Paraneoplastic Pemphigus/Paraneoplastic Autoimmune Multiorgan Syndrome: Part II. Diagnosis and Management.

In the second part of this Continuing Medical Education article on paraneoplastic pemphigus/paraneoplastic autoimmune multiorgan syndrome (PNP/PAMS), its diagnostic criteria, investigative work-up, and management are reviewed. PNP/PAMS is a rare autoimmune blistering disorder associated with high morbidity and mortality. Recognizing PNP/PAMS's key features and its diagnostic criteria is critical in initiating appropriate work-up. Evaluating PNP/PAMS requires knowledge of its findings on histopathology, direct immunofluorescence, indirect immunofluorescence, and enzyme-linked immunosorbent assay. Lastly, treatments for PNP/PAMS are reviewed with suggestions based on case reports and expert opinions in the literature. LEARNING OBJECTIVES: After completing this learning objective, the reader will be able to identify the criteria necessary for diagnosing paraneoplastic pemphigus (PNP/PAMS), learn how to work-up a diagnosis of PNP/PAMS, and understand important principles in the management of PNP/PAMS.

6-Aryl-quinazolines as novel GABAB receptor positive allosteric modulators.

The systemic use of GABAB orthosteric agonist baclofen might be limited due to its detrimental properties: sedation and motor impairment. In contrast, GABAB positive allosteric modulators produce less adverse effects. Using BHF-177 as a starting point, we found a new active scaffold: the 6-aryl-quinazoline scaffold. Further elaborating the scaffold, we identified several in vitro and in vivo active compounds.

Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits.

Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits. Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases.

Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.

Cytidine base editors, which are composed of a cytidine deaminase fused to clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) nickase, enable the efficient conversion of the C·G base pair to T·A in various organisms. However, the currently used rat apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1(rA1)-based BE3 is often inefficient in target Cs that are immediately downstream of a G (GC context). Here, we observed that, with an 11-nt editing window, an optimized activation-induced cytidine deaminase (AID)-Cas9 fusion can efficiently convert C to T in a variety of sequence contexts in rabbits. Strikingly, the enhanced AID-Cas9 fusion (eAID-BE4max) has significant effectiveness of inducing Tyr p.R299H mutation in GC contexts (from 16.67 to 83.33%) in comparison with BE3 in founder rabbits. Furthermore, the engineered AID-Cas9 variants were produced with reduced bystander activity [eAID (N51G)-BE4max] and increased genome-targeting scope (eAID-NG-BE4max). Overall, this work provides a series of improved tools that were generated using optimized AID-Cas9 fusions and associated engineered variants that can be used for efficient and versatile C-to-T base editing, especially in GC contexts.-Liu, Z., Shan, H., Chen, S., Chen, M., Zhang, Q., Lai, L., Li, Z. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.

Small molecules block the interaction between porcine reproductive and respiratory syndrome virus and CD163 receptor and the infection of pig cells.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the pork industry globally. PRRS is caused by PRRS virus (PRRSV). Currently there are no effective treatments against this swine disease. METHODS: Through artificial intelligence molecular screening, we obtained a set of small molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which is a cell surface receptor specific for PRRSV infection. These compounds were screened using a cell-based bimolecular fluorescence complementation (BiFC) assay, and the function of positive hit was further evaluated and validated by PRRSV-infection assay using porcine alveolar macrophages (PAMs). RESULTS: Using the BiFC assay, we identified one compound with previously unverified function, 4-Fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide (designated here as B7), that significantly inhibits the interaction between the PRRSV glycoprotein (GP2a or GP4) and the CD163-SRCR5 domain. We further demonstrated that compound B7 inhibits PRRSV infection of PAMs, the primary target of PRRSV in a dose-dependent manner. B7 significantly inhibited the infection caused by both type I and type II PRRSV strains. Further comparison and functional evaluation of chemical compounds structurally related to B7 revealed that the 3-(morpholinosulfonyl)aniline moiety of B7 or the 3-(piperidinylsulfonyl)aniline moiety in a B7 analogue is important for the inhibitory function against PRRSV infection. CONCLUSIONS: Our study identified a novel strategy to potentially prevent PRRSV infection in pigs by blocking the PRRSV-CD163 interaction with small molecules.

