Hierarchical antibiotic delivery system based on calcium phosphate cement/montmorillonite-gentamicin sulfate with drug release pathways.

Antibiotic-loaded calcium phosphate cement (CPC) has emerged as a promising biomaterial for drug delivery in orthopedics. However, there are problems such as the burst release of antibiotics, low cumulative release ratio, inappropriate release cycle, inferior mechanical strength, and poor anti-collapse properties. In this research, montmorillonite-gentamicin (MMT-GS) was fabricated by solution intercalation method and served as the drug release pathways in CPC to avoid burst release of GS, achieving promoted cumulative release ratios and a release cycle matched the time of inflammatory response. The results indicated that the highest cumulative release ratio and release concentration of GS in CPC/MMT-GS was 94.1 ± 2.8 % and 1183.05 µg/mL, and the release cycle was up to 504 h. In addition, the hierarchical GS delivery system was divided into three stages, and the kinetics followed the Korsmeyer-Peppas model, the zero-order model, and the diffusion-dissolution model, respectively. Meanwhile, the compressive strength of CPC/MMT-GS was up to 51.33 ± 3.62 MPa. Antibacterial results demonstrated that CPC/MMT-GS exhibited excellent in vitro long-lasting antibacterial properties to E. coli and S. aureus. Furthermore, CPC/MMT-GS promoted osteoblast proliferation and exhibited excellent in vivo histocompatibility. Therefore, CPC/MMT-GS has favorable application prospects in the treatment of bone defects with bacterial infections and inflammatory reactions.

Intelligent salivary biosensors for periodontitis: in vitro simulation of oral oxidative stress conditions.

ASTRACT: One of the most common oral diseases affecting millions of people worldwide is periodontitis. Usually, proteins in body fluids are used as biomarkers of diseases. This study focused on hydrogen peroxide, lipopolysaccharide (LPS), and lactic acid as salivary non-protein biomarkers for oxidative stress conditions of periodontitis. Electrochemical analysis of artificial saliva was done using Gamry with increasing hydrogen peroxide, bLPS, and lactic acid concentrations. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were conducted. From EIS data, change in capacitance and CV plot area were calculated for each test condition. Hydrogen peroxide groups had a decrease in CV area and an increase in percentage change in capacitance, lipopolysaccharide groups had a decrease in CV area and a decrease in percentage change in capacitance, and lactic acid groups had an increase of CV area and an increase in percentage change in capacitance with increasing concentrations. These data showed a unique combination of electrochemical properties for the three biomarkers. Scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS) employed to observe the change in the electrode surface and elemental composition data present on the sensor surface also showed a unique trend of elemental weight percentages. Machine learning models using hydrogen peroxide, LPS, and lactic acid electrochemical data were applied for the prediction of risk levels of periodontitis. The detection of hydrogen peroxide, LPS, and lactic acid by electrochemical biosensors indicates the potential to use these molecules as electrochemical biomarkers and use the data for ML-driven prediction tool for the periodontitis risk levels.

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies.

African swine fever virus (ASFV) poses a significant threat to the global swine industry. Currently, there are no effective vaccines or treatments available to combat ASFV infection in pigs. The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig. Recently, a lower virulent ASFV isolate with a deleted EP402R gene (CD2v-deleted) has been reported in China, which further complicates the control of ASFV infection the pig farms. Furthermore, an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain. Therefore, it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections. In this study, two recombinant ASFV-p72 and -CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels. Subsequently, eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened. Following the production of these nanobodies with horse radish peroxidase (HRP) fusion proteins, the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs (cELISAs) to detect anti-ASFV antibodies. The two cELISAs exhibited high sensitivity, good specificity, repeatability, and stability. The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6% and 97.6%, respectively. Collectively, the two cELISA for detecting antibodies against ASFV demonstrated ease of operation, a low cost, and a simple production process. The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV, and they play an important role in monitoring ASFV infections in pig farms.

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.

Early Detection of Fretting Corrosion in Hip Replacement by Acoustic Emission Non-Invasive Technique.

