the chromosomal mouse Kdr exon 4 locus to obtain an hKDR humanized mouse that can be used to evaluate the marketed Ab ramucirumab. In addition, the humanized mAb VEGFR‐HK19 was developed, and a series of comparative assays with ramucirumab as the benchmark revealed that VEGFR‐HK19 has higher affinity and superior antiproliferation activity. Moreover, VEGFR‐HK19 selectively inhibited tumor growth in the hKDR mouse model but not in WT mice. The most important binding epitopes of VEGFR2‐HK19 are D257, L313, and T315, located in the VEGF binding region. Therefore, the VEGFR2‐HK19 Ab inhibits tumor growth by blocking VEGF‐induced angiogenesis, inflammation, and promoting apoptosis. To our best knowledge, this novel humanized KDR mouse fills the gaps both in an animal model and the suitable in vivo evaluation method for developing antiangiogenesis therapies in the future, and the newly established humanized Ab is expected to be a drug candidate possibly benefitting tumor patients. [ABSTRACT FROM AUTHOR]

Human coronaviruses (HCoVs) were first described in 1960s for patients experiencing common cold. Since then, increasing number of HCoVs have been discovered, including those causing severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the circulating coronavirus disease 2019 (COVID‐19), which can cause fatal respiratory disease in humans on infection. HCoVs are believed to spread mainly through respiratory droplets and close contact. However, studies have shown that a large proportion of patients with HCoV infection develop gastrointestinal (GI) symptoms, and many patients with confirmed HCoV infection have shown detectable viral RNA in their faecal samples. Furthermore, multiple in vitro and in vivo animal studies have provided direct evidence of intestinal HCoV infection. These data highlight the nature of HCoV GI infection and its potential faecal‐oral transmission. Here, we summarise the current findings on GI manifestations of HCoVs. We also discuss how HCoV GI infection might occur and the current evidence to establish the occurrence of faecal‐oral transmission. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants, particularly those with multiple mutations in receptor‐binding domain (RBD), pose a critical challenge to the efficacy of coronavirus disease 2019 (COVID‐19) vaccines and therapeutic neutralizing monoclonal antibodies (mAbs). Omicron sublineages BA.1, BA.2, BA.3, as well as the recent emergence of C.1.2, B.1.630, B.1.640.1, and B.1.640.2, have multiple mutations in RBD and may lead to severe neutralizing antibody evasion. It is urgent to evaluate the antigenic change of the above seven variants against mAbs and sera from guinea pigs immunized with variants of concern (VOCs) (Alpha, Beta, Gamma, Delta, Omicron) and variants of interest (VOIs) (Lambda, Mu) immunogens. Only seven out of the 24 mAbs showed no reduction in neutralizing activity against BA.1, BA.2, and BA.3. However, among these seven mAbs, the neutralization activity of XGv337 and XGv338 against C.1.2, B.1.630, B.1.640.1, and B.1.640.2 were decreased. Therefore, only five neutralizing mAbs showed no significant change against these seven variants. Using VOCs and VOIs as immunogens, we found that the antigenicity of variants could be divided into three clusters, and each cluster showed similar antigenicity to different immunogens. Among them, D614G, B.1.640.1, and B.1.630 formed a cluster, C.1.2 and B.1.640.2 formed a cluster, and BA.1, BA.2, and BA.3 formed a cluster. [ABSTRACT FROM AUTHOR]

Variants of SARS‐CoV‐2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high‐frequency naturally occurring amino acid changes in the receptor‐binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2‐overexpressing 293T cells of different species, LLC‐MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high‐frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high‐frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies. Highlights: Infectivity increased by adding three VOCs of V367F, S494P, or A520S.The infectivity of the three VOCs with multiple high‐frequency mutations decreased.Almost all of the possible variants of the three VOCs did not show severe antigenic drift. