Keap1 recognizes EIAV early accessory protein Rev to promote antiviral defense.

The Nrf2/Keap1 axis plays a complex role in viral susceptibility, virus-associated inflammation and immune regulation in host cells. However, whether or how the Nrf2/Keap1 axis is involved in the interactions between equine lentiviruses and their hosts remains unclear. Here, we demonstrate that the Nrf2/Keap1 axis was activated during EIAV infection. Mechanistically, EIAV-Rev competitively binds to Keap1 and releases Nrf2 from Keap1-mediated repression, leading to the accumulation of Nrf2 in the nucleus and promoting Nrf2 responsive genes transcription. Subsequently, we demonstrated that the Nrf2/Keap1 axis represses EIAV replication via two independent molecular mechanisms: directly increasing antioxidant enzymes to promote effective cellular resistance against EIAV infection, and repression of Rev-mediated RNA transport through direct interaction between Keap1 and Rev. Together, these data suggest that activation of the Nrf2/Keap1 axis mediates a passive defensive response to combat EIAV infection. The Nrf2/Keap1 axis could be a potential target for developing strategies for combating EIAV infection.

SYNJ2BP Improves the Production of Lentiviral Envelope Protein by Facilitating the Formation of Mitochondrion-Associated Endoplasmic Reticulum Membrane.

Equine infectious anemia virus (EIAV) and HIV are both members of the Lentivirus genus and are similar in major virological characters. EIAV endangers the horse industry. In addition, EIAV can also be used as a model for HIV research. The maturation of the lentiviral Env protein, which is necessary for viral entry, requires Env to be folded in the endoplasmic reticulum (ER). It is currently unclear how this process is regulated. Mitochondrion-associated endoplasmic reticulum membrane (MAM) is a specialized part of the close connection between the ER and mitochondria, and one of the main functions of MAM is to promote oxidative protein production in the ER. SYNJ2BP is one of the key proteins that make up the MAM, and we found that SYNJ2BP is essential for EIAV replication. We therefore constructed a SYNJ2BP knockout HEK293T cell line in which the number of MAMs is significantly reduced. Moreover, overexpression of SYNJ2BP could increase the number of MAMs. Our study demonstrates that SYNJ2BP can improve the infectivity of the EIAV virus with elevated production of the viral Env protein through increased MAM formation. Interestingly, SYNJ2BP was able to improve the production of not only EIAV Env but also HIV. Further investigation showed that MAMs can provide more ATP and calcium ions, which are essential factors for Env production, to the ER and can also reduce ER stress induced by HIV or EIAV Envs to increase the Env production level in cells. These results may help us to understand the key production mechanisms of lentiviral Env. IMPORTANCE Lentiviral Env proteins, which are rich in disulfide bonds, need to be fully folded in the ER; otherwise, misfolded Env proteins will induce ER stress and be degraded by ER-associated protein degradation (ERAD). To date, it is still unclear about Env production mechanism in the ER. MAM is the structure of closely connection between the ER and mitochondria. MAMs play important roles in the calcium steady state and oxidative stress, especially in the production of oxidative protein. For the first time, we found that SYNJ2BP can promote the production of lentiviral Env proteins by providing the ATP and calcium ions required for oxidative protein production in the ER and by reducing ER stress through facilitating formation of MAMs. These studies shed light on how MAMs improve lentiviral Env production, which will lay the foundation for the study of replication mechanisms in other lentiviruses from the perspective of the cellular organelle microenvironment.

Identification of a Novel Post-transcriptional Transactivator from the Equine Infectious Anemia Virus.