The present study aimed to investigate the feasibility of using acoustic emission (AE) as a detection method for identifying failure mechanisms at the modular junction interface in total hip replacements (THRs) subjected to fretting corrosion. The experimental setup involved simulating fretting corrosion using a Ti6Al4V disc representing the femoral neck and a ZrO2 pin representing the femoral head. Mechanical testing provided insights into the wear and frictional behavior occurring at the modular junction interface. The results revealed that for all three potential conditions, a fretting condition of partial slip was observed. These findings highlight the importance of understanding the mechanical interactions and their influence on the overall performance and longevity of THRs. Electrochemical analysis shed light on the corrosion behavior under different potentiostatic conditions. High potentials in the anodic condition led to increased corrosion and ion transfer due to the breakdown of the passive oxide layer. Conversely, the cathodic potential condition exhibited a regrowth of the passive oxide layer, protecting the Ti6Al4V surface from further corrosion. The mid-range corrosion potential condition showed a dynamic equilibrium between corrosion and passivation processes. These electrochemical insights enhance our understanding of the mechanisms involved in fretting corrosion. The AE data proved to be promising in detecting and monitoring the onset and progression of failure mechanisms. The AE signals exhibited distinctive patterns that correlated with the severity of fretting corrosion. Notably, the hit driven data results, derived from AE signals, demonstrated the ability to differentiate between different levels of fretting conditions. This suggests that AE can serve as a valuable diagnostic tool for early detection and continuous monitoring of implant failure in THRs.

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.

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.

In Vitro Tribocorrosion Evaluation of Carbide-derived Carbon (CDC) for Hip Implants.

Carbide-derived carbon (CDC) was previously proposed as a surface modification method for hip implant applications since it showed excellent tribocorrosion performance under open-circuit potential (OCP) conditions. Nonetheless, a systematic evaluation of CDC's tribocorrosion properties was still missing. Therefore, our objective is to test CDC's tribocorrosion performance under various electrochemical conditions and to identify the synergism between wear and corrosion. Based on the findings, the variations in OCP for CDC (0.626 mV) is smaller than Ti6Al4V (1.91 mV), and CDC showed lower induced current than T6Al4V for all potentials, suggesting CDC is more stable than Ti6Al4V under tribocorrosive conditions. Eventually, the weight loss of Ti6Al4V (50.662±5.19 µg) was found to be significantly higher than that of CDC (4.965±5.19 µg), which agrees with the electrochemical results. In summary, CDC showed better tribocorrosion performance than Ti6Al4V and was determined as an Antagonism regime.

Fretting-corrosion Apparatus with Low Magnitude Micro-motion (≤5 µm): Development and Preliminary Outcome.

Fretting-corrosion is one of the failure processes in many applications, including biomedical implants. For example, the modern design of hip implants with multiple components offers better flexibility and inventory storage. However, it will trigger the fretting at the implant interfaces with a small displacement amplitude (< 5 µm) and usually in a partial slip region. Although many studies have been reported on the fretting, they have high displacement amplitude and are in the gross slip region. It is imperative to have an apparatus to overcome such limitations, specifically for hip implant applications. Therefore, this study describes the development of a fretting-corrosion apparatus with low micro-motion (≤ 5 µm) that can simultaneously monitor the corrosion process. Initial experiments with Ti6Al4V-Ti6Al4V in 0.9% saline, Ti6Al4V-Ti6Al4V in bovine calf serum (BCS), and ZrO2-Ti6Al4V in BCS were conducted to validate the system. As a result, the fretting regime of all groups remained partially slip region throughout the 3600 cycles, and the possible failure mechanisms are proposed in this manuscript.

Survivability of Titanium Implant Materials: In Vitro Simulated Inflammatory and Infectious Environment.

Titanium-based implants utilized in total joint arthroplasties could restore primary musculoskeletal function to patients suffering from osteoarthritis and other conditions. Implants are susceptible to failure stemming from aseptic loosening and infection at the joint site, eventually requiring revision surgery. We hypothesized that there might be a feedback loop by which metal degradation particles and ions released from the implant decrease cell viability and increase immune response, thereby creating biochemical conditions that increase the corrosion rate and release more metal ions. This study focused on the synergistic process through cell viability assays and electrochemical tests. From the results, inflammatory conditions from ion release resulting in cell death would further increase the corrosion rate at the metal implant site. The synergistic interaction in the implant surroundings in which infectious conditions produce Ti ions that contribute to more infection, creating a potential cycle of accelerating corrosion.

Amino acids 1811-1960 of myosin heavy chain 9 is involved in murine gammaherpesvirus 68 infection.

