Hematopoietic stem cells and lineage cells undergo dynamic alterations under microgravity and recovery conditions.

Spaceflight leads to health risks including bone demineralization, skeletal muscle atrophy, cardiovascular dysfunction, and disorders of almost all physiologic systems. However, the impacts of microgravity on blood lineage cells and hematopoietic stem cells (HSCs) in vivo are largely unknown. In this study, we analyzed peripheral blood samples from 6 astronauts who had participated in spaceflight missions and found significant changes in several cell populations at different time points. These dynamic alterations of lineage cells and the role of HSCs were further studied in a mouse model, using hindlimb unloading (HU) to simulate microgravity. Large reductions in the frequency of NK cells, B cells, and erythrocyte precursors in the bone marrow of the HU mice were observed, together with an increased frequency of T cells, neutrophils, and HSCs. T cell levels recovered faster than those of B cells and erythrocyte precursors, whereas the recovery rates of NK cells and granulocytes were slow. In addition, competitive reconstitution experiments demonstrated the impaired function of HSCs, although these changes were reversible. Deep sequencing showed changes in the expression of regulatory molecules important for the differentiation of HSCs. This study provides the first determination of altered HSC function under simulated microgravity in vivo. The impairment of HSC function and differentiation provides an explanation for the immune disorders that occur under simulated microgravity. Thus, our findings demonstrated that spaceflight and simulated microgravity disrupt the homeostasis of immune system and cause dynamic alterations on both HSCs and lineage cells.-Cao, D., Song, J., Ling, S., Niu, S., Lu, L., Cui, Z., Li, Y., Hao, S., Zhong, G., Qi, Z., Sun, W., Yuan, X., Li, H., Zhao, D., Jin, X., Liu, C., Wu, X., Kan, G., Cao, H., Kang, Y., Yu, S., Li, Y. Hematopoietic stem cells and lineage cells undergo dynamic alterations under microgravity and recovery conditions.

Lrp5 and Lrp6 are required for maintaining self-renewal and differentiation of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have the capacity for self-renewal to maintain the HSCs' pool and the ability for multilineage differentiation, which are responsible for sustained production of multiple blood lineages. The regulation of HSC development is controlled precisely by complex signal networks and hematopoietic microenvironment, which has been termed the HSCs' niche. The Wnt signaling pathway is one of a variety of signaling pathways that have been involved in HSC self-renewal and maintenance. Previous studies are indeterminant on the regulation of adult HSCs upon canonical Wnt signaling pathways because of the different experimental systems and models used. In this study, we generated the conditional knockout Wnt coreceptor low-density lipoprotein receptor-related protein 5 (Lrp5) and low-density lipoprotein receptor-related protein 6 (Lrp6) mice in adult hematopoiesis via Vav-Cre Loxp system. Inactivation of Lrp5 and -6 in a hematopoietic system diminished the pool of HSCs, but there were no obvious defects in mature immune cells. Lrp5 and -6 double deficiency HSCs showed intrinsic defects in self-renewal and differentiation due to reduced proliferation and increased quiescence of the cell cycle. Analysis of HSC gene expression suggested that the quiescence regulators were significantly up-regulated, such as Egr1, Cdkn1a, Nr4a1, Gata2, Junb and Btg2, and the positive cell cycle regulators were correspondingly down-regulated, such as Ccna2 and Ranbp1. Taken together, we investigated the roles of Lrp5 and -6 in HSCs by functional and bioinformatic assays, and we demonstrated that Lrp5 and -6 are required for the self-renewal and differentiation of adult HSCs. The canonical Wnt pathway may contribute to maintaining the HSC pool and regulate the differentiation of adult HSCs by controlling cell cycle gene regulatory module.-Liu, J., Cui, Z., Wang, F., Yao, Y., Yu, G., Liu, J., Cao, D., Niu, S., You, M., Sun, Z., Lian, D., Zhao, T., Kang, Y., Zhao, Y., Xue, H.-H., Yu, S. Lrp5 and Lrp6 are required for maintaining self-renewal and differentiation of hematopoietic stem cells.