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection and the associated COVID‐19 diseases are an emerging threat to global public health. Although considerable scientific research on the immune, especially antibody, responses to SARS‐CoV‐2 infection have been conducted, additional dominant epitopes and protective antibodies are needed for diagnosis and treatment of COVID‐19 patients. Here, two different phage libraries are used to identify immunogenic epitopes across the spike protein and monoclonal antibodies from COVID‐19 patients. Three peptides are further characterized in the receptor‐binding motif (RBM) and measured their antibody levels in COVID‐19 patients, from which one identifies one most immunodominant epitope with the highest antibody response in COVID‐19 patients and in immunized mice. More importantly, monoclonal antibodies specifically binding to this peptide isolated from COVID‐19 patients have therapeutic potential to neutralize SARS‐CoV‐2 infection. Thus, the approaches to systemically identify immunogenic peptides and directly identify human monoclonal antibodies from patients will provide useful diagnostic and therapeutic tools for COVID‐19 and other emerging infectious diseases. [ABSTRACT FROM AUTHOR]

The development of therapies for human smallpox is needed due to the increasing concern over the potential use of smallpox virus as a biological weapon. Here, we report a high‐throughput screening for anti‐smallpox virus drugs from a 767‐small‐molecule library, employing two vaccinia virus (VACV) strains containing firefly luciferase (VTT‐Fluc and VG9‐Fluc) as surrogate viruses. Using an eight‐point dose response format assay, 26 compounds of different pharmacological classes were identified with in vitro anti‐VACV activities. Mycophenolate mofetil (MMF) and tranilast (TRA) were detected to possess the highest anti‐VACV potency (selectivity index values of >334 and >74, respectively); they could inhibit VTT‐Fluc replication in nude mice at 5 days post‐infection by 99% (10 mg/kg, P < .01) and 59% (45 mg/kg, P = .01), respectively, as indicated by bioluminescent intensity. In conclusion, MMF and TRA are promising anti‐smallpox virus candidates for further optimization and repurposing for use in clinical practice. Highlight: 26 compounds were identified with in vitro anti‐vaccinia virus potency.Both MMF and TRA exhibited high anti‐vaccinia virus potency in a nude mouse model.MMF and TRA took effects by blocking the fusion and intracellular biosynthesis. [ABSTRACT FROM AUTHOR]

Nucleotide-based methods are conventionally used to classify the hepatitis E virus (HEV) genotypes. A serological enzyme immunoassay (EIA) using open reading frame 3 (ORF3) C-terminal peptides was developed to conveniently and accurately classify and evaluate the genotypes of HEV. The sera of mice immunized with HEV genotype 1, 3, and 4 reacted highly specifically to the peptides of the corresponding genotypes. Most (84.2%) clinical sera infected with HEV genotype 4 were positive for anti-HEV antibodies when tested with the ORF3 peptides of genotype 4, but were negative for genotypes 1 and 3. Monkey and clinical serial sera infected with HEV reacted strongly to the homologous genotype ORF3 peptides. The indirect EIAs were more sensitive, with stronger reactivity, than commercial anti-HEV immunoglobulin G assays when serial sera from monkeys infected with HEV genotype 1 or 4 were tested. All our results indicate that the serological typing EIA assays described in this study are more effective and convenient for the classification of HEV genotypes than molecular approaches, and can be used to screen large numbers of serum samples and differentiate genotypes for the diagnosis of HEV infections. [ABSTRACT FROM AUTHOR]

Summary: Emerging and reemerging infectious diseases have a strong negative impact on public health. However, because many of these pathogens must be handled in biosafety level, 3 or 4 containment laboratories, research and development of antivirals or vaccines against these diseases are often impeded. Alternative approaches to address this issue have been vigorously pursued, particularly the use of pseudoviruses in place of wild‐type viruses. As pseudoviruses have been deprived of certain gene sequences of the virulent virus, they can be handled in biosafety level 2 laboratories. Importantly, the envelopes of these viral particles may have similar conformational structures to those of the wild‐type viruses, making it feasible to conduct mechanistic investigation on viral entry and to evaluate potential neutralizing antibodies. However, a variety of challenging issues remain, including the production of a sufficient pseudovirus yield and the inability to produce an appropriate pseudotype of certain viruses. This review discusses current progress in the development of pseudoviruses and dissects the factors that contribute to low viral yields. [ABSTRACT FROM AUTHOR]