Myosin heavy chain 9 (MYH9) has been identified as a crucial factor in gammaherpesvirus infection. Murine gammaherpesvirus 68 (MHV-68) was used as an appropriate viral model for investigating gammaherpesviruses in vivo and developing antiviral treatments. However, the roles of MYH9 in MHV-68 infection have not been documented. In the study, the relationship between the expression of MYH9 and MHV-68 infection and MYH9 as the antiviral target were analyzed. The results revealed that MYH9 was enriched on the cell surface and co-localized with MHV-68 upon viral infection. Knocking down MYH9 with siRNA or using the specific inhibitor of MYH9 activity, Blebbistatin, resulted in the decreasing of MHV-68 infection. Furthermore, polyclonal antibodies against MYH9 reduced infection by approximately 74% at a dose of 100 µg/ml. The study determined that MYH9 contributes to MHV-68 infection by interacting with viral glycoprotein 150 (gp150) in the BHK-21 cell membrane. The specific region of MYH9, amino acids 1811-1960 (C-150), was identified as the key domain involved in the interaction with MHV-68 gp150 and was found to inhibit MHV-68 infection. Moreover, C-150 was also shown to decrease HSV-1 infection in Vero cells by approximately 73%. Both C-150 and Blebbistatin were found to inhibit MHV-68 replication and reduce histopathological lesions in vivo in C57BL/6J mice. Taken together, these findings suggested that MYH9 is crucial for MHV-68 infection through its interaction with viral gp150 and that C-150 may be a promising antiviral target for inhibiting MHV-68 infection in vitro and in vivo.

Bioactive graphene oxide-functionalized self-expandable hydrophilic and osteogenic nanocomposite for orthopaedic applications.

Polymethyl methacrylate (PMMA) bone cement (PBC) is commonly used in orthopaedic surgery. However, polymerization volumetric shrinkage, exothermic injury, and low bioactivity prevent PBC from being an ideal material. The developed expandable P(MMA-AA-St) well overcomes the volumetric shrinkage of PBC. However, its biomechanical properties are unsatisfactory. Herein, graphene oxide (GO), a hydrophilic material with favourable biomechanics and osteogenic capability, was added to P(MMA-AA-St) to optimize its biomechanics and bioactivity. The GO-modified self-expandable P(MMA-AA-St)-GO nanocomposite (PGBCs) exhibited outstanding compressive strength (>70 â€‹MPa), water absorption, and volume expansion, as well as a longer handling time and a reduced setting temperature. The cytocompatibility of PGBCs was superior to that of PBC, as demonstrated by CCK-8 assay, live-dead cell staining, and flow cytometry. In addition, better osteoblast attachment was observed, which could be attributed to the effects of GO. The improved level of osteogenic gene and protein expression further illustrated the improved cell-material interactions between osteoblasts and PGBCs. The results of an in vivo study performed by filling bone defects in the femoral condyles of rabbits with PGBCs demonstrated promising intraoperative handling properties and convenient implantation. Blood testing and histological staining demonstrated satisfactory in vivo biosafety. Furthermore, bone morphological and microarchitecture analyses using bone tissue staining and micro-CT scanning revealed better bone-PGBCs contact and osteogenic capability. The results of this study indicate that GO modification improved the physiochemical properties, cytocompatibility, and osteogenic capability of P(MMA-AA-St) and overcame the drawbacks of PBC, allowing its material derivatives to serve as effective implantable biomaterials.

The Progress in Tribocorrosion Research (2010-21): Focused on the Orthopedics and Dental Implants.

Tribocorrosion is an integration of two areas-tribology and corrosion. It can be defined as the material degradation caused by the combined effect of corrosion and tribological process at the material interfaces. Significant development has occurred in the field of tribocorrosion over the past years. This development is due to its applications in various fields, such as aerospace, marine, biomedical, and space. Focusing on biomedical applications, tribocorrosion finds its applications in the implants used in cardiovascular, spine, orthopedics, trauma, and dental areas. It was reported that around 7.2 million Americans are living with joint implants. Implant surgery is a traumatic and expensive procedure. Tribocorrosion can affect the lifespan of the implants, thus leading to implant failure and a potential cause of revision surgery. Hence, it is essential to understand how tribocorrosion works, its interaction with the implants, and what procedures can be implemented to protect materials from tribocorrosion. This paper discusses how tribocorrosion research has evolved over the past 11 years (2010-2021). This is a comprehensive overview of tribocorrosion research in biomedical applications.

Corrigendum: Screening and identification of nucleocapsid protein-nanobodies that inhibited Newcastle disease virus replication in DF-1 cells.

[This corrects the article DOI: 10.3389/fmicb.2022.956561.].

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.

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus.

African swine fever virus (ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds. Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase (nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA (cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7% by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.

Screening and identification of nucleocapsid protein-nanobodies that inhibited Newcastle disease virus replication in DF-1 cells.