Prostaglandin-E2 deficiency during late pregnancy and the associated increase in interleukin-1ß derived from periaortic lymph nodes lead to abortion.

Prostaglandin E2 (PGE2) is a hormone with many physiological functions. During pregnancy, it is generally believed that there is a high level of PGE2 at the final stage of pregnancy, which induces the contraction of uterine smooth muscle and promotes the occurrence of childbirth. However, we find that high PGE2 levels are present throughout late pregnancy in mice, not just during childbirth, and that PGE2 deficiency induced by indomethacin during late pregnancy causes damage to the placental labyrinth and eventually leads to abortion. Interestingly, the damage is closely related to inflammation, which involves the role of inflammatory factors produced by the periaortic lymph nodes (PLNs) near the uterus. Further, through RNA sequencing, we reveal that PLNs produce a large amount of interleukin-1ß (IL-1ß) when exposed to PGE2 deficiency, which causes damage to the placental labyrinth, probably via destroying the extracellular matrix. Finally, events leading to abortion following indomethacin administration are effectively prevented by supplementing PGE2 or by PLN removal. These results suggest that high levels of PGE2 during late pregnancy protect fetuses from inflammatory damage related to IL-1ß. This work suggests a new role of PGE2 during late pregnancy and may provide potential therapeutic strategies for pathological pregnancy.

Long noncoding RNA Malat1 is not essential for T cell development and response to LCMV infection.

Long noncoding RNAs (lncRNAs) are emerging as critical mediators of various biological processes in the immune system. The current data showed that the lncRNA Malat1 is highly expressed in T cell subsets, but the function of Malat1 in T cell remains unclear. In this study, we detected the T cell development and both CD8+ and CD4+ T cell response to LCMV infection using Malat1-/- mice model. To our surprise, there were no significant defects in thymocytes at different developmental stages and the peripheral T cell pool with ablation of Malat1. During LCMV infection, Malat1-/- mice exhibited normal effector and memory CD8+ T cells as well as TFH cells differentiation. Our results indicated that Malat1 is not essential for T cell development and T cell-mediated antiviral response though it expresses at very high level in different T cell populations.

Dexamethasone promotes tolerance in vivo by enriching CD11clo CD40lo tolerogenic macrophages.

We previously showed that antigen immunization in the presence of the immunosuppressant dexamethasone (a strategy we termed "suppressed immunization") could tolerize established recall responses of T cells. However, the mechanism by which dexamethasone acts as a tolerogenic adjuvant has remained unclear. In the present study, we show that dexamethasone enriches CD11c(lo) CD40(lo) macrophages in a dose-dependent manner in the spleen and peripheral lymph nodes of mice by depleting all other CD11c(+) CD40(+) cells including dendritic cells. The enriched macrophages display a distinct MHC class II (MHC II)(lo) CD86(hi) phenotype. Upon activation by antigen in vivo, CD11c(lo) CD40(lo) macrophages upregulate IL-10, a classic marker for tolerogenic antigen-presenting cells, and elicit a serum IL-10 response. When presenting antigen in vivo, these cells do not elicit recall responses from memory T cells, but rather stimulate the expansion of antigen-specific regulatory T cells. Moreover, the depletion of CD11c(lo) CD40(lo) macrophages during suppressed immunization diminishes the tolerogenic efficacy of the treatment. These results indicate that dexamethasone acts as a tolerogenic adjuvant partly by enriching the CD11c(lo) CD40(lo) tolerogenic macrophages.

Tolerogenic DNA vaccine for prevention of autoimmune ovarian disease.

DNA vaccines have been widely used to induce immune responses against molecular targets. In this study, we explored the possibility of using DNA vaccine combined with the immunosuppressant FK506 (tacrolimus) to antigen-specifically suppress unwanted immune responses and prevent autoimmune ovarian disease. To that end, we immunized C57BL/6 mice with a DNA vaccine encoding mouse zona pellucida 3 (ZP3) together with FK506. The immunization induced ZP3-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), which suppressed the induction of ZP3-specific delayed-type hypersensitivity in the animals. Significantly, the immunization also protected the animals from experimentally induced autoimmune ovarian disease. These results suggest that DNA vaccination in the presence of FK506 may be used to induce Treg cells and prevent AOD.