All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in size, comprises the first 18 amino acids (aa) of Gag protein together with the last 82 aa of Rev, and was detected in EIAV-infected cells. Similar to Rev, Grev is localized to the nucleus, and both are able to mediate the expression of Mat (a recently identified viral protein of unknown function from EIAV), but Rev can mediate the expression of EIAV Gag/Pol, while Grev cannot. We also demonstrate that Grev, similar to Rev, specifically binds to rev-responsive element 2 (RRE-2, located in the first exon of mat mRNAs) to promote nuclear export of mat mRNA via the chromosome region maintenance 1 (CRM1) pathway. However, unlike Rev, whose function depends on its multimerization, we could not detect multimerization of Grev using coimmunoprecipitation (co-IP) or bimolecular fluorescence complementation (BiFC) assays. Together, these data suggest that EIAV encodes two post-transcriptional transactivators, Rev and Grev, with similar, but not identical, functions. IMPORTANCE Nuclear export of viral transcripts is a crucial step for viral gene expression and viral replication in lentiviruses, and this export is regulated by a post-transcriptional transactivator, Rev, that is shared by all lentiviruses. Here, we report that the equine infectious anemia virus (EIAV) encodes a novel viral protein, Grev, and demonstrated that Grev, like Rev, mediates the expression of the viral protein Mat by binding to the first exon of mat mRNAs via the chromosome region maintenance 1 (CRM1) pathway. Grev is encoded by a single-spliced transcript containing two exons, whereas Rev is encoded by a multiple-spliced transcript containing four exons. Moreover, Rev is able to mediate EIAV Gag/Pol expression by binding to rev-responsive element (RRE) located within the Env-coding region, while Grev cannot. Therefore, the present study demonstrates that EIAV encodes two post-transcriptional regulators, Grev and Rev, suggesting that post-transcriptional regulation patterns in lentivirus are diverse and complex.

A Novel, Fully Spliced, Accessory Gene in Equine Lentivirus with Distinct Rev-Responsive Element.

All lentiviruses encode the accessory protein Rev, whose main biological function is to mediate the nuclear export of unspliced and incompletely spliced viral transcripts by binding to a viral cis-acting element (termed the Rev-responsive element, RRE) within the env-encoding region. Equine infectious anemia virus (EIAV) is a member of the lentivirus genus in the Retroviridae family and is considered an important model for the study of lentivirus pathogenesis. Here, we identified a novel transcript from the EIAV genome that encoded a viral protein, named Mat, with an unknown function. The transcript mat was fully spliced and comprised parts of the coding regions of MA and TM. Interestingly, the expression of Mat depended on Rev and the chromosome region maintenance 1 (CRM1) pathway. Rev could specifically bind to Mat mRNA to promote its nuclear export. We further identified that the first exon of Mat mRNA, which was located within the Gag-encoding region, acted as an unreported RRE. Altogether, we identified a novel fully spliced transcript mat with an unusual RRE, which interacted with Rev for nuclear export through the CRM1 pathway. These findings updated the EIAV genome structure, highlighted the diversification of posttranscriptional regulation patterns in EIAV, and may help to expand the understanding of gene transcription and expression of lentivirus. IMPORTANCE In lentiviruses, the nuclear export of viral transcripts is an important step in controlling viral gene expression. Generally, the unspliced and incompletely spliced transcripts are exported via the CRM1-dependent export pathway in a process mediated by the viral Rev protein by binding to the Rev-responsive element (RRE) located within the Env-coding region. However, the completely spliced transcripts are exported via an endogenous cellular pathway, which was Rev independent. Here, we identified a novel fully spliced transcript from EIAV and demonstrated that it encoded a viral protein, termed Mat. Interestingly, we determined that the expression of Mat depended on Rev and identified that the first exon of Mat mRNA could specifically bind to Rev and be exported to the cytoplasm, which suggested that the first exon of Mat mRNA was a second RRE of EIAV. These findings provided important insights into the Rev-dependent nuclear export of completely spliced transcripts in lentiviruses.

Development and evaluation of a blocking ELISA for serological diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) is an important viral disease characterized by persistent infection in equids worldwide. Most EIA cases are life-long virus carriers with low antibody reactions and without the appearance of clinical symptoms. A serological test with high sensitivity and specificity is required to detect inapparent infection. In this study, a B-cell common epitope-based blocking ELISA (bELISA) was developed using a monoclonal antibody together with the EIAV p26 protein labelled with HRP. The test has been evaluated against the standard and with field serum samples globally. This bELISA test can be completed within 75 min, and the sensitivity is higher than those of either the AGID or one commercial cELISA kit. This bELISA assay was 8-16 times more analytically sensitive than AGID, and 2 to 4 times more analytically sensitive than one cELISA kit by testing three sera from the USA, Argentina, and China, respectively. The 353 serum samples from Argentina were tested, in comparison with AGID, the diagnostic sensitivity and specificity of our bELISA assay were 100% (154/154) and 97.0% (193/199), respectively, and the accuracy of the bELISA test was 98.3%. The bELISA test developed in this study is a rapid, sensitive, specific method for the detection of EIAV infection, and could be a promising candidate for use in the monitoring of the EIA epidemic worldwide. KEY POINTS: • A universal epitope-based blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies to EIAV. • The bELISA assay can be used to test EIAV serum samples from different regions of the world including North America, South America, Europe, and Asia. • The bELISA assay was evaluated in three different international labs and showed a better performance than other commercial kits.

