A Broad-specificity Neutralizing Nanobody against Hepatitis E Virus Capsid Protein.

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.

A nanobody inhibiting porcine reproductive and respiratory syndrome virus replication via blocking self-interaction of viral nucleocapsid protein.

Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.

A novel strategy for an anti-idiotype vaccine: nanobody mimicking neutralization epitope of porcine circovirus type 2.

Vaccination is the most effective method to protect humans and animals from diseases. Anti-idiotype vaccines are safer due to their absence of pathogens. However, the commercial production of traditional anti-idiotype vaccines using monoclonal and polyclonal antibodies (mAb and pAb) is complex and has a high failure rate. The present study designed a novel, simple, low-cost strategy for developing anti-idiotype vaccines with nanobody technology. We used porcine circovirus type 2 (PCV2) as a viral model, which can result in serious economic loss in the pig industry. The neutralizing mAb-1E7 (Ab1) against PCV2 capsid protein (PCV2-Cap) was immunized in the camel. And 12 nanobodies against mAb-1E7 were screened. Among them, Nb61 (Ab2) targeted the idiotype epitope of mAb-1E7 and blocked mAb-1E7's binding to PCV2-Cap. Additionally, a high-dose Nb61 vaccination can also protect mice and pigs from PCV2 infection. Epitope mapping showed that mAb-1E7 recognized the 75NINDFL80 of PCV2-Cap and 101NYNDFLG107 of Nb61. Subsequently, the mAb-3G4 (Ab3) against Nb61 was produced and can neutralize PCV2 infection in the PK-15 cells. Structure analysis showed that the amino acids of mAb-1E7 and mAb-3G4 respective binding to PCV2-Cap and Nb61 were also similar on the amino acids sequences and spatial conformation. Collectively, our study first provided a strategy for producing nanobody-based anti-idiotype vaccines and identified that anti-idiotype nanobodies could mimic the antigen on amino acids and structures. Importantly, as more and more neutralization mAbs against different pathogens are prepared, anti-idiotype nanobody vaccines can be easily produced against the disease with our strategy, especially for dangerous pathogens.IMPORTANCEAnti-idiotype vaccines utilize idiotype-anti-idiotype network theory, eliminating the need for external antigens as vaccine candidates. Especially for dangerous pathogens, they were safer because they did not contact the live pathogenic microorganisms. However, developing anti-idiotype vaccines with traditional monoclonal and polyclonal antibodies is complex and has a high failure rate. We present a novel, universal, simple, low-cost strategy for producing anti-idiotype vaccines with nanobody technology. Using a neutralization antibody against PCV2-Cap, a nanobody (Ab2) was successfully produced and could mimic the neutralizing epitope of PCV2-Cap. The nanobody can induce protective immune responses against PCV2 infection in mice and pigs. It highlighted that the anti-idiotype vaccine using nanobody has a very good application in the future, especially for dangerous pathogens.

Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release. IMPORTANCE: Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.

GUGA-based MRCI approach with core-valence separation approximation (CVS) for the calculation of the core-excited states of molecules.

We develop and demonstrate how to use the Graphical Unitary Group Approach (GUGA)-based MRCISD with Core-Valence Separation (CVS) approximation to compute the core-excited states. First, perform a normal Self-Consistent-Field (SCF) or valence MCSCF calculation to optimize the molecular orbitals. Second, rotate the optimized target core orbitals and append to the active space, form an extended CVS active space, and perform a CVS-MCSCF calculation for core-excited states. Finally, construct the CVS-MRCISD expansion space and perform a CVS-MRCISD calculation to optimize the CI coefficients based on the variational method. The CVS approximation with GUGA-based methods can be implemented by flexible truncation of the Distinct Row Table. Eliminating the valence-excited configurations from the CVS-MRCISD expansion space can prevent variational collapse in the Davidson iteration diagonalization. The accuracy of the CVS-MRCISD scheme was investigated for excitation energies and compared with that of the CVS-MCSCF and CVS-CASPT2 methods using the same active space. The results show that CVS-MRCISD is capable of reproducing well-matched vertical core excitation energies that are consistent with experiments by combining large basis sets and a rational reference space. The calculation results also highlight the fact that the dynamic correlation between electrons makes an undeniable contribution in core-excited states.