SRSF1 Deficiency Impairs the Late Thymocyte Maturation and the CD8 Single-Positive Lineage Fate Decision.

The underlying mechanisms of thymocyte development and lineage determination remain incompletely understood, and the emerging evidences demonstrated that RNA binding proteins (RBPs) are deeply involved in governing T cell fate in thymus. Serine/arginine-rich splicing factor 1 (SRSF1), as a classical splicing factor, is a pivotal RBP for gene expression in various biological processes. Our recent study demonstrated that SRSF1 plays essential roles in the development of late thymocytes by modulating the T cell regulatory gene networks post-transcriptionally, which are critical in response to type I interferon signaling for supporting thymocyte maturation. Here, we report SRSF1 also contributes to the determination of the CD8+ T cell fate. By specific ablation of SRSF1 in CD4+CD8+ double positive (DP) thymocytes, we found that SRSF1 deficiency impaired the maturation of late thymocytes and diminished the output of both CD4+ and CD8+ single positive T cells. Interestingly, the ratio of mature CD4+ to CD8+ cells was notably altered and more severe defects were exhibited in CD8+ lineage than those in CD4+ lineage, reflecting the specific function of SRSF1 in CD8+ T cell fate decision. Mechanistically, SRSF1-deficient cells downregulate their expression of Runx3, which is a crucial transcriptional regulator in sustaining CD8+ single positive (SP) thymocyte development and lineage choice. Moreover, forced expression of Runx3 partially rectified the defects in SRSF1-deficient CD8+ thymocyte maturation. Thus, our data uncovered the previous unknown role of SRSF1 in establishment of CD8+ cell identity.

Efficient induction of CD25- iTreg by co-immunization requires strongly antigenic epitopes for T cells.

BACKGROUND: We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40 low IL-10 high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg). However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction. RESULTS: In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes. CONCLUSIONS: Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.

Tcf1 Sustains the Expression of Multiple Regulators in Promoting Early Natural Killer Cell Development.

T cell factor 1 (Tcf1) is known as a critical mediator for natural killer (NK) cell development and terminal maturation. However, its essential targets and precise mechanisms involved in early NK progenitors (NKP) are not well clarified. To investigate the role of Tcf1 in NK cells at distinct developmental phases, we employed three kinds of genetic mouse models, namely, Tcf7fl/flVavCre/+, Tcf7fl/flCD122Cre/+ and Tcf7fl/flNcr1Cre/+ mice, respectively. Similar to Tcf1 germline knockout mice, we found notably diminished cell number and defective development in BM NK cells from all strains. In contrast, Tcf7fl/flNcr1Cre/+ mice exhibited modest defects in splenic NK cells compared with those in the other two strains. By analyzing the published ATAC-seq and ChIP-seq data, we found that Tcf1 directly targeted 110 NK cell-related genes which displayed differential accessibility in the absence of Tcf1. Along with this clue, we further confirmed that a series of essential regulators were expressed aberrantly in distinct BM NK subsets with conditional ablating Tcf1 at NKP stage. Eomes, Ets1, Gata3, Ikzf1, Ikzf2, Nfil3, Runx3, Sh2d1a, Slamf6, Tbx21, Tox, and Zeb2 were downregulated, whereas Spi1 and Gzmb were upregulated in distinct NK subsets due to Tcf1 deficiency. The dysregulation of these genes jointly caused severe defects in NK cells lacking Tcf1. Thus, our study identified essential targets of Tcf1 in NK cells, providing new insights into Tcf1-dependent regulatory programs in step-wise governing NK cell development.

CD40-expressing plasmid induces anti-CD40 antibody and enhances immune responses to DNA vaccination.