Attenuation of Equine Lentivirus Alters Mitochondrial Protein Expression Profile from Inflammation to Apoptosis.

Equine infectious anemia virus (EIAV) is an equine lentivirus similar to HIV-1, targets host immune cells, and causes a life-long infection in horses. The Chinese live EIAV vaccine is attenuated from long-term passaging of a highly virulent strain in vitro The parent pathogenic strain (EIAVDLV34) induces a host inflammatory storm to cause severe pathological injury of animals. However, the vaccine strain (EIAVDLV121) induces a high level of apoptosis to eliminate infected cells. To investigate how these processes are regulated, we performed a comparative proteomics analysis and functional study in equine monocyte-derived macrophages (eMDMs) and found that the divergent mitochondrial protein expression profiles caused by EIAV strains with different virulence led to disparate mitochondrial function, morphology, and metabolism. This in turn promoted the distinct transformation of macrophage inflammatory polarization and intrinsic apoptosis. In EIAVDLV34-infected cells, a high level of glycolysis and increased mitochondrial fragmentation were induced, resulting in the M1-polarized proinflammatory-type transformation of macrophages and the subsequent production of a strong inflammatory response. Following infection with EIAVDLV121, the infected cells were transformed into M2-polarized anti-inflammatory macrophages by inhibition of glycolysis. In this case, a decrease in the mitochondrial membrane potential and impairment of the electron transport chain led to increased levels of apoptosis and reactive oxygen species. These results correlated with viral pathogenicity loss and may help provide an understanding of the key mechanism of lentiviral attenuation.IMPORTANCE Following viral infection, the working pattern and function of the cell can be transformed through the impact on mitochondria. It still unknown how the mitochondrial response changes in cells infected with viruses in the process of virulence attenuation. EIAVDLV121 is the only effective lentiviral vaccine for large-scale use in the world. EIAVDLV34 is the parent pathogenic strain. Unlike EIAVDLV34-induced inflammation storms, EIAVDLV121 can induce high levels of apoptosis. For the first time, we found that, after the mitochondrial protein expression profile is altered, EIAVDLV34-infected cells are transformed into M1-polarized-type macrophages and cause inflammatory injury and that the intrinsic apoptosis pathway is activated in EIAVDLV121-infected cells. These studies shed light on how the mitochondrial protein expression profile changes between cells infected by pathogenic lentivirus strains and cells infected by attenuated lentivirus strains to drive different cellular responses, especially from inflammation to apoptosis.

Characterization of Equine Infectious Anemia Virus Long Terminal Repeat Quasispecies In Vitro and In Vivo.

The equine infectious anemia virus (EIAV) attenuated vaccine was developed by long-term passaging of a field-isolated virulent strain in cross-species hosts, followed by successive cultivation in cells in vitro To explore the molecular mechanism underlying the evolution of the EIAV attenuated vaccine, a systematic study focusing on long-terminal-repeat (LTR) variation in numerous virus strains ranging from virulent EIAV to attenuated EIAV was performed over time both in vitro and in vivo Two hypervariable regions were identified within the U3 region in the enhancer region (EHR) and the negative regulatory element (NRE) and within the R region in the transcription start site (TSS) and the Tat-activating region (TAR). Among these sites, variation in the U3 region resulted in the formation of additional transcription factor binding sites; this variation of the in vitro-adapted strains was consistent with the loss of pathogenicity. Notably, the same LTR variation pattern was observed both in vitro and in vivo Generally, the LTR variation in both the attenuated virus and the virulent strain fluctuated over time in vivo Interestingly, the attenuated-virus-specific LTR variation was also detected in horses infected with the virulent strain, supporting the hypothesis that the evolution of an attenuated virus might have involved branching from EIAV quasispecies. This hypothesis was verified by phylogenetic analysis. The present systematic study examining the molecular evolution of attenuated EIAV from EIAV quasispecies may provide an informative model reflecting the evolution of similar lentiviruses.IMPORTANCE The attenuated EIAV vaccine was the first lentiviral vaccine used to successfully control for equine infectious anemia in China. This vaccine provides an important reference for studying the relationship between EIAV gene variation and changes in biological characteristics. Importantly, the vaccine provides a model for the investigation of lentiviral quasispecies evolution. This study followed the "natural" development of the attenuated EIAV vaccine by use of a systematic analysis of LTR evolution in vitro and in vivo The results revealed that the increase in LTR variation with passaging was accompanied by a decrease in virulence, which indicated that LTR variability might parallel the attenuation of virulence. Interestingly, the attenuated-virus-specific LTR variation was also detected in virulent-strain-infected horses, a finding consistent with those of previous investigations of gp90 and S2 evolution. Therefore, we present a hypothesis that the evolution of the attenuated virus may involve branching from EIAV quasispecies present in vivo.