Although the biological and epidemiological features of hepatitis E virus (HEV) have been studied extensively in recent years, the mechanism by which HEV infects cells is still poorly understood. In this study, coimmunoprecipitation, pull-down, and ELISA were used to show that the HEV ORF2 protein interacts directly with the ectodomain of both ASGR1 and ASGR2. Susceptibility to HEV correlated positively with the expression level of surface asialoglycoprotein receptor (ASGPR) in cell lines. ASGPR-directed small interfering RNA (siRNA) in HEV-infected PLC/PRF/5 cells had no significant effect on HEV release, suggesting that ASGPR mainly regulates the viral binding and entry steps. Both the purified ASGPR ectodomain and anti-ASGPR antibodies disturbed the binding of HEV to PLC/PRF/5 cells. The classic ASGPR ligands asialofetuin, asialoganglioside, and fibronectin competitively inhibited the binding of HEV to hepatocytes in the presence of calcium. HeLa cell lines stably expressing ASGPR displayed increased HEV-binding capacity, whereas ASGPR-knockout PLC/PRF/5 cell lines had lower HEV-binding capacity. Thus, our study demonstrates that ASGPR is involved in and facilitates HEV infection by binding to ORF2. J. Med. Virol. 88:2186-2195, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

To investigate the multi-infection patterns and type competition for human papillomavirus (HPV) in Chinese clinic attending women and evaluate the association between the infection status and cancer risk. Three hundred and thirty-two HPV-DNA-positive samples were genotyped for 21 HPVs and tested for 13 types of HPV neutralizing antibody using pseudoviron-based assay. Odds ratios (ORs) were calculated to evaluate the coinfection patterns for both DNA and neutralizing antibody (NAb), and the associations of HSIL+ with HPV DNA and NAb. Of the 332 HPV-DNA-positive subjects, 279 (84.0%) were detected as NAb positive. Multi-positive results were identified in 23.2% (77/332) HPV DNA tests and 60.2% (168/279) HPV NAb assays. The NAb titers for the multiple-positive samples (geometric mean 214) were significantly higher than the single-positive samples (geometric mean 114) ( P < 0.01). HPV16-HPV52 was identified as a type-competition pair for both HPV DNA (OR = 0.09; 95%CI = 0.02-0.42) and NAb (OR = 0.27; 95%CI = 0.11-0.69) data. Compared to other types, HPV16 DNA was associated significantly with high risk of HSIL+ (OR = 2.57; 95%CI = 1.23-5.38). HPV16-HPV52 was identified as a potential type-competition pair, which might result in the type modulation with the implementation of the approved bi- or quadri-valent vaccines. J. Med. Virol. 88:1989-1998, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The hepatitis E virus (HEV) capsid antigen expressed in insect cell has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. However, the expression and purification of HEV virus-like particles (VLPs) from insect cells have not been explored. We aimed to optimize the procedure to obtain HEV VLPs. In this study, two conformations of the HEV capsid proteins were expressed in insect cells, VLPs and non-VLPs, and they were purified separately. The physicochemical properties and the humoral immune responses induced by the two forms were analyzed and compared. We found that HEV VLPs were more immunogenic in mice than HEV non-VLPs. Therefore, we optimized the conditions that yielded high VLPs expression in insect cell cultures and developed an efficient purification method. The results suggest that the distinction and isolation of VLPs from non-VLPs are essential to generate a more immunogenic vaccine. [ABSTRACT FROM AUTHOR]