A new nanobody-enzyme fusion protein-linked immunoassay for detecting antibodies against influenza A virus in different species.

Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAV infection in different species. Recently, nanobodies, which show advantages of easy gene editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nanobodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can crossreact with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen and dilutions of the fusion and testing sera were 100 ng/well, 1:4000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. In addition, we found that the cELISA and hemagglutination inhibition test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species.

Identification and pathogenicity of hepatitis E Virus from laboratory Bama miniature pigs.

BACKGROUND: Hepatitis E virus (HEV) genotypes 3 and 4 are zoonotic. In this study, HEV infection in laboratory Bama miniature pigs in Sichuan Province of China was investigated. Firstly, one hundred rectal swabs were collected for HEV RNA testing, and chose positive samples for sequence analysis. Concurrently, for pathogenicity study, six healthy Bama miniature pigs were randomly divided into two groups of 3 pigs each. A total of 500 µL of HEV stock (positive fecal samples identified in this study) was inoculated intravenously into each pig in the experimental group, and the three pigs in the other group served as negative controls. Serum and fecal samples were collected at 1 to 10 weeks post-inoculation (wpi) for alanine aminotransferase (ALT) levels, anti-HEV antibodies and HEV RNA detection, respectively. During necropsies, liver lesions and HEV antigen in liver were observed at 10 wpi. RESULTS: The rate of fecal sample HEV RNA-positivity was 12% (12/100). Sequence comparisons indicated that partial ORF1 and ORF2 gene sequences of this isolate shared highest identities with corresponding sequences of genotype 4a HEV isolates (81.4%-96.1% and 89.9%-97.1%, respectively). Phylogenetic tree analysis further demonstrated that sequences of this isolate clustered together with sub-genotype 4a HEV isolate sequences. Experimentally, the pathogenicity of Bama miniature pigs infected with this isolate exhibited viremia, fecal virus shedding, seroconversion, ALT level increasing, liver lesions and HEV antigen in liver. CONCLUSIONS: This is the first study to confirm that HEV is currently circulating in laboratory Bama miniature pigs in China and this isolate can successfully infect Bama miniature pigs experimentally. More importantly, this study suggested HEV screening of laboratory pigs should be conducted to prevent research personnel from acquiring zoonotic HEV infections.

Development of a Nanobody-Based Competitive Enzyme-Linked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes considerable economic loss to the global pig industry. Efficient detection assay is very important for the prevention of the virus infection. Nanobodies are the advantages of small molecular weight, simple genetic engineering, and low production cost for promising diagnostic application. In this study, to develop a nanobody-based competitive ELISA (cELISA) for specifically detecting antibodies against PRRSV, three nanobodies against PRRSV-N protein were screened by camel immunization, library construction, and phage display. Subsequently, a recombinant HEK293S cell line stably secreting nanobody-horseradish peroxidase (HRP) fusion protein against PRRSV-N protein was successfully constructed using the lentivirus transduction assay. Using the cell lines, the fusion protein was easily produced. Then, a novel cELISA was developed using the nanobody-HRP fusion protein for detecting antibodies against PRRSV in pig sera, exhibiting a cut-off value of 23.19% and good sensitivity, specificity, and reproducibility. Importantly, the cELISA specifically detect anti-genotype 2 PRRSV antibodies. The cELISA showed more sensitive than the commercial IDEXX ELISA kit by detecting the sequential sera from the challenged pigs. The compliance rate of cELISA with the commercial IDEXX ELISA kit was 96.4%. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera. Collectively, the developed nanobody-based cELISA showed advantages of simple operation and low production cost and can be as an assay for epidemiological investigation of genotype 2 PRRSV infection in pigs and evaluation after vaccination.

Development of a competitive ELISA for detecting antibodies against genotype 1 hepatitis E virus.

