Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies.

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as 48WDCQDGICKNKITESRFIDS67, and 122GDHQQLSIKQ131, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1.

SARS-CoV-2 and filovirus enter cells via the cell surface angiotensin-converting enzyme 2 (ACE2) or the late-endosome Niemann-Pick C1 (NPC1) as a receptor. Here, we screened 974 natural compounds and identified Tubeimosides I, II, and III as pan-coronavirus and filovirus entry inhibitors that target NPC1. Using in-silico, biochemical, and genomic approaches, we provide evidence that NPC1 also binds SARS-CoV-2 spike (S) protein on the receptor-binding domain (RBD), which is blocked by Tubeimosides. Importantly, NPC1 strongly promotes productive SARS-CoV-2 entry, which we propose is due to its influence on fusion in late endosomes. The Tubeimosides' antiviral activity and NPC1 function are further confirmed by infection with SARS-CoV-2 variants of concern (VOC), SARS-CoV, and MERS-CoV. Thus, NPC1 is a critical entry co-factor for highly pathogenic human coronaviruses (HCoVs) in the late endosomes, and Tubeimosides hold promise as a new countermeasure for these HCoVs and filoviruses.

Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

A cell line expressing the CD2v protein of ASFV was generated. The efficient expression of CD2v protein was determined by immunofluorescence and Western blotting. The CD2v protein was Ni-affinity purified from the supernatant of cell cultures. The CD2v-expressing cells showed properties of hemadsorption, and the secreted CD2v protein exhibited hemagglutinating activity. The antigenicity and immunoprotection ability of CD2v were evaluated by immunizing pigs alone, combined with a cell-line-expressed p30 protein or triple combined with p30 and K205R protein. Immunized pigs were challenged with the highly virulent ASFV strain HLJ/18. Virus challenge results showed that CD2v immunization alone could provide partial protection at the early infection stage. Protein p30 did not show synergistic protection effects in immunization combined with CD2v. Interestingly, immunization with the triple combination of CD2V, p30 and K205R reversed the protection effect. The viremia onset time was delayed, and one pig out of three recovered after the challenge. The pig recovered from ASFV clinical symptoms, the rectal temperature returned to normal levels and the viremia was cleared. The mechanism of this protection effect warrants further investigation.

A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it challenging to develop broad-spectrum prophylactic vaccines and therapeutic antibodies. Here, we have identified a broad-spectrum neutralizing antibody and its highly conserved epitope in the receptor-binding domain (RBD) of the spike protein (S) S1 subunit of SARS-CoV-2. First, nine monoclonal antibodies (MAbs) against the RBD or S1 were generated; of these, one RBD-specific MAb, 22.9-1, was selected for its broad RBD-binding abilities and neutralizing activities against SARS-CoV-2 variants. An epitope of 22.9-1 was fine-mapped with overlapping and truncated peptide fusion proteins. The core sequence of the epitope, 405D(N)EVR(S)QIAPGQ414, was identified on the internal surface of the up-state RBD. The epitope was conserved in nearly all variants of concern of SARS-CoV-2. MAb 22.9-1 and its novel epitope could be beneficial for research on broad-spectrum prophylactic vaccines and therapeutic antibody drugs. IMPORTANCE The continuous emergence of new variants of SARS-CoV-2 has caused great challenge in vaccine design and therapeutic antibody development. In this study, we selected a broad-spectrum neutralizing mouse monoclonal antibody which recognized a conserved linear B-cell epitope located on the internal surface of RBD. This MAb could neutralize all variants until now. The epitope was conserved in all variants. This work provides new insights in developing broad-spectrum prophylactic vaccines and therapeutic antibodies.

Comprehensive mapping of antigenic linear B-cell epitopes on K205R protein of African swine fever virus with monoclonal antibodies.