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 × 106 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. J. Med. Virol. 86: 1736-1744, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The pseudoviron-based neutralization assay is accepted as the gold standard to evaluate the functional humoral immune response against HPV. The goal of this study was to develop and optimize a human papillomavirus (HPV) neutralization assay using HPV pseudovirons with Gaussia luciferase (Gluc) as the reporter gene. For this purpose, high-titers Gluc pseudovirons were generated by cotransfecting 293TT cells with HPV structural genes and Gluc expressing plasmids. Six types of neutralizing monoclonal antibodies, vaccines immunized serum samples and WHO international antibody standard were used to validate the new developed assay. The ideal circumstances of the assay were identified for cell counts (30,000/well for 96-well plate), pseudoviron inoculating size (100 times RLU above background) and incubation time (72 hr). The sensitivity of the Gluc assay was comparable to secreted alkaline phosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay. The non-specific background for different types of sample was significantly different (rabbit sera>human sera>mouse sera, P<0.01). The non-specific neutralization effects were not attributed to IgG antibody. The cutoff value for this assay was determined as 50% inhibition at a dilution of 1:40. Without requirements of sample dilution and different incubation times at different temperature before processing, the detection time was shortened from more than 90 min to less than 5 min for a 96-well plate compared with the SEAP-based assay. With the advantages of short detection time and easy-to-use procedure, the newly developed assay is more suitable for large sero-epidemiological studies or clinical trials and more amenable to automation. [ABSTRACT FROM AUTHOR]

Background The risk of hepatitis E virus ( HEV) infection from blood transfusion has aroused increasing concern in many countries. The aim of this study was to analyze the potential risk of HEV infection through blood transfusion in China. Study Design and Methods Qualified blood donations and donations with isolated alanine aminotransferase ( ALT) elevations from five geographically diverse Chinese regions were tested for anti- HEV immunoglobulin ( Ig) M and IgG and HEV antigen. The positive samples for anti- HEV IgM and HEV antigen were tested for HEV RNA. HEV open reading frame ( ORF)2 partial sequences were analyzed from HEV RNA-positive samples. Results The seroprevalence rates of HEV antigen and anti- HEV IgM and IgG among qualified donations were 0.06% (6/10,741), 1.02% (109/10,741), and 27.42% (2945/10,741), respectively. Samples with isolated ALT elevations had higher prevalence of HEV markers, namely, HEV antigen of 0.25% (2/797), anti- HEV IgM of 2.76% (22/797), and anti- HEV IgG of 40.02% (319/797). The HEV antibody prevalence varied significantly by age, sex, and geographic region. All 131 samples that were anti- HEV IgM positive were negative for HEV RNA, whereas four of eight (50%) samples positive for HEV antigen were HEV RNA positive. HEV ORF2 sequences from three of four HEV RNA-positive samples were determined and grouped with Genotype 4. Conclusion Qualified donations after routine blood donor screening still carry potential risk for transmitting HEV. HEV antigen screening could be one measure to reduce the risk of HEV transmission by blood transfusion. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is a hepatotropic virus that causes acute and, occasionally, chronic viral hepatitis. At least four recognized genotypes of mammalian HEV have been identified. Genotypes 3 and 4 are zoonotic in humans and pigs are the major host. The 7.2 kb genome of HEV contains three open reading frames: ORF1, ORF2, and ORF3. ORF1 encodes a nonstructural protein and the ORF2 protein is the main capsid protein but the precise functions of ORF3 protein remain obscure. To explore the role of ORF3 in the porcine host, the genotype 4 ORF3 protein was used for yeast two-hybrid screening to find cellular binding partners encoded by a pig liver cDNA library, two porcine liver specific proteins, plasminogen and α2-antiplasmin were identified. Their interactions with the ORF3 protein were validated by chemiluminescent Co-Immunoprecipitation assay and by Western blotting in Co-IP and His pull-down assay. The biological significance of these interactions and their possible role in the HEV infection are discussed. J. Med. Virol. 86:487-495, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Front Cover Caption: The cover image is based on the Research Article Aggregation of high‐frequency RBD mutations of SARS‐CoV‐2 with three VOCs did not cause significant antigenic drift by Tao Li et al., https://doi.org/10.1002/jmv.27596. [ABSTRACT FROM AUTHOR]