BACKGROUND: Various approaches have been used to improve the efficacy of DNA vaccination, including the incorporation of molecular adjuvants. Because the CD40 ligand-CD40 interaction plays a major role in initiating immune responses, we sought to develop a molecular adjuvant targeting this interaction. METHODS AND RESULTS: We immunized mice with a foot-and-mouth disease virus DNA vaccine, pcD-VP1, together with a CD40-expressing plasmid, pcD-CD40. We found that pcD-CD40 induced anti-CD40 antibodies, which temporally correlated with the augmented production of anti-VP1 antibody. pcD-CD40 similarly augmented the humoral response of another DNA vaccine that targets hepatitis B virus, and passive transfer of anti-CD40 antisera also showed a similar effect. Furthermore, the pcD-CD40-elicited anti-CD40 antibodies were able to activate the CD40 signal pathway in antigen-presenting cells in vitro, which led to the maturation of dendritic cells (DCs) and DC-mediated T cell activation. Thus, pcD-CD40 augments DNA vaccination by inducing anti-CD40 antibodies, which in turn promotes T cell activation. CONCLUSIONS: This is the first reported 'proadjuvant' that augments DNA vaccination indirectly by eliciting agonistic antibodies.

Increasing a robust antigen-specific cytotoxic T lymphocyte response by FMDV DNA vaccination with IL-9 expressing construct.

Various chemokines and cytokines as adjuvants can be used to improve efficacy of DNA vaccination. In this study, we sought to investigate if a DNA construct expressing IL-9 (designed as proV-IL9) as a molecular adjuvant enhance antigen specific immune responses elicited by the pcD-VP1 DNA vaccination. Mice immunized with pcD-VP1 combined with proV-IL9 developed a strong humoral response. In addition, the coinoculation induced significant higher level of antigen-specific cell proliferation and cytotoxic response. This agreed well with higher expression level of IFN-gamma and perforin in CD8+ T cells, but not with IL-17 in these T cells. The results indicate that IL-9 induces the development of IFN-gamma-producing CD8+ T cells (Tc1), but not the IL-17-producing CD8+ T cells (Tc17). Up-regulated expressions of BCL-2 and BCL-XL were exhibited in these Tc1 cells, suggesting that IL-9 may trigger antiapoptosis mechanism in these cells. Together, these results demonstrated that IL-9 used as molecular adjuvant could enhance the immunogenicity of DNA vaccination, in augmenting humoral and cellular responses and particularly promoting Tc1 activations. Thus, the IL-9 may be utilized as a potent Tc1 adjuvant for DNA vaccines.

FK506 as an adjuvant of tolerogenic DNA vaccination for the prevention of experimental autoimmune encephalomyelitis.

BACKGROUND: DNA vaccination is a strategy that has been developed primarily to elicit protective immunity against infection and cancer. METHODS: DNA vaccine was used, in conjunction with an immunosuppressant, to tolerize harmful autoimmunity. RESULTS: Immunization of C57BL/6 mice with MOG(35-55), a myelin oligodendrocyte glycoprotein-derived peptide, and FK506 (Tacrolimus) as a tolerogenic adjuvant stimulated regulatory dendritic cells, induced antigen-specific regulatory T cells (Treg), and protected the animals from subsequent induction of experimental autoimmune encephalomyelitis (EAE). After EAE induction, there were fewer lymphocytes, including fewer T helper 17 cells, and more Treg infiltrating the spinal cord in the immunized mice compared to in control mice. Furthermore, at the peak of the EAE manifestation, CD4 T cells in the immunized mice showed decreased expression of interferon-gamma and interleukin (IL)-17, but not IL-4, in treated mice. CONCLUSIONS: DNA vaccination, when applied with an immunosuppressant as adjuvant, can induce antigen-specific tolerance and prevent autoimmune disease.

Depleting intratumoral CD4+CD25+ regulatory T cells via FasL protein transfer enhances the therapeutic efficacy of adoptive T cell transfer.