African swine fever virus causes an acute, highly contagious swine disease with high mortality, leading to enormous losses in the pig industry. The K205R, a nonstructural protein of African swine fever virus, is abundantly expressed in the cytoplasm of infected cells at the early stage of infection and induces a strong immune response. However, to date, the antigenic epitopes of this immunodeterminant have not been characterized. In the present study, the K205R protein was expressed in a mammalian cell line and purified using Ni-affinity chromatography. Furthermore, three monoclonal antibodies (mAbs; 5D6, 7A8, and 7H10) against K205R were generated. Indirect immunofluorescence assay and western blot results showed that all three mAbs recognized native and denatured K205R in African swine fever virus (ASFV)-infected cells. To identify the epitopes of the mAbs, a series of overlapping short peptides were designed and expressed as fusion proteins with maltose-binding protein. Subsequently, the peptide fusion proteins were probed with monoclonal antibodies using western blot and enzyme-linked immunosorbent assay. The three target epitopes were fine-mapped; the core sequences of recognized by the mAbs 5D6, 7A8, and 7H10 were identified as 157FLTPEIQAILDE168, 154REKFLTP160, and 136PTNAMFFTRSEWA148, respectively. Probing with sera from ASFV-infected pigs in a dot blot assay demonstrated that epitope 7H10 was the immunodominant epitope of K205R. Sequence alignment showed that all epitopes were conserved across ASFV strains and genotypes. To our knowledge, this is the first study to characterize the epitopes of the antigenic K205R protein of ASFV. These findings may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

Generation of A Stable GFP-reporter Zika Virus System for High-throughput Screening of Zika Virus Inhibitors.

Zika virus (ZIKV) is associated with severe birth defects and Guillain-Barré syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine (HHT), bruceine D (BD), dihydroartemisinin (DHA) and digitonin (DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549. The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B.

Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Here, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which the host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. The loss of SPCS1 function markedly reduced intracellular virion assembly and the production of infectious JEV particles but did not affect cell entry, RNA replication, or translation of the virus. SPCS1 was found to interact with nonstructural protein 2B (NS2B), which is involved in posttranslational protein processing and virus assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B(1-49) and NS2B(84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B(1-49) and P112A in NS2B(84-131) weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1(91-169), which contains two transmembrane domains, was involved in interactions with both NS2B(1-49) and NS2B(84-131). Taken together, these results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing the posttranslational processing of JEV proteins and the assembly of virions.IMPORTANCE Understanding virus-host interactions is important for elucidating the molecular mechanisms of virus propagation and identifying potential antiviral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the posttranslational protein processing and viral assembly stages of the JEV life cycle but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate virus assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. These findings expand our understanding of how host factors participate in the flavivirus replication life cycle and identify potential antiviral targets for combating flavivirus infection.

West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

West Nile virus (WNV) is a neurotropic pathogen which causes zoonotic disease in humans. Recently, there have been an increasing number of infected cases and there are no clinically approved vaccines or effective drugs to treat WNV infections in humans. The purpose of this study was to facilitate vaccine and antiviral drug discovery by developing a packaging cell line-restricted WNV infectious replicon particle system. We constructed a DNA-based WNV replicon lacking the C-prM-E coding region and replaced it with a GFP coding sequence. To produce WNV replicon particles, cell lines stably-expressing prM-E and C-prM-E were constructed. When the WNV replicon plasmid was co-transfected with a WNV C-expressing plasmid into the prM-E-expressing cell line or directly transfected the C-prM-E expressing cell line, the replicon particle was able to replicate, form green fluorescence foci, and exhibit cytopathic plaques similar to that induced by the wild type virus. The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells. Thus, this system provides a safe and convenient reporter WNV manipulating tool which can be used to study WNV viral invasion mechanisms, neutralizing antibodies and antiviral efficacy.

Generation and characterization of a monoclonal antibody against prM protein of West Nile virus.

West Nile virus (WNV), which is an emerging pathogenic flavivirus with increasing distribution worldwide, is the cause of major human and animal health concerns. The pre-membrane (prM) protein of WNV is cleaved during maturation by the furin protease into the structural protein M and a pr-segment. In this study we generated and characterized a monoclonal antibody (MAb) against the WNV prM protein. Western blot analysis showed that the MAb reacted with WNV prM specifically. Immunohistochemistry assays demonstrated that the MAb recognized native prM protein in transfected BHK-21 cells. Preliminary studies were performed to identify the epitope recognized by the MAb using a set of synthesized overlapping peptides spanning the whole length of the prM protein. The MAb reported here may provide a valuable tool for the further exploration of the biological properties and functions of the prM protein and may also be developed for potential clinical applications.

Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1:40 to 1:320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1:10,240 to 1:81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1:80 to 1:10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1 × 10(5) TCID50. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3-4 days after challenge and were euthanized from 7-9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.

Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate.

BACKGROUND: Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine. RESULTS: We generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15-20 µg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4-6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. CONCLUSION: These results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.

Comprehensive mapping of a novel NS1 epitope conserved in flaviviruses within the Japanese encephalitis virus serocomplex.

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues ²²7ETHTLW²³². Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(348). Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation.

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a -1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.

A monoclonal antibody against PrM/M protein of Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a major public health threat in the Asia-Pacific region. The pre-membrane (PrM) protein of Japanese encephalitis virus is cleaved during maturation by the cellular protease into the structural protein M and a pr-segment. Here, we describe a procedure to generate monoclonal antibody (MAb) against JEV PrM/M protein and investigate its characteristics. Western blot analysis showed that the MAbs produced in this study were against JEV PrM/M specifically. Indirect immunofluorescence assay demonstrated that they could recognize native PrM/M protein in JEV-infected BHK-21 cells. Preliminary studies identified the epitope of the MAb with a set of synthesized overlapping peptides covering the whole length of PrM protein of JEV. The MAbs reported here may provide valuable tools for the further exploration of biological properties and functions of PrM/M protein and may also be developed for potential clinical applications.

Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats.

Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.

Identification and characterization of a virus-specific continuous B-cell epitope on the PrM/M protein of Japanese Encephalitis Virus: potential application in the detection of antibodies to distinguish Japanese Encephalitis Virus infection from West Nile Virus and Dengue Virus infections.

BACKGROUND: Differential diagnose of Japanese encephalitis virus (JEV) infection from other flavivirus especially West Nile virus (WNV) and Dengue virus (DV) infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV. RESULTS: To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120) was detected by enzyme-linked immunosorbent assay (ELISA). By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118). This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera. CONCLUSION: Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.

Identification of a virus-specific and conserved B-cell epitope on NS1 protein of Japanese encephalitis virus.

NS1 protein of Japanese encephalitis virus (JEV) is an important non-structural protein, which is able to induce protective immune response in target animals and can be used as specific serological diagnosis tool, but the epitopes on NS1 of JEV have not been identified. For epitope mapping, in this study, a series of 51 partially overlapping fragments covering entire NS1 protein were expressed with a GST-tag and then screened by a monoclonal antibody (mAb). Through enzyme-linked immunosorbent assay (ELISA), linear epitope-containing fragment, the overlapping region of NS1-18 and NS1-19 (residues 145-152), was located. Then a set of peptides derived from that overlapping region with deletions were expressed and subjected to ELISA and Western blot for further mapping purpose. Results indicated that the motif of (146)EHARW(150) is the minimal unit of the linear epitope recognized by that monoclonal antibody (mAb). Western blot showed that this epitope could be recognized by JEV-positive serum from pigs. Furthermore, it was found that the epitope is highly conserved among JEV strains through sequence alignments analysis. Notably, none of the homologous regions on NS1 proteins of other flavivirus could react with the mAb when they were tested for cross-reactivity, suggesting the potential clinical application of this epitope in differential diagnosis.

Isolation and genetic characterization of avian origin H9N2 influenza viruses from pigs in China.

As pigs are susceptible to infection with both avian and human influenza A viruses, they have been proposed to be an intermediate host for the adaptation of avian influenza viruses to humans. In April 2006, a disease caused by highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) occurred in several pig farms and subsequently overwhelmed almost half of China with more than 2,000,000 cases of pig infection. Here we report a case in which four swine H9N2 influenza viruses were isolated from pigs infected by highly pathogenic PRRSVs in Guangxi province in China. All the eight gene segments of the four swine H9N2 viruses are highly homologous to A/Pigeon/Nanchang/2-0461/00 (H9N2) or A/Wild Duck/Nanchang/2-0480/00 (H9N2). Phylogenetic analyses of eight genes show that the swine H9N2 influenza viruses are of avian origin and may be the descendants of A/Duck/Hong Kong/Y280/97-like viruses. Molecular analysis of the HA gene indicates that our H9N2 isolates might have high-affinity binding to the alpha2,6-NeuAcGal receptor found in human cells. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially after the emergence of highly pathogenic PRRSVs in pigs in China.

Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.