Hepatitis E, a significant global public health issue in China, is caused by sporadic infections with regional hepatitis E virus (HEV) genotypes 1, 3, and 4. To date, most immunoassays currently used to test human sera for the presence of anti-HEV antibodies cannot identify HEV at the genotype level. However, such information would be useful for identifying the source of infecting virus. Therefore, here we describe the development of a competitive enzyme-linked immunosorbent assay (ELISA) for detecting anti-genotype 1 HEV antibodies in human sera. Using recombinant genotype 1 HEV ORF3 protein as immunogen, traditional hybridoma technology was employed to generate seven monoclonal antibodies (mAbs), of which two mAbs specifically reacted with the immunogen. One of these two mAbs, 1D2, was labeled with horseradish peroxidase (HRP) for use in competitive ELISA (cELISA). After cELISA optimization using a checkerboard assay design, the amount of ORF3SAR-55 as coating antigen (100 ng/well), HRP-1D2 mAb concentration (1 µg/mL), and test serum dilution (1:10) were selected and a result ≥ 19.5 was used as the cutoff for a positive result. Importantly, cross-genotype cELISA results indicated that the cELISA could not detect anti-genotype 3 rabbit and 4 swine HEV antibodies. Moreover, human sera confirmed as negative for anti-HEV antibodies using the commercial ELISA kit were all negative via cELISA. However, because the commercial ELISA kit detects anti-all genotypes HEV antibodies and the cELISA only detects anti-genotype 1 HEV antibodies, the consistence rate of two assays detecting positive sera is low. In summary, here a cELISA for detecting anti-genotype 1 HEV antibodies was developed for use in epidemiological investigations of genotype 1 HEV infections in humans. KEY POINTS: • Seven mAbs were produced using genotype 1 HEV ORF3 protein as immunogen. • One mAb that specifically bound to genotype 1 HEV ORF3 protein was selected and labeled for use in a cELISA to detect anti-genotype 1 HEV antibodies. • The competitive ELISA developed here will aid clinical diagnosis of HEV infections and will be useful for large-scale serological testing of genotype 1 HEV infections in humans.

A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2.

BACKGROUND: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)­horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. RESULTS: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15­HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti­PCV2 antibodies. The cut­off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. CONCLUSIONS: In brief, the newly developed cELISA based PCV2-Nb15­HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

Development of a double monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus.

Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)-conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 µL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores. KEY POINTS: • Three CDV mAbs that recognized different epitopes and bound to virion were generated. • The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed. • The sandwich ELISA is an ideal method for detecting CDV infections in the field.

Nanobody­horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays.

BACKGROUND: Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV. RESULTS: In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni-NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests. CONCLUSIONS: For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.

Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus.

BACKGROUND: Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. RESULTS: A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. CONCLUSIONS: In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625DFCP628, 458PSRPF462, and 407EPTV410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design.IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied in vitro and in vivo Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.

Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay.

BACKGROUND: Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera. RESULTS: In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R2 = 0.914) was found between the percent competitive inhibition values of cELISA and HI titers. CONCLUSIONS: A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.

Influence of soil and water conservation measures on soil fertility in the Beijing mountain area.

Soil and water conservation (SWC) measures can be adopted to conserve soil and water and improve soil fertility. The degree to which SWC measures improve soil fertility is affected by the type of SWC measure, soil type, climate, etc. The purpose of this study was to study the effect of the main SWC measures implemented in the Beijing mountain area on soil fertility. Six runoff plots, including a fish pit (fallow) (FPF), fish pit (Platycladus orientalis L. Franco) (FPP), narrow terrace (fallow) (NTF), narrow terrace (Juglans regia L.) (NTJ), tree pan (Juglans regia L.) (TPJ), and fallow land (FL), were established to analyze the differences in soil fertility in the Beijing mountain area. Soil samples were collected in 2005 and 2015 from the six runoff plots. Soil particle size; soil total nitrogen (TN), total phosphorous (TP), total potassium (TK), alkali-hydrolysable nitrogen (Ah-N), available P (Av-P), and available K (Av-K); and soil organic matter (SOM) were measured. The soil integrated fertility index (IFI) was calculated. The results showed that the soil nutrient content and IFI significantly decreased from 2005 to 2015 in the FL plot and significantly increased in the five runoff plots with SWC measures. Compared to the other runoff plots with SWC measures, the FPP plot more significantly improved the soil nutrient content and IFI. The TN, Ah-N, Av-K, SOM, and IFI in the FPP plots increased by 98%, 113%, 61%, 69 and 47%, respectively, from 2005 to 2015. The IFI for the FPP, NTJ, and TPJ exceeded the average IFI of the farmland soil in the study region. The results indicated that the combination of engineering practices and vegetative measures effectively improved soil fertility. These results may be helpful for selecting SWC measures, land-use planning and monitoring and assessing soil fertility.