One strategy for improving adoptive therapy is preconditioning the host immune environment by depleting CD4(+)CD25(+) regulatory T cells (Treg) suppressive to antitumor responses. Given that Treg increase, or selectively accumulate, within tumors and are sensitive to FasL-mediated apoptosis, we test here the hypothesis that inducing apoptosis of intratumoral Treg using FasL may improve adoptive T cell therapy. We show that FasL applied intratumorally via protein transfer decreases intratumoral Treg via inducing apoptosis in these cells. Significantly, we show that the use of FasL prior to the infusion of tumor-reactive CD8(+) T cells enhances the therapeutic efficacy of adoptive T cell transfer against established tumors, which is mediated by persistent, systemic antitumor immunity. Intratumoral FasL protein transfer also results in neutrophil infiltration of tumor. However, we show that intratumoral immunodepletion of neutrophils does not abolish the effect of FasL on adoptive transfer. Rather, the effect of FasL is completely abolished by cotransfer of Treg, isolated from the tumor-draining lymph nodes. Hence, our study shows for the first time that using FasL to predeplete intratumoral Treg provides a useful means for optimizing adoptive therapy.

[Suppression of the antigen-specific T cell immune response by co-immunization with the HBV DNA vaccine and recombinant HBsAg].

OBJECTIVE: To explore a new therapeutic strategy against acute hepatitis B and fulminant hepatitis B, we studied effect of co-immunization with HBV DNA and HBsAg on the T cell proliferation reaction. METHODS: We immunized the BALB/ c mice with HBV DNA vaccine (pcDS2) plus HBsAg by intramuscular injection. The immunization was performed on week 0, 2 and 4. The anti-HBs(IgG)antibody titer, T lymphocyte proliferation reaction , and the expression of IL-10 and Foxp3 in CD3 T cell were detected on week 6. RESULTS: The anti-HBs IgG titer induced by pcDS2 plus HBsAg group was higher than that induced by pcDS2, or HBsAg alone. Compared to mice immunized with pcDS2, or HBsAg alone, the stimulated index (SI) of T cell proliferation induced by the pcDS2 plus HBsAg group tested by MTH methods decreased. Besides, the immune suppression of T cell proliferation response induced by co-immunization group was further confirmed by flow cytometry. Finally, the expression of IL-10 and Foxp3 in CD3+ T cell was up-regulated in the co-immunization group significantly. CONCLUSION: The co-immunization of HBV DNA vaccine and HBsAg can induce the humoral immune response, but cannot induce antigen specific T cell proliferation reaction. Besides, the immune suppression induced by co-immunization may be correlated with the expression of IL-10 and Foxp3.

Induction of regulatory T cells by physiological level estrogen.

Naturally occurring CD4+CD25+ regulatory T cells (Treg) exert an important role in mediating maternal tolerance to the fetus during pregnancy, and this effect might be regulated via maternal estrogen secretion. Although estrogen concentration in the pharmaceutical range has been shown to drive expansion of CD4+CD25+ Treg cells, little is known about how and through what mechanisms E2 within the physiological concentration range of pregnancy affects this expansion. Using in vivo and in vitro mouse models in these experiments, we observed that E2 at physiological doses not only expanded Treg cell in different tissues but also increased expression of the Foxp3 gene, a hallmark for CD4+CD25+ Treg cell function, and the IL-10 gene as well. Importantly, our results demonstrate that E2, at physiological doses, stimulated the conversion of CD4+CD25- T cells into CD4+CD25+ T cells which exhibited enhanced Foxp3 and IL-10 expression in vitro. Such converted CD4+CD25+ T cells had similar regulatory function as naturally occurring Treg cells, as demonstrated by their ability to suppress naïve T cell proliferation in a mixed lymphocyte reaction. We also found that the estrogen receptor (ER) exist in the CD4+CD25- T cells and the conversion of CD4+CD25- T cells into CD4+CD25+ T cells stimulated by E2 could be inhibited by ICI182,780, a specific inhibitor of ER(s). This supports that E2 may directly act on CD4+CD25- T cells via ER(s). We conclude that E2 is a potential physiological regulatory factor for the peripheral development of CD4+CD25+ Treg cells during the implantation period in mice.

Protective immune responses against foot-and-mouth disease virus by vaccination with a DNA vaccine expressing virus-like particles.