The integration of a macrophage-adapted live vaccine strain of equine infectious anaemia virus (EIAV) in the horse genome.

Integration is an important feature of retroviruses and retrovirus-based therapeutic transfection vectors. The non-primate lentivirus equine infectious anaemia virus (EIAV) primarily targets macrophages/monocytes in vivo. Investigation of the integration features of EIAVDLV121 strains, which are adapted to donkey monocyte-derived macrophages (MDMs), is of great interest. In this study, we analysed the integration features of EIAVDLV121 in equine MDMs during in vitro infection. Our previously published integration sites (IS) for EIAVFDDV13 in fetal equine dermal (FED) cells were also analysed in parallel as references. Sequencing of the host genomic regions flanking the viral IS showed that reference sequence (RefSeq) genes were preferentially targeted for integration by EIAVDLV121. Introns, AT-rich regions, long interspersed nuclear elements (LINEs) and DNA transposons were also predominantly biased toward viral insertion, which is consistent with EIAVFDDV13 integration into the horse genome in FED cells. In addition, the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, specifically gag junctions for EIAVDLV121 and tight junctions for EIAVFDDV13, are regulators of metabolic function, which is consistent with the common bioprocesses, specifically cell cycle and chromosome/DNA organization, identified by gene ontology (GO) analysis. Our results demonstrate that EIAV integration occurs in regions that harbour structural and topological features of local chromatin in both macrophages and fibroblasts. Our data on EIAV will facilitate further understanding of lentivirus infection and the development of safer and more effective gene therapy vectors.

Genetic Evolution during the development of an attenuated EIAV vaccine.

BACKGROUND: The equine infectious anemia virus (EIAV) vaccine is the only attenuated lentiviral vaccine applied on a large scale that has been shown to be effective in controlling the prevalence of EIA in China. This vaccine was developed by successive passaging of a field-isolated virulent strain in different hosts and cultivated cells. To explore the molecular basis for the phenotype alteration of this vaccine strain, we systematically analyzed its genomic evolution during vaccine development. RESULTS: Sequence analysis revealed that the genetic distance between the wild-type strain and six representative strains isolated from key development stages gradually increased with the number of passages. Env gene, but not gag and pol, showed a clear evolutionary flow similar to that of the whole genomes of different generations during the attenuation. Stable mutations were identified in multiple regions of multiple genes along with virus passaging. The adaption of the virus to the growth environment of cultured cells with accumulated genomic and genetic variations was positively correlated with the reduction in pathogenicity and rise of immunogenicity. Statistical analyses revealed significant differences in the frequency of the most stable mutations between in vivo and ex vivo-adapted strains and between virulent and attenuated strains. CONCLUSIONS: These data indicate that EIAV evolution during vaccine development generated an accumulation of mutations under the selective drive force, which helps to better understand the molecular basis of lentivirus pathogenicity and immunogenicity.

Proteomic alteration of equine monocyte-derived macrophages infected with equine infectious anemia virus.

Similar to the well-studied viruses human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV) is another member of the Lentivirus genus in the family Retroviridae. Previous studies revealed that interactions between EIAV and the host resulted in viral evolution in pathogenicity and immunogenicity, as well as adaptation to the host. Proteomic analysis has been performed to examine changes in protein expression and/or modification in host cells infected with viruses and has revealed useful information for virus-host interactions. In this study, altered protein expression in equine monocyte-derived macrophages (eMDMs, the principle target cell of EIAV in vivo) infected with the EIAV pathogenic strain EIAV(DLV34) (DLV34) was examined using 2D-LC-MS/MS coupled with the iTRAQ labeling technique. The expression levels of 210 cellular proteins were identified to be significantly upregulated or downregulated by infection with DLV34. Alterations in protein expression were confirmed by examining the mRNA levels of eight selected proteins using quantitative real-time reverse-transcription PCR, and by verifying the levels of ten selected proteins using parallel reaction monitoring (PRM). Further analysis of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG)-Pathway enrichment demonstrated that these differentially expressed proteins are primarily related to the biological processes of oxidative phosphorylation, protein folding, RNA splicing, and ubiquitylation. Our results can facilitate a better understanding of the host response to EIAV infection and the cellular processes required for EIAV replication and pathogenesis.