Porcine interferon lambda 3 (IFN-λ3) shows potent anti-PRRSV activity in primary porcine alveolar macrophages (PAMs).

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious viral disease of swine. At present, there are vaccines for the control of PRRSV infection, but the effect is not satisfactory. The recombination of attenuated vaccines causes significant difficulties with the prevention and control of PRRSV. Type III interferons (IFNs), also called IFN-λs, were newly identified and showed potent antiviral activity within the mucosal surface and immune organs. RESULTS: Therefore, primary porcine alveolar macrophages (PAMs) were used for this investigation. To this end, we found that the replication of PRRSV in PAMs was significantly reduced after pre-treatment with IFN-λ3, and such inhibition was dose- and time-dependent. The plaque formation of PRRSV abrogated entirely, and virus yields were reduced by four orders of magnitude when the primary PAMs were treated with IFN-λ3 at 1000 ng/ml. In addition, IFN-λ3 in our study was able to induce the expression of interferon-stimulated genes 15 (ISG15), 2'-5'-oligoadenylate synthase 1 (OAS1), IFN-inducible transmembrane 3 (IFITM3), and myxoma resistance protein 1(Mx1) in primary PAMs. CONCLUSIONS: IFN-λ3 had antiviral activity against PRRSV and can stimulate the expression of pivotal interferon-stimulated genes (ISGs), i.e., ISG15, Mx1, OAS1, and IFITM3. So, IFN-λ3 may serve as a useful antiviral agent.

Health mediators as members of multidisciplinary group practice: lessons learned from a primary health care model programme in Hungary.

BACKGROUND: A Model Programme of primary care group practices was implemented in Hungary between 2013 and 2017 - where virtually all GPs had worked in single practices - aiming to increase preventive service uptake and reduce inequalities based on a bilateral agreement between the Swiss and Hungarian governments. Group practices employed a wide variety of health professionals as well as support workers called health mediators. Employment of the latter was based on two decades of European experience of health mediators who specifically facilitate access to and use of health services in Roma minority groups. Health mediators had been recruited from local communities, received training on the job, and were tasked to increase uptake of new preventive services provided by the group practices by personal contacts in the local minority populace. The paper describes the contribution of the work of health mediators to the uptake of two new services provided by group practices. METHODS: Quantitative analysis of depersonalized administrative data mandatorily reported to the Management of the Programme during 43 months of operation was carried out on the employment of health mediators and their contribution to the uptake of two new preventive services (health status assessment and community health promoting programmes). RESULTS: 80% of all clients registered with the GPs participated at health status assessment by invitation that was 1.3-1.7 times higher than participation at the most successful national screening programmes in the past 15 years. Both the number of mediator work minutes per client and participation rate at health status assessment, as well as total work time of mediators and participants at community health events showed high correlation. Twice as many Roma minority patients were motivated for service use by health mediators compared to all patients. The very high participation rate reflects the wide impact of health mediators who probably reached not only Roma minority, but vulnerable population groups in general. CONCLUSION: The future of general practices lays in multidisciplinary teams in which health mediators recruited from the serviced communities can be valuable members, especially in deprived areas.

Evaluation of phytotoxic effects of cationic polyacrylamide polymers: implication for the use of sludges as organic soil amendments in assisted phytostabilization.

Sludges, as biosolids, are organic soil amendments commonly used in assisted phytostabilitation. Extensive studies on their environmental impacts exists, particularly for improper land application and contents of trace elements, organic compounds and pathogens, but not for their content of cationic polyacrylamide polymers (C-PAMs). Direct toxicity of C-PAMs on aquatic organisms has been demonstrated but scarce information about plant toxicity is available. In this study, the effect of C-PAMs on early plant growth was evaluated by means of standard toxicity assays. Firstly, increasing doses of C-PAMs were applied as solutions to seeds of Avena sativa, Lactuca sativa and Solanum lycopersycum to evaluate germination and root elongation. Secondly, the effect of increasing doses of C-PAMs spiked in pig manure and mixed with sand (75 t ha-1 dry base) was evaluated on the emergence, radicle elongation and biomass of A. sativa. Results showed high phytotoxicity of C-PAMs in solutions above 1,000 mg L-1, but no effect was detected when spiked into experimental substrate; a significant effect was only observed above 5,000 mg L-1 for radicle elongation, aerial biomass, and radicle biomass. Results demonstrate direct phytotoxic effects of C-PAMs, which can be mitigated when spiked into an organic (pig manure) and mineral (sand) matrix.