ABSTRACT Recombinant adeno-associated virus serotype 1 (AAV1) has attracted tremendous interest as a promising vector for gene therapy and vaccine applications. However, the presence of AAV1 neutralizing antibodies as a consequence of exposure to wild type AAV1 can limit significantly effective gene transfer for biologics based AAV1 vector. Prior studies have reported that a prevalence of AAV1 neutralizing antibodies ranged from 10% to 50% in different countries around the world, and up to 79% in Dutch subjects. However, few studies have reported on the AAV1 neutralizing antibody prevalence in Chinese subjects. In this study, a high-throughput luciferase-based virus neutralization assay was established and standardized for critical parameters, including the appropriate cell line, and the optimal viral infection dose, and the infection time with homologous AAV1 vaccinated mice and guinea pig sera. Then, a total of 500 healthy individual serum samples from two separate regions of China were screened for the AAV1 neutralizing antibodies by conducting a non-randomized, cross-sectional analysis. Interestingly, a high prevalence of AAV1 neutralizing antibody (69.8%) was found in all individuals. There was significant difference observed for prevalence by gender ( P = 0.042), age range ( P = 0.011) and geographic origin ( P < 0.001). The percentage of positive AAV1 neutralizing antibodies (NT50 > 10) in teenagers (year <18, as of 2012) was significant lower than that of adults (19-56, as of 2012) ( P = 0.011), indicating the optimal vaccination period of childhood. The current study provides a useful insight for the future development of AAV1-based vaccination and gene therapy strategies in Beijing and Anhui provinces of China. J. Med. Virol. 85:1550-1556, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein product remains unclear but it is able to induce a strong antibody response following HEV infection. Therefore, it has been used in some enzyme immunoassays (EIAs) for detecting anti-HEV antibody. In order to evaluate the difference in antigenicity of HEV ORF3 polypeptides derived from genotypes 1 and 4, two EIAs were developed, based on ORF3 polypeptides from genotypes 1 and 4 HEV. Serial weekly serum samples from two rhesus monkeys vaccinated with ORF3 antigens derived from the genotype 4 ORF3 protein and nine rhesus monkeys experimentally infected with genotypes 1 and 4 HEV were tested for anti-HEV using the assays. HEV ORF3 antigens derived from viruses of genotypes 1 and 4 showed different patterns of reactivity with sera obtained from monkeys immunized with ORF3 antigens or infected experimentally with HEV. The genotype 1 ORF3 polypeptide exhibited stronger reactivity with the sera from monkeys infected with genotype 1 than the genotype 4 ORF3 polypeptide. The genotype 4 ORF3 polypeptide demonstrated stronger reactivity with the sera from monkeys infected with genotype 4 than did the genotype 1 ORF3 polypeptide. The HEV ORF3 polypeptide contains genotype-specific antigens and the antigen-antibody reactions between the same genotypes were stronger than those between different genotypes. J. Med. Virol. 83:827-832, 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. J. Med. Virol. 81:1371-1379, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Following infection with hepatitis E virus (HEV), anti-HEV immunoglobulin (Ig) M is thought to develop before anti-HEV IgG and to be a better marker for differentiating between the acute and convalescent phases of infection. In order to select polypeptides for improved detection of anti-HEV IgM, six and three overlapping polypeptides from open reading frames (ORFs) 2 and 3, respectively, of HEV genotypes 1 and 4 were expressed as fusion proteins in Escherichia coli. The reactivities of the polypeptides with anti-HEV IgM were evaluated using immunoblotting and enzyme immunoassays (EIAs). The