Construction and characterization of a full-length infectious clone of an emerging senecavirus A strain.

Senecavirus A (SVA) is an emerging virus that causes vesicular disease in pigs. Construction of a full-length SVA cDNA clone is crucial for understanding its replication and pathogenesis. Here, we successfully constructed a CMV-promoter-driven infectious cDNA clone of the SVA isolate SVA/GX/CH/2018, which we named rSVA GX01. Sequence comparison between the pSVA GX01 and the parental isolate (SVA/GX/CH/2018) revealed three single-nucleotide differences. Four-week-old piglets were experimentally infected with either the parental virus or the cloned virus. The results showed that the cloned rSVA GX01 displayed weak pathogenicity in 4-week-old pigs compared to the parental virus SVA CH-GX-01-2018. The infectious clone of SVA will serve as a valuable tool for studying the viral replication cycle and for functional analysis of the viral genome.

Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

Near-field antenna measurement based on Rydberg-atom probe.

Current near-field antenna measurement methods are commonly based on metal probes, with the accuracy limited and hard to be optimized due to the drawbacks they suffered, such as large volume, severe metal reflection/interference and complex circuit signal processing in parameter extracting. In this work, a novel method is proposed based on Rydberg atom in the near-field antenna measurement, which can offer a higher accuracy due to its intrinsic character of traceability to electric field. Replacing the metal probe in near-field measurement system by Rydberg atoms contained in a vapor cell (probe), amplitude- and phase- measurements on a 2.389 GHz signal launched out from a standard gain horn antenna are conducted on a near-field plane. They are transformed to far-field pattern and agree well with simulated results and measured results by using a traditional metal probe method. A high precision in longitudinal phase testing with an error below 1.7% can be achieved.

Tunable frequency of a microwave mixed receiver based on Rydberg atoms under the Zeeman effect.

Researchers are interested in the sensor based on Rydberg atoms because of its broad testing frequency range and outstanding sensitivity. However, the discrete frequency detection limits its further employment. We expand the frequency range of microwaves using Rydberg atoms under the Zeeman effect. In such a scheme, the magnetic field is employed as a tool to split and modify adjacent Rydberg level intervals to realize tunable frequency measurement over 100 MHz under 0-31.5 Gauss magnetic field. In this frequency range, the microwave has a linear dynamic variation range of 63 dB, and has achieved a sensitivity of 11.72 µV cm-1Hz-1/2 with the minimum detectable field strength of 17.2 µV/cm.. Compared to the no magnetic field scenario, the sensitivity would not decrease. By theoretical analysis, in a strong magnetic field, the tunable frequency range can be much larger than 100 MHz. The proposed method for achieving tunable frequency measurement provides a crucial tool in radars and communication.

Molecular epidemiological and genetic characterization of pseudorabies virus in Guangxi, China.

Pseudorabies virus (PRV) is an important pathogen that can cause harm to the pig population. Since 2011, there have been a number of large-scale outbreaks of pseudorabies on Chinese farms where animals had been vaccinated with the Bartha-K61 vaccine. In order to understand the epidemiological trend and genetic variations of PRV in Guangxi province, China, 819 tissue samples were collected from swine farms where PRV infection was suspected from 2013 to 2019, and these were tested for infectious wild strains of PRV. The results showed a positive rate of PRV in Guangxi province of 28.21% (231/819). Thirty-six wild-type PRV strains were successfully isolated from PRV-positive tissue samples, and a genetic evolutionary analysis was performed based on the gB, gC, gD, gE, and TK genes. Thirty of the PRV strains were found to be closely related to the Chinese variant strains HeN1-China-2012 and HLJ8-China-2013. In addition, five PRV strains were genetically related to Chinese classical strains, and one isolate was a recombinant of the PRV variant and the vaccine strain Bartha-K61. Amino acid sequence analysis showed that all 36 PRV strains had characteristic variant sites in the amino acid sequences of the gB, gC, gD, and gE proteins. Pathogenicity analysis showed that, compared to classical PRV strains, the PRV variant strains were more pathogenic in mice and had a lower LD50. Taken together, our results show that wild-type PRV infections are common on pig farms in Guangxi province of China and that the dominant prevalent strains were those of the PRV variants. The PRV variant strains also had increased pathogenicity in mice. Our data will provide a useful reference for understanding the prevalence and genetic evolution of PRV in China.