Examining the Effects of (α4)3(ß2)2 Nicotinic Acetylcholine Receptor-Selective Positive Allosteric Modulator on Acute Thermal Nociception in Rats.

Neuronal nicotinic acetylcholine receptor (nAChR)-based therapeutics are sought as a potential alternative strategy to opioids for pain management. In this study, we examine the antinociceptive effects of 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrazol-4-yl)isoxazole (CMPI), a novel positive allosteric modulator (PAM), with preferential selectivity to the low agonist sensitivity (α4)3(ß2)2 nAChR and desformylflustrabromine (dFBr), a PAM for α4-containing nAChRs. We used hot plate and tail flick tests to measure the effect of dFBr and CMPI on the latency to acute thermal nociceptive responses in rats. Intraperitoneal injection of dFBr, but not CMPI, dose-dependently increased latency in the hot plate test. In the tail flick test, the effect achieved at the highest dFBr or CMPI dose tested was only <20% of the maximum possible effects reported for nicotine and other nicotinic agonists. Moreover, the coadministration of dFBr did not enhance the antinociceptive effect of a low dose of nicotine. Our results show that the direct acute effect of dFBr is superior to that for CMPI, indicating that selectivity to (α4)3(ß2)2 nAChR is not advantageous in alleviating responses to acute thermal nociceptive stimulus. However, further studies are necessary to test the suitability of (α4)3(ß2)2 nAChR-selective PAMs in chronic pain models.

A technique to measure respirator protection factors against aerosol particles in simulated workplace settings using portable instruments.

The aim of this study was to develop a new method to measure respirator protection factors for aerosol particles using portable instruments while workers conduct their normal work. The portable instruments, including a set of two handheld condensation particle counters (CPCs) and two portable aerosol mobility spectrometers (PAMSs), were evaluated with a set of two reference scanning mobility particle sizers (SMPSs). The portable instruments were mounted to a tactical load-bearing vest or backpack and worn by the test subject while conducting their simulated workplace activities. Simulated workplace protection factors (SWPFs) were measured using human subjects exposed to sodium chloride aerosols at three different steady state concentration levels: low (8x103 particles/cm3), medium (5x104 particles/cm3), and high (1x105 particles/cm3). Eight subjects were required to pass a quantitative fit test before beginning a SWPF test for the respirators. Each SWPF test was performed using a protocol of five exercises for 3 min each: (1) normal breathing while standing; (2) bending at the waist; (3) a simulated laboratory-vessel cleaning motion; (4) slow walking in place; and (5) deep breathing. Two instrument sets (one portable instrument {CPC or PAMS} and one reference SMPS for each set) were used to simultaneously measure the aerosol concentrations outside and inside the respirator. The SWPF was calculated as a ratio of the outside and inside particles. Generally, the overall SWPFs measured with the handheld CPCs had a relatively good agreement with those measured with the reference SMPSs, followed by the PAMSs. Under simulated workplace activities, all handheld CPCs, PAMSs, and the reference SMPSs showed a similar GM SWPF trend, and their GM SWPFs decreased when simulated workplace movements increased. This study demonstrated that the new design of mounting two handheld CPCs in the tactical load-bearing vest or mounting one PAMS unit in the backpack permitted subjects to wear it while performing the simulated workplace activities. The CPC shows potential for measuring SWPFs based on its light weight and lack of major instrument malfunctions.

An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam.

OBJECTIVE: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. METHODS: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. RESULTS: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). CONCLUSION: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

Acute Administration of Desformylflustrabromine Relieves Chemically Induced Pain in CD-1 Mice.

Neuronal nicotinic acetylcholine receptors are cell membrane-bound ion channels that are widely distributed in the central nervous system. The α4ß2 subtype of neuronal nicotinic acetylcholine receptor plays an important role in modulating the signaling pathways for pain. Previous studies have shown that agonists, partial agonists, and positive allosteric modulators for the α4ß2 receptors are effective in relieving pain. Desformylflustrabromine is a compound that acts as an allosteric modulator of α4ß2 receptors. The aim of this study was to assess the effects of desformylflustrabromine on chemically induced pain. For this purpose, the formalin-induced pain test and the acetic acid-induced writhing response test were carried out in CD-1 mice. Both tests represent chemical assays for nociception. The results show that desformylflustrabromine is effective in producing an analgesic effect in both tests used for assessing nociception. These results suggest that desformylflustrabromine has the potential to become a clinically used drug for pain relief.

In vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. From a previous small-scale screen we identified an effective DNA-based short antisense oligonucleotide (AS-ON) targeting viral NSP9, which could inhibit PRRSV replication in both Marc-145 cells and pulmonary alveolar macrophages (PAMs). The objective of this study was to explore the strategy of incorporating locked nucleic acids (LNAs) to achieve better inhibition of PRRSV replication in vitro. METHODS: The effective DNA-based AS-ON (YN8) was modified with LNAs at both ends as gap-mer (LNA-YN8-A) or as mix-mer (LNA-YN8-B). Marc-145 cells or PAMs were infected with PRRSV and subsequently transfected. RESULTS: Compared with the DNA-based YN8 control, the two AS-ONs modified with LNAs were found to be significantly more effective in decreasing the cytopathic effect (CPE) induced by PRRSV and thus in maintaining cell viability. LNA modifications conferred longer lifetimes to the AS-ON in the cell culture model. Viral ORF7 levels were more significantly reduced at both RNA and protein levels as shown by quantitative PCR, western blot and indirect immunofluorescence staining. Moreover, transfection with LNA modified AS-ON reduced the PRRSV titer by 10-fold compared with the YN8 control. CONCLUSION: Taken together, incorporation of LNA into AS-ON technology holds higher therapeutic promise for PRRS control.

Discovery of dual positive allosteric modulators (PAMs) of the metabotropic glutamate 2 receptor and CysLT1 antagonists for treating migraine headache.

Pyridylmethylsulfonamide series were the first reported example of positive allosteric modulators (PAM) of the mGlu2 receptor. The hydroxyacetophenone scaffold is a second series of mGlu2 PAMs we have identified. This series of molecules are potent mGlu2 potentiators and possess significant CysLT1 (cysteinyl leukotriene receptor 1) antagonist activity, showing in vivo efficacy in a dural plasma protein extravasation (PPE) model of migraine. In this paper, we describe the dual SAR, pharmacokinetics and preclinical in vivo efficacy data for a tetrazole containing hydroxyacetophenone scaffold.

Successful use of Bruton's kinase inhibitor, ibrutinib, to control paraneoplastic pemphigus in a patient with paraneoplastic autoimmune multiorgan syndrome and chronic lymphocytic leukaemia.

We present the case of a 51-year-old man who developed paraneoplastic pemphigus (PNP) in the context of chronic lymphocytic leukemia (CLL). His CLL was successfully controlled with ibrutinib. Concurrently, there was significant improvement of his PNP, suggesting that ibrutinib may be a very useful addition to the treatment options in this potentially life-threatening autoimmune disorder.

Difluorocyclobutylacetylenes as positive allosteric modulators of mGluR5 with reduced bioactivation potential.

Schizophrenia is a serious illness that affects millions of patients and has been associated with N-methyl-d-aspartate receptor (NMDAR) hypofunction. It has been demonstrated that activation of metabotropic glutamate receptor 5 (mGluR5) enhances NMDA receptor function, suggesting the potential utility of mGluR5 positive allosteric modulators (PAMs) in the treatment of schizophrenia. Herein we describe the optimization of an mGluR5 PAM by replacement of a phenyl with aliphatic heterocycles and carbocycles as a strategy to reduce bioactivation in a biaryl acetylene chemotype. Replacement with a difluorocyclobutane followed by further optimization culminated in the identification of compound 32, a low fold shift PAM with reduced bioactivation potential. Compound 32 demonstrated favorable brain uptake and robust efficacy in mouse novel object recognition (NOR) at low doses.

7TM X-ray structures for class C GPCRs as new drug-discovery tools. 1. mGluR5.