Newcastle disease (ND) is an acute and highly contagious infectious disease found in poultry. Although commercial ND virus (NDV) vaccines are universally used, some case reports persistently documented vaccination failure. Therefore, novel strategies are still required to control the occurrence of the disease in chickens. Recently, nanobodies (Nbs), which have the advantages of small molecular weight and low production costs, have been shown to be promising therapeutics against viral infection. In the present study, a total of 16 Nbs against NDV nucleocapsid protein (NP) were screened from two libraries against NDV using phage display technology. Of the 16 screened Nbs, eight were prevented from binding to NDV NP protein through administering positive chicken sera for anti-NDV antibodies, indicating that the epitopes recognized by these eight Nbs were able to induce the immune response after the chickens were infected with NDV stock. Subsequently, transfection assay, construction of recombinant DF-1 cells capable of expressing different nanobodies and viral inhibition assay were used to screen the nanobodies inhibiting NDV replication. The results demonstrated that Nb18, Nb30, and Nb88 significantly inhibited the replication of Class I and different genotypes of Class II NDV strains in DF-1 cells when they were expressed in the cytoplasm. Collectively, these nanobodies provided new tools for researching the functions of NDV NP protein and may be used as a novel strategy for designing drugs against NDV infection in chickens.

Isolation and Genetic Characterization of Parvoviruses From Dogs, Cats, Minks, and Raccoon Dogs in the Eastern Region of Shandong Province, China.

The eastern region of Shandong province, China, is an intensive economic mink and raccoon dog breeding area. To investigate the molecular variations of parvovirus in cats, dogs, minks, and raccoon dogs from this region, feline panleukopenia virus (FPV), canine parvovirus 2 (CPV-2), mink enteritis virus (MEV), and raccoon dog parvovirus (RDPV) were separately isolated and characterized from the respective animals with gastroenteritis. PCR amplification showed that there were 15/18 (83.3%), 9/13 (69.2%), 8/11 (72.7%), and 3/7 (42.9%) samples from the diseased animals separately positive for FPV, CPV-2, MEV, and RDPV. Of these, a total of six FPV, six MEV, four CPV-2, and three RDPV strains were successfully isolated using F81 cells. Next, the near-complete genomes of 19 parvovirus isolates were amplified and analyzed. The viral particle 2 (VP2) sequence alignment showed that they shared 97.2-100% nucleotide similarity. Phylogenetic analysis showed that the five FPV isolates were in the same branch, and an FPV isolate was closely related with MEV and RDPV isolates obtained in this study. These suggested that cross-species infection occurred in the Shandong region between the FPV, MEV, and RDPV. For the four CPV-2 isolates, three were antigenic variant strains CPV-2a, and the other was antigenic variant strain CPV-2c. Additionally, the mutations that had emerged in the VP2 amino acids of CPV-2 also occurred in the VP2 from the FPV, MEV, and RDPV isolates. This study suggested that the continuous evolution of the parvovirus may be accelerated in areas with a high density of economic animal trading/breeding, and controlling parvovirus infection in these animals remains a challenge.

Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization.

Avian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro. By using CaHEV capsid as mimic of virion to treat cell in vitro, it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/ß1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells. IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/ß1 integrins. The triggering of the signaling pathway-associated Integrin α5/ß1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.

A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing DsRed Protein Based on Bacterial Artificial Chromosome System.

Recombinant viruses possessing reporter proteins as tools are widely applied in investigating viral biology because of the convenience for observation. Previously, we generated a recombinant pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with enhanced green fluorescent protein (EGFP) reporter for monitoring virus spread and screening of neutralizing antibodies. PRRSV with different kinds of reporters can support more application scenarios. Here, we described a new genetically stable infectious clones of a highly pathogenic PRRSV (HP-PRRSV) harboring the DsRed (a red fluorescent protein isolated from the coral Discosoma) gene. In the recombinant infectious clone, the transcription regulatory sequence 2 (TRS2) of PRRSV was inserted between the open reading frame 7 (ORF7) and 3'UTR to drive the transcription of DsRed gene, which makes it a separate transcription unit in the viral genome. Using the bacterial artificial chromosome (BAC) system and cytomegalovirus (CMV) promoter, the recombinant HP-PRRSV with the DsRed insertion was successfully rescued and showed similar growth and replication patterns compared with the wild-type virus in the MARC-145 cells. In addition, the DsRed protein was stably expressed in the recombinant virus for at least 10 passages with consistent fluorescence intensity and density. Using the recombinant HP-PRRSV with DsRed protein, the virus tracking in MARC-145 was observed by live-cell imaging. Meanwhile, quantification of the DsRed fluorescence positive cells by flow cytometry provides an alternative to standard methods for testing the level of PRRSV infection. This recombinant PRRSV with DsRed fluorescence protein expression could be a useful tool for fundamental research on the viral biology and shows the new design for stable expression of foreign genes in PRRSV.