Mice transgenic for equine cyclin T1 and ELR1 are susceptible to equine infectious anemia virus infection.

BACKGROUND: As a member of the tumor necrosis factor receptor (TNFR) protein superfamily, equine lentivirus receptor 1 (ELR1) has been shown to be expressed in various equine cells that are permissive for equine infectious anemia virus (EIAV) replication. The EIAV Tat protein (eTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter through a unique mechanism that requires the recruitment of the equine cyclin T1 (eCT1) cofactor into the viral TAR RNA target element. In vitro studies have demonstrated that mouse fibroblast cell lines (e.g., NIH 3T3 cells) that express the EIAV receptor ELR1 and eCT1 support the productive replication of EIAV. Therefore, we constructed transgenic eCT1- and ELR1-expressing mice to examine whether they support in vivo EIAV replication. FINDINGS: For the first time, we constructed mice transgenic for ELR1 and eCT1. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis confirmed that ELR1 and eCT1 were expressed in the transgenic mouse tissues, particularly in the intestines, spleen and lymph nodes. Consistent with the results of EIAV infection in NIH 3T3 cells expressing ELR1 and eCT1, mouse embryonic fibroblasts (MEFs) from the transgenic mice could support EIAV replication. More importantly, this virus could infect and replicate in mouse blood monocyte-derived macrophages (mMDMs). Macrophages are the principle target cell of EIAV in its natural hosts. Furthermore, after the transgenic mice were inoculated with EIAV, the virus could be detected not only in the plasma of the circulating blood but also in multiple organs, among which, the spleen and lymph nodes were the predominant sites of EIAV replication. Finally, we found that consistent with high viral replication levels, the relevant pathological changes occurred in the spleen and lymph nodes. CONCLUSIONS: Our results show that mice transgenic for ELR1 and eCT1 are susceptible to EIAV infection and replication. Further, EIAV infection can cause lesions on the spleen and lymph nodes, similar to those frequently observed in horses, the natural hosts. Therefore, ELR1 and eCT1 are essential in vivo for EIAV invasion and replication.

Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env, and receptor via distortion of the endoplasmic reticulum.

UNLABELLED: Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the α-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin α-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the α-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level. IMPORTANCE: In previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms, including the reduction of EIAV Gag budding and Env expression, and these activities are dependent on different viperin domains. We especially demonstrate that the overexpression of viperin inhibits EIAV entry by decreasing the level of virus receptor. Therefore, viperin restriction of viruses is determined largely by the dependence of virus on the cellular membrane transportation system.

TRIMe7-CypA, an alternative splicing isoform of TRIMCyp in rhesus macaque, negatively modulates TRIM5α activity.

The existence of innate, host-specific restriction factors is a major obstacle to the development of nonhuman primate models for AIDS studies, and TRIM5α is one of the most important of these restriction factors. In recent years, a TRIM5 chimeric gene that was retrotransposed by a cyclophilin A (CypA) cDNA was identified in certain macaque species. The TRIM5α-CypA fusion protein, TRIMCyp, which was expressed in these monkeys, had lost its restriction ability toward HIV-1. We previously found that TRIMe7-CypA, an alternative splicing isoform of the TRIMCyp transcripts, was expressed in pig-tailed and rhesus macaques but absent in long-tailed macaques. In this study, the anti-HIV-1 activity of TRIMe7-CypA in the rhesus macaque (RhTRIMe7-CypA) was investigated. The over-expression of RhTRIMe7-CypA in CrFK, HeLa and HEK293T cells did not restrict the infection or replication of an HIV-1-GFP reporter virus in these cells. As a positive control, rhesus (rh)TRIM5α strongly inhibited the reporter virus. Intriguingly, the anti-HIV-1 activity of RhTRIM5α was significantly reduced in a dose-dependent manner by the co-repression of RhTRIMe7-CypA. Our data indicate that although the RhTRIMe7-CypA isoform does not appear to restrict HIV-1, it may act as a negative modulator of TRIM family proteins, presumably by competitive inhibition.

Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain.

The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAVFDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAVUK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon ß (IFNß) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAVFDDV13 and was correlated with enhanced TLR3, sELR1 and IFNß expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNß and lowered the level of infection resistance induced by EIAVFDDV13. These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNß, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

Development of antigen capture ELISA for the quantification of EIAV p26 protein.

An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9-62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3-8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3-8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R (2) =0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium.

Avian ANP32A incorporated in avian influenza A virions promotes interspecies transmission by priming early viral replication in mammals.

Species-specific differences in acidic nuclear phosphoprotein 32 family member A (ANP32A) determine the restriction of avian-signature polymerase in mammalian cells. Mutations that evade this restriction, such as PB2-E627K, are frequently acquired when avian influenza A viruses jump from avian hosts to mammalian hosts. However, the mechanism underlying this adaptation process is still unclear. Here, we report that host factor ANP32 proteins can be incorporated into influenza viral particles through combination with the viral RNA polymerase (vPol) and then transferred into targeted cells where they support virus replication. The packaging of the ANP32 proteins into influenza viruses is dependent on their affinity with the vPol. Avian ANP32A (avANP32A) delivered by avian influenza A virions primes early viral replication in mammalian cells, thereby favoring the downstream interspecies transmission event by increasing the total amount of virus carrying adaptive mutations. Our study clarifies one role of avANP32A where it is used by avian influenza virus to help counteract the restriction barrier in mammals.

Comprehensive analysis of the overall codon usage patterns in equine infectious anemia virus.

BACKGROUND: Equine infectious anemia virus (EIAV) is an important animal model for understanding the relationship between viral persistence and the host immune response during lentiviral infections. Comparison and analysis of the codon usage model between EIAV and its hosts is important for the comprehension of viral evolution. In our study, the codon usage pattern of EIAV was analyzed from the available 29 full-length EIAV genomes through multivariate statistical methods. FINDING: Effective number of codons (ENC) suggests that the codon usage among EIAV strains is slightly biased. The ENC-plot analysis demonstrates that mutation pressure plays a substantial role in the codon usage pattern of EIAV, whereas other factors such as geographic distribution and host translation selection also take part in the process of EIAV evolution. Comparative analysis of codon adaptation index (CAI) values among EIAV and its hosts suggests that EIAV utilize the translational resources of horse more efficiently than that of donkey. CONCLUSION: The codon usage bias in EIAV is slight and mutation pressure is the main factor that affects codon usage variation in EIAV. These results suggest that EIAV genomic biases are the result of the co-evolution of genome composition and the ability to evade the host's immune response.

Development and evaluation of a real-time quantitative PCR for the detection of equine infectious anemia virus.

IMPORTANCE: Equine infectious anemia (EIA) has a worldwide distribution and causes significant losses to the equine industry worldwide. A reliable detection method is necessary to control the transmission of EIA virus (EIAV). Currently, most of the available real-time PCR assays, including the qPCR of recommended by WOAH, are developed according to the sequences of European or American EIAV strains; however, the primers and probe sequences have low homology with Asian EIAV strains. To the best of our knowledge, no qPCR method capable of the well detection of Asian EIAV strains, especially Chinese EIAV strains, has been published to date. The development of a sensitive, specific, and rapid qPCR assay for the detection of the EIAV strains is therefore of great importance.

A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC.