To display antigenic protein on the surface of virus-like particles (VLPs) presents a potentially powerful strategy for vaccine development. We genetically engineered the major capsid protein VP1 of foot-and-mouth disease virus (FMDV) into the predominant epitope C of HBV core gene to yield a chimeric core-VP1 VLP. The VLP was successfully expressed in HeLa cells transfected with core-VP1 DNA construct. Compared with a regular VP1 DNA construct, immunization with core-VP1 DNA induced significantly higher levels of antigen-specific IgG production, T cell proliferation, cytotoxic T lymphocyte response, and cytokine production in mice. Most importantly, the level of neutralizing antibody elicited by core-VP1 immunization was significantly higher than that with VP1 DNA immunization, which correlated well with animal protection level from subsequent live FMDV challenge. Thus, immunization with chimeric VLP induces higher efficacy and provides an attractive DNA vaccine strategy for controlling FMDV infection in future.

Enhanced protective efficacy and reduced viral load of foot-and-mouth disease DNA vaccine with co-stimulatory molecules as the molecular adjuvants.

To improve efficacy of DNA vaccination, various approaches have been developed, including the use of plasmid expressing co-stimulatory molecules as molecular adjuvants. In this study, we investigated whether co-inoculation of a construct expressing either 4-1BBL or OX40L as the molecular adjuvant with FMDV DNA vaccine, pcD-VP1, can increase immune responses and protective efficacies. Compared to the group immunized with pcD-VP1 alone, the co-inoculation of either molecular adjuvant induced a higher ratio of IgG2a/IgG1, higher levels of expression of IFN-gamma in CD4(+) and CD8(+) T cells and antigen-specific CTL responses, and more importantly provided an enhanced protection against the live FMDV challenge in animals. Concurrently, 4-1BBL as the molecular adjuvant dramatically reduced the viral loads of FMDV in vivo after the challenge. Together, the results demonstrate that co-stimulatory molecules 4-1BBL and OX40L can enhance the antigen-specific cell-mediated responses elicited by VP1 DNA vaccine and provide an enhanced protective efficacy with the reduced viral loads.

Mushroom lectin overcomes hepatitis B virus tolerance via TLR6 signaling.

Currently, chronic hepatitis B virus (HBV) infection remains a serious public health problem in the world. Recombinant HBV vaccine, as a preventive strategy against HBV infection, generates high antibody level, but it is not effective to activate innate and cellular immunity for chronic HBV infection therapy. Lectins from mushroom are natural and active proteins which have been shown important biological functions. However, little is known about the immunological mechanism engaged by mushroom lectins. Here we report that, lectin from Pleurotus ostreatus (POL) stimulated innate response by activating Toll-like receptor 6 signal pathway of dendritic cells. Subsequently POL enhanced HBV specific antibody level and follicular helper T cells response which overcame HBV tolerance in transgenic mice. This study suggests a novel mechanism for POL acting on immune response and a therapeutic approach to break HBV tolerance.

Efficacy of modified levamisole adjuvant on inactivated virus vaccine.

To improve efficacy, especially for the cell-mediated response to inactivated viral vaccines, a modified levamisole (LMS) adjuvant formulation, designated LMS+, was evaluated for its efficacy in mice and chickens, using Newcastle Disease Virus (NDV) as a model pathogen. Compared with oil adjuvant, the killed NDV in LMS+ induced a significantly higher helper T cell type 1 response, as shown by higher levels of interleukin-2, interferon-gamma, T cell proliferation, and delayed-type hypersensitivity responses, without sacrificing the level of IgG production in mice. In addition, vaccine in LMS+ formulation increased the expression of MHC and costimulatory molecules as well as the number of CD11c+ dendritic cells, suggesting that the better response to the LMS+ formulation occurs partly via the maturation of dendritic cells and activation of MHC-antigen presentation and costimulation. Furthermore, this formulation provides 100% protection in chickens after challenge with a lethal dose of virulent NDV strain F48E9 at 1000 ELD50 (50% egg lethal dose). These results demonstrated that modified LMS+ adjuvant could be used to improve both humoral and cell-mediated responses for inactivated viral vaccines and its development as an effective inactivated viral vaccine is warranted.

Correction: Developing a Triple Transgenic Cell Line for High-Efficiency Porcine Reproductive and Respiratory Syndrome Virus Infection.

[This corrects the article DOI: 10.1371/journal.pone.0154238.].