We illustrate, with a focus on mGluR5, how the recently published, first X-ray structures of mGluR 7TM domains, specifically those of mGluR1 and mGluR5 complexed with negative allosteric modulators (NAMs), will begin to influence ligand- (e.g., drug- or sweetener-) discovery efforts involving class C GPCRs. With an extensive docking study allowing full ligand flexibility and full side chain flexibility of all residues in the ligand-binding cavity, we have predicted and analyzed the binding modes of a variety of structurally diverse mGluR5 NAM ligands, showing how the X-ray structures serve to effectively rationalize each ligand's binding characteristics. We demonstrated that the features that are inherent in our earlier overlay model are preserved in the protein structure-based docking models. We identified structurally diverse compounds, which potentially act as mGluR NAMs, and revealed binding-site differences by performing high-throughput docking using a database of approximately six million structures of commercially available compounds and the mGluR1 and mGluR5 X-ray structures. By comparing the 7TM domains of the mGluR5 and mGluR1 X-rays structures, we identified selectivity factors within group I of the mGluRs. Similarly, using homology models that we built for mGluR2 and mGluR4, we have identified the factors leading to the selectivity between group I and groups II and III for ligands occupying the deepest portion of the mGluR5 binding cavity. Finally, we have proposed a structure-based explanation of the pharmacological switching within a set of positive allosteric modulators (PAMs) and their corresponding, very close NAM analogs.

Bio-remediation of Pb and Cd polluted soils by switchgrass: A case study in India.

INTRODUCTION: In the present study bioremediation potential of a high biomass yielding grass, Panicum virgatum (switchgrass), along with plant associated microbes (AM fungi and Azospirillum), was tested against lead and cadmium in pot trials. METHODS: A pot trial was set up in order to evaluate bioremediation efficiency of P. virgatum in association with PAMs (Plant Associated Microbes). Growth parameters and bioremediation potential of endomycorrhizal fungi (AMF) and Azospirillum against different concentrations of Pb and Cd were compared. RESULTS: AM fungi and Azospirillum increased the root length, branches, surface area, and root and shoot biomass. The soil pH was found towards neutral with AMF and Azospirillum inoculations. The bioconcentration factor (BCF) for Pb (12 mg kg(-1)) and Cd (10 mg kg(-1)) were found to be 0.25 and 0.23 respectively and translocation index (Ti) was 17.8 and 16.7 respectively (approx 45% higher than control). CONCLUSIONS: The lower values of BCF and Ti, even at highest concentration of Pb and Cd, revealed the capability of switchgrass of accumulating high concentration of Pb and Cd in the roots, while preventing the translocation of Pb and Cd to aerial biomass.

Non-infectious immune complexes downregulate the production of interferons and tumor necrosis factor-α in primary porcine alveolar macrophages in vitro.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) has been harming the pig industry worldwide for nearly 40 years. Although scientific researchers have made substantial efforts to explore PRRSV pathogenesis, the immune factors influencing PRRSV infection still need to be better understood. Infectious virus-antibody immune complexes (ICs) formed by PRRSV and sub-or non-neutralizing antibodies specific for PRRSV may significantly promote the development of PRRS by enhancing PRRSV replication through antibody-dependent enhancement. However, nothing is known about whether PRRSV infection is affected by non-infectious ICs (NICs) formed by non-pathogenic/infectious antigens and corresponding specific antibodies. Here, we found that PRRSV significantly induced the transcripts and proteins of interferon-α (IFN-α), IFN-ß, IFN-γ, IFN-λ1, and tumor necrosis factor-α (TNF-α) in vitro primary porcine alveolar macrophages (PAMs) in the early stage of infection. Our results showed that NICs formed by rabbit-negative IgG (RNI) and pig anti-RNI specific IgG significantly reduced the transcripts and proteins of IFN-α, IFN-ß, IFN-γ, IFN-λ1, and TNF-α in vitro PAMs and significantly elevated the transcripts and proteins of interleukine-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) in vitro PAMs. NICs-mediated PRRSV infection showed that NICs not only significantly decreased the induction of IFN-α, IFN-ß, IFN-γ, IFN-λ1, and TNF-α by PRRSV but also significantly increased the induction of IL-10 and TGF-ß1 by PRRSV and considerably enhanced PRRSV replication in vitro PAMs. Our data suggested that NICs could downregulate the production of antiviral cytokines (IFN-α/ß/γ/λ1 and TNF-α) during PRRSV infection in vitro and facilitated PRRSV proliferation in its host cells by inhibiting innate antiviral immune response. This study elucidated one novel immune response to PRRSV infection, which would enhance our understanding of the pathogenesis of PRRSV.