The quadrilateral reassortant IAV A/(H1N1) pdm09 is the pathogen responsible for the first influenza pandemic of the 21st century. The virus spread rapidly among hosts causing high mortality within human population. Efficient accumulation of virions is known to be important for the rapid transmission of virus. However, the mechanism by which A/(H1N1) pdm09 promotes its rapid replication has not been fully studied. Here, we found the NS1 of A/(H1N1) pdm09 mediated complete macroautophagy/autophagy, and then facilitated self-replication, which may be associated with the more rapid spread of this virus compared with H1N1WSN and H3N8JL89. We found that the promotion of self-replication could be mainly attributed to NS1pdm09 strongly antagonizing the inhibitory effect of LRPPRC on autophagy. The interaction between NS1pdm09 and LRPPRC competitively blocked the interaction of LRPPRC with BECN1/Beclin1, resulting in increased recruitment of BECN1 for PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and induction of the initiation of autophagy. In conclusion, we uncover the unique molecular mechanism by which A/(H1N1) pdm09 utilizes autophagy to promote self-replication, and we provide theoretical basics for the analysis of the etiological characteristics of the A/(H1N1) pdm09 pandemic and the development of anti-influenza drugs and vaccines.Abbreviations: 293T: human embryonic kidney 293 cells; 293T_LRPPRC: stable LRPPRC expression 293T cells; 3-MA: 3-methyladenine; A549 cells: human non-small cell lung cancer cells; AA: amino acid; ACTB: actin beta; BECN1: beclin 1; BECN1 KO: BECN1 knockout 293T cells; Cal: calyculin A; Co-IP: co-immunoprecipitation; CQ: chloroquine; DC: dendritic cell; Eug: eugenol; GFP: green fluorescent protein; HA: hemagglutinin; HIV: human immunodeficiency virus; IAVs: Influenza A viruses; IFN: interferon; JL89: A/equine/Jilin/1/1989 (H3N8); LAMP2: lysosomal associated membrane protein 2; LRPPRC: leucine rich pentatriicopeptide repeat containing; LRPPRC KO: LRPPRC knockout 293T cells; M2: matrix 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDCK: Madin-Darby canine kidney cells; MOI: multiplicity of infection; MS: mass spectrometry; NP: nucleoprotein; NS1: non-structural protein 1; NS1JL89: non-structural protein 1 of A/equine/Jilin/1/1989 (H3N8); NS1pdm09: non-structural protein 1 of A/(H1N1) pdm09; NS1SC09: non-structural protein 1 of A/Sichuan/2009 (H1N1); NS1WSN: non-structural protein 1 of A/WSN/1933 (H1N1); PB1: polymerase basic protein 1; PB1-F2: alternate reading frame discovered in PB1 gene segment; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PR8: A/PR/8/34 (H1N1); Rapa: rapamycin; RFP: red fluorescent protein; SC09: A/Sichuan/2009 (H1N1); SQSTM1/p62: sequestosome 1; STK4/MST1: serine/threonine kinase 4; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; WHO: World Health Organization; WSN: A/WSN/1933 (H1N1); WSN-NS1JL89: WSN recombinant strain in which NS1 was replaced with that of JL89; WSN-NS1SC09: WSN recombinant strain in which NS1 was replaced with that of SC09.

Comparison of health-related quality of life in different polycystic ovary syndrome phenotypes: A cross-sectional study.

OBJECTIVE: The goal of this research was to evaluate the Health-Related Quality of Life (HRQoL) of Chinese women with varying phenotypes of Polycystic Ovary Syndrome (PCOS) and related factors in order to guide clinical treatment. METHODS: We employed a cross-sectional design that included 221 women with an initial diagnosis of PCOS at the Department of Obstetrics and Gynecology, Tianjin Medical University General Hospital. All subjects were stratified, according to the Rotterdam criteria, into three sub-cohorts: phenotype A (n = 100), phenotype B (n = 60), and phenotype D (n = 61). All patients completed the Modified Polycystic Ovary Syndrome Health Related Quality of Life Questionnaire (MPCOSQ). RESULTS: The three PCOS phenotypes showed no obvious differences in terms of the overall mean MPCOSQ score. However, patients in phenotype D exhibited markedly elevated weight, hirsutism, and acne scores, as compared to phenotypes A and B (p＜0.05). Among all phenotypes, A and B exhibited the worst weight-related HRQoL (3.58 ± 1.89, 3.42 ± 1.99). In contrast, phenotype D displayed the worst emotional disturbance (3.74 ± 1.25), followed by infertility (3.86 ± 1.99). According to the multiple linear regression analysis, worse HRQoL in Phenotypes A and B was significantly associated with excess body weight (Beta = -0.387,-0.626,p＜0.001), hirsutism (Beta = -0.309,-0.269p＜0.01), and abnormal menstruation (Beta = -0.283,-0.304,p＜0.01). In contrast, there was no marked association between HRQoL and hirsutism in Phenotype D. CONCLUSION: Personalized treatment and management must be provided, based on various factors that influence HRQoL among the three PCOS phenotypes. Based on our analysis, the interventions that greatly improve HRQoL of PCOS patients are significant weight loss, combined with anti-androgen administration for phenotypes A and B, and fertility enhancement, combined with menstruation regulation, for phenotype D.