data indicated that polypeptides from the N-terminus of ORF3 and middle region of ORF2 were weakly or not reactive with anti-HEV IgM, while those from the remaining regions of ORF2 and ORF3 contained reactive epitopes. Anti-HEV IgM against the N- or C-terminus of ORF2 appeared earlier and disappeared faster than that against polypeptides from the C-terminus of ORF3, based on serum samples from rhesus monkeys infected experimentally, and from patients infected naturally, with HEV. The N- and C-terminal polypeptides from ORF2 complemented one another in detecting anti-HEV IgM and EIA sensitivity was improved significantly with a combination of these polypeptides. The reactivities of ORF2 polypeptides from genotypes 1 and 4 were similar but that of ORF3 differed with sera from monkeys infected by the two genotypes. Thus, a combination of N- and C-terminal polypeptides of ORF2 from one genotype may be effective in EIAs to detect anti-HEV IgM. J. Med. Virol. 81:1052-1061, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

A total of 82 samples from patients with cervical cancer (Group 1) and 50 samples from patients with other genital diseases (Group 2) were collected in Gansu, China. All 132 samples were tested for HPV DNA with a typing kit that can detect 21 types of HPV, and also tested for neutralizing antibodies against HPV-16, -18, -58, -45, -6, and -11 using pseudovirus-based neutralization assays. The results revealed that 28% (23/82) of sera in Group 1 were positive for type-specific neutralizing antibodies with a titer range of 160-640, of which 23.2% (19/82), 2.4% (2/82), 2.4% (2/82), 1.2% (1/82), and 1.2% (1/82) were against HPV-16, -58, -6, -18, and -45, respectively. Only one serum (2%) in Group 2 was positive for neutralizing antibodies, which were against HPV-6 with a titer of 2,560. Overall, 85.4% (70/82) of samples in Group 1 were HPV DNA-positive, compared with 28% (14/50) of samples in Group 2. The seven most common types detected in Group 1 were HPV-16 (80%), HPV-52 (7.1%), HPV-66 and HPV-11 (5.7% each), and HPV-58, HPV-18, and HPV-33 (4.3% each), while the four most common types in Group 2 were HPV-16 (12%), HPV-52 and HPV-11 (6% each), and HPV-68 (4%). The concordance between HPV DNA and corresponding neutralizing antibodies was 32.9% (27/82) with a significant difference ( P < 0.005). More specifically, the concordance was 42.7% (35/82) for HPV-16 in Group 1. The full-length sequences of six HPV types (HPV-16, -58, -33, -59, -11, and -68) were determined and showed 99% identities with their reported genomes. J. Med. Virol. 81:693-702, 2009 © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Nine hundred and four serum samples were collected from pigs from 16 pig farms in China's Hunan province and tested for anti-hepatitis E virus (HEV) antibody and the HEV capsid antigen using EIAs. Of the 904 samples, 617 (68.3%) and 57 (6.3%) were positive for anti-HEV antibody and antigen, respectively. The prevalence of HEV antibodies and detection of antigen varied significantly among pigs from different farms ( P < 0.01). The positivity rate for anti-HEV antibody and occurrence of high titer antibody were significantly higher in pigs above, than in those below, 3 months of age, whilst the detection of antigen did not differ significantly between the two groups. Based on these data, 481 serum samples that were positive for HEV antigen or with an S/CO ≤10 for anti-HEV were tested for HEV RNA using real-time RT-PCR; 28 of 481 (5.8%) were positive. The positivity rate of HEV RNA was much higher for the HEV antigen positive sera (40.1%) than the anti-HEV antibody positive (1.4%) or negative (1.1%), antigen negative samples. Sixteen of the 28 samples were positive for HEV RNA using nested RT-PCR and their products were cloned and sequenced. These 16 isolates belonged to HEV genotype 4, including 12 that did not belong to any subtype of HEV genotype 4 reported previously. Thus, the infection rate of HEV in pigs is high and the HEV antigen has a close relationship with HEV RNA. The HEV genotype infecting the pigs was genotype 4 and a novel subtype may exist in Hunan province. J. Med. Virol. 