Ammonia in the presence of nano titanium dioxide (nano-TiO2) induces greater oxidative damage in the gill and liver of female zebrafish.

Water pollution caused by a highly hazardous chemical ammonia and a widespread application nanomaterials-nano titanium dioxide (n-TiO2) in nature water has attracted extensive concern of the world. However, the potential joint effects of the two factors are unknown. Aim to investigate the potential interactive effects of ammonia and n-TiO2 and the behind mechanisms, adult female zebrafish (Danio rerio) were co-exposed for 8 weeks by total ammonia nitrogen (TAN; 0, 3, 30 mg/L) and n-TiO2 (0, 0.1, 1 mg/L) in different combination conditions based on a full-factorial design. The analysis of absorption kinetics confirmed that n-TiO2 could absorb free ammonia (NH3) in aqueous solution and the loss rate of free NH3 increased with the rise of n-TiO2 concentration. Consistent with this, free NH3 concentrations in the gill and liver were higher in the presence of n-TiO2 compared to TAN exposure alone. The increases of MDA and PC concentrations in the gill and liver of fish indicated that TAN and n-TiO2 alone or in combination caused oxidative stress. Simultaneously, the activity and transcription of antioxidant enzymes (T-SOD, CuZn-SOD, Mn-SOD, CAT, GPx and GST) as well as antioxidant GSH contents were extensively inhibited by TAN and n-TiO2 via Nrf2-Keap1 signaling. The significant interactive effects of TAN and n-TiO2 were detected on levels of GSH, GST and gstr1 mRNA in the gill, and on levels of GSH, T-SOD, Mn-SOD, CAT levels as well as gpx1a and keap1 mRNAs in the liver, implying synergistic toxic risk of TAN and n-TiO2. The more severe histopathological alterations and higher IBR analysis in co-treatment groups further proved that the existence of n-TiO2 excavated ammonia-induced toxicity in the gill and liver, especially in liver. In conclusion, ammonia and n-TiO2 have a synergistic toxic risk of fish health because ammonia and n-TiO2 cause oxidative-antioxidative imbalance by inducing ROS overproduction.

Multiple resistance mechanisms to penoxsulam in Echinochloa crus-galli from China.

Penoxsulam is an important herbicide for the control of Echinochloa crus-galli (L.) P. Beauv. Two resistant populations 17GA (R1) and 16NXB (R2) showed 17- and 3-fold resistance to penoxsulam, respectively. A known resistance mutation of Trp-574-Leu in ALS gene and enhanced rates of penoxsulam metabolism likely involving GST contribute to penoxsulam resistance in R1 population. This population had resistance to the ALS-inhibitors pyribenzoxim and bispyribac­sodium and the auxin herbicide quinclorac, but was susceptible to ACCase-inhibitors quizalofop-p-ethyl and cyhalofop-butyl. No known mutations in the ALS gene conferring target site resistance to ALS-inhibiting herbicides were presented in R2 population. However, penoxsulam metabolism in R2 plants was about 4-fold greater than in susceptible population 14YC (S0) plants. The enzyme inhibitors piperonyl butoxide, malathion and 4-chloro-7-nitrobenzoxadiazole reversed penoxsulam resistance in this population. GST and P450 enzyme activities and the genes of GST1-1, GST1-2, GST1-3, CYP81A18, CYP81A12, CYP81A21 were increased significantly in R2 population. These results indicate that multiple resistance mechanisms had occurred in E. crus-galli populations in central China and resistance needs to be managed effectively by diverse chemical and non-chemical methods.

Evaluation of Ammonia Nitrogen Exposure in Immune Defenses Present on Spleen and Head-Kidney of Wuchang Bream (Megalobrama amblycephala).