Effect of housing arrangement on fecal-oral transmission of avian hepatitis E virus in chicken flocks.

BACKGROUND: Avian hepatitis E virus (HEV) infection is common in chicken flocks in China, as currently no measures exist to prevent the spread of the disease. In this study, we analyzed the effect of caged versus cage-free housing arrangements on avian HEV transmission. First, 127 serum and 110 clinical fecal samples were collected from 4 chicken flocks including the two arrangements in Shaanxi Province, China and tested for HEV antibodies and/or virus. Concurrently, 36 specific-pathogen-free chickens were divided equally into four experimental living arrangement groups, designated cage-free (Inoculated), caged (Inoculated), cage-free (Negative) and caged (Negative) groups. In caged groups, three cages contained 3 chickens each. Three chickens each from cage-free (Inoculated) and caged (Inoculated) groups (one chicken of each cage) were inoculated by cutaneous ulnar vein with the same dose of avian HEV, respectively. The cage-free (Negative) and caged (Negative) groups served as negative control. Serum and fecal samples were collected at 1 to 7 weeks post-inoculation (wpi) and liver lesions were scored at 7 wpi. RESULTS: The results of serology showed that the avian HEV infection rate (54.10%) of the cage-free chickens was significantly higher than the one (12.12%) for caged chickens (P < 0.05). Also, the rate of detection of avian HEV RNA in the clinical fecal samples was significantly higher in the cage-free (22.80%, 13/57) than caged birds (5.66%, 3/53). Moreover, under experimental conditions, the infected number of uninoculated cage-free chickens (6) was significantly higher than the one for the uninoculated caged birds (2), as evidenced by seroconversion, fecal virus shedding, viremia and gross and microscopic liver lesions. CONCLUSIONS: These results suggest that reduction of contact with feces as seen in the caged arrangement of housing chickens can reduce avian HEV transmission. This study provides insights for prevention and control of avian HEV infection in chicken flocks.

Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs.

UNLABELLED: Antisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure. IMPORTANCE: More and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.

Seroprevalence of avian hepatitis E virus and avian leucosis virus subgroup J in chicken flocks with hepatitis syndrome, China.

BACKGROUND: From 2014 to 2015 in China, many broiler breeder and layer hen flocks exhibited a decrease in egg production and some chickens developed hepatitis syndrome including hepatomegaly, hepatic necrosis and hemorrhage. Avian hepatitis E virus (HEV) and avian leucosis virus subgroup J (ALV-J) both cause decreasing in egg production, hepatomegaly and hepatic hemorrhage in broiler breeder and layer hens. In the study, the seroprevalence of avian HEV and ALV-J in these flocks emerging the disease from Shandong and Shaanxi provinces were investigated. RESULTS: A total of 1995 serum samples were collected from 14 flocks with hepatitis syndrome in Shandong and Shaanxi provinces, China. Antibodies against avian HEV and ALV-J in these serum samples were detected using iELISAs. The seroprevalence of anti-avian HEV antibodies (35.09%) was significantly higher than that of anti-ALV-J antibodies (2.16%) (p = 0.00). Moreover, the 43 serum samples positive for anti-ALV-J antibodies were all also positive for anti-avian HEV antibodies. In a comparison of both provinces, Shandong chickens exhibited a significantly higher seroprevalence of anti-avian HEV antibodies (42.16%) than Shaanxi chickens (26%) (p = 0.00). In addition, the detection of avian HEV RNA and ALV-J cDNA in the liver samples from the flocks of two provinces also showed the same results of the seroprevalence. CONCLUSIONS: In the present study, the results showed that avian HEV infection is widely prevalent and ALV-J infection is endemic in the flocks with hepatitis syndrome from Shandong and Shaanxi provinces of China. These results suggested that avian HEV infection may be the major cause of increased egg drop and hepatitis syndrome observed during the last 2 years in China. These results should be useful to guide development of prevention and control measures to control the diseases within chicken flocks in China.