80:1391-1396, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine cross-protection between HEV genotypes 1 and 4, which are prevalent in China. Fecal suspensions of genotypes 1 and 4 from patients, as well as genotype 4 from swine, were inoculated intravenously into rhesus macaques. Each inoculum contained 5 × 104 genome equivalents of HEV. After infection, serum and fecal samples were collected serially and the levels of alanine aminotransferase (ALT) and anti-HEV IgG and IgM in sera, and HEV RNA in fecal samples, were measured. Liver biopsies were carried out. All the infected monkeys (12/12) developed anti-HEV IgG and exhibited fecal shedding of virus. IgM was detected in 11 of 12, and ALT elevation occurred about 2-6 weeks post-inoculation in 10 of 12, infected monkeys. Hepatic histopathology was consistent with acute viral hepatitis and the ORF2 antigen of HEV was detected in the granular cytoplasm of hepatocytes by immunohistochemistry. After recovery from their initial HEV infection, the monkeys were challenged with a heterologous genotype or heterologous source of HEV and monitored for hepatitis and fecal shedding. Previous infection with HEV completely or partially protected against subsequent challenge with a heterologous virus, because 7 of 11 monkeys did not develop HEV infection or shed virus in the feces, and none of them developed hepatitis or exhibited ALT elevation or liver biopsy findings of hepatitis. In conclusion, previous HEV infection may give rise to cross-genotype and cross-host-species protection. J. Med. Virol. 80:824-832, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In this study, 190 HIV-positive samples were collected from different regions of China. The HIV clades of 153 samples were determined successfully based on env sequencing. Specifically, 48, 5, 87, and 13 isolates belonged to clades B′, B, BC, and AE, respectively. The viral loads in all samples were measured using three commercial assays, Amplicor HIV-1 monitor v1.5, Nuclisens HIV-1 QT and NucliSens EasyQ HIV-1 assays. The differences and linear correlations for individual assays were compared, with expected 1:1 relationships. Significant differences were found for the following viral loads: clade BC measured by any two assays ( P < 0.001); clade AE between Amplicor 1.5 and Easy Q ( P = 0.005); clade B′ between Amplicor 1.5 and Nuclisens QT ( P = 0.002); clade AE between Amplicor 1.5 and Nuclisens QT ( P = 0.025); and clade B′ between Amplicor 1.5 and EasyQ ( P = 0.04). The largest mean difference in the log10 values was 0.9518, which was found between Amplicor 1.5 and Nuclisens QT. However, the viral loads for clades AE and B′ measured by EasyQ and Nuclisens QT, and those for clade B measured by any two assays did not differ significantly. The degrees of correlation for clades B and B′ between any two assays (R > 0.8) were higher that those for clades AE and BC between any two assays (R < 0.7), except for clade AE between Amplicor 1.5 and Easy Q. Thus, the clade types, especially clades BC and AE, are most likely to impact on the quantitation of viral load using differentassays. J. Med. Virol. 80:1-8, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Pressure‐induced electronic structure transition from insulating phase to metal state is a potential new paradigm for halide perovskites. The metallization based on these materials may afford a novel motif toward realizing new electronic properties even superconductivity phenomenon. Herein, how static compression modulates the crystal and electronic structure of typical perovskite semiconductors cesium lead iodine (CsPbI3) by both experimental and theoretical studies is reported. The comprehensive studies discover the insulator–metal transition of CsPbI3 at 39.3 GPa, and reveal the key information behind the electronic transition. The perovskite's precise structural evolution is tracked upon compression, from orthorhombic Pnma phase to monoclinic C2/m structure before the metallic transition. More interestingly, the C2/m phase has the most distorted octahedra and the shortest Pb–I bond length relative to the average bond length that is ever reported in a halide perovskite structure. The electronic transition stems from the structural changes accompanied by the anomalously self‐distorted octahedra. These studies show that pressure can significantly alter the structural and electronic properties of these technologically important perovskites. [ABSTRACT FROM AUTHOR]