Ammonia is one of the most important environmental factors in aquatic ecosystems. However, there are limited studies on the effects of chronic or long-term ammonia stress and its potential molecular mechanism in fish. This study aimed to investigate the immune response and molecular mechanisms in the spleen and head-kidney of fish following chronic ammonia exposure. Megalobrama amblycephala (9.98 ± 0.48 g) were exposed to different concentrations of total ammonia nitrogen (0-30 mg/L) for 30 days. Ammonia exposure caused significant increases in cortisol levels and decreases in lysozyme and complement 3/4 concentrations in the serum, indicating inhibitory effects of ammonia stress on innate immune responses. Ammonia exposure also induced concentration-dependent increases in ammonia concentrations in tissue, pathological damage and indexes of spleen and head-kidney. Additionally, the contents of immunoglobulin M (IgM), interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) as well as mRNA levels of toll-like receptors (TLRs)/Myeloid differentiation factor 88 (MyD88)-independent signaling molecules in the spleen and head-kidney were significantly downregulated after ammonia exposure. Our findings suggested that chronic ammonia exposure caused the suppression of innate and adaptive immune responses through downregulating TLR/MyD88-independent signaling. Adverse influences of chronic ammonia stress were more severe in the spleen than in the head-kidney.

Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a.

In recent years, the availability of reverse genetics systems for porcine reproductive and respiratory syndrome virus (PRRSV) has created new perspectives for the use of recombinant viruses as expression vectors. Most of these recombinant PRRSV vectors express foreign genes through either an independent transcription unit inserted in ORF1b and ORF2, or in ORF7 and the 3' UTR. The aim of this study was to find an alternative site for foreign gene insertion into the PRRSV genome. Here, we constructed an infectious cDNA clone for a cell-adapted PRRSV strain, GXNN1396-P96. This cDNA-clone-derived recombinant virus (rGXAM) was comparable in its growth kinetics in MARC-145 cells to the parental virus, GX1396-P96. Using the infectious cDNA-clone, we inserted an independent transcription unit in ORF4 and ORF5a to generate a novel PRRSV-based recombinant virus expressing the green fluorescent protein (GFP) gene. Biological characterization of the recombinant virus, rGX45BSTRS-GFP, showed that it maintained similar growth characteristics but produced fewer infectious virions than the parental PRRSV. These data demonstrate that the ORF4 and ORF5a site is able to tolerate the insertion of foreign genes.

Genetic analysis of porcine circovirus type 2 (PCV2) strains between 2002 and 2016 reveals PCV2 mutant predominating in porcine population in Guangxi, China.

BACKGROUND: Porcine circovirus 2-associated disease (PCVAD) is acknowledged as one of the most economically important diseases for the swine industry worldwide. The aim of this study was to characterize and determine the genetic diversity of PCV2 in the porcine population of Guangxi, China. METHODS: The full length genome and open reading frame 2 (ORF2) of 95 PCV2 strains collected from the tissues and sera of pigs that had either died as a result of PCVAD or did not exhibit disease symptoms were analyzed. RESULTS: The results of multiple sequence alignments showed that there is considerable diversity among the PCV2 ORF2 sequences. Phylogenetic analyses based on the complete genome showed that current PCV2 strains in this study could be divided into PCV2a (1/95), PCV2b (39/95), PCV2d (43/95), PCV2e (10/95) and PCV2h (2/95). Among the 5 sub-genotypes, PCV2b was dominant in the porcine population from 2002 to 2008. The newly identified sub-genotype, PCV2d, was seen from 2003 and has increased every year. PCV2b and PCV2d formed two predominant genetic groups circulating in southern China between 2009 and 2013 and the sub-genotype PCV2d has become the dominant virus in China since 2014. CONCLUSIONS: This study reveals the complex genetic diversity of PCV2 and improves our understanding regarding the epidemiological trends of PCV2 sub-genotypes in China.

Molecular epidemiology and viremia of porcine astrovirus in pigs from Guangxi province of China.

BACKGROUND: Porcine astroviruses (PAstVs) are common in pigs worldwide. There are five distinct lineages with each lineage representing a different ancestral origin. Recently, multiple reports have demonstrated the evidence of extra-intestinal infection of PAstVs, but little is known about viremia. RESULTS: In this study, a total of 532 fecal samples and 120 serum samples from healthy pigs were collected and tested from 2013 to 2015 in Guangxi province, China; of these 300/532 (56.4%) and 7/120 (5.8%) of fecal samples tested positive for PAstVs, respectively. Our study revealed that there was wide genetic diversity and high prevalence of the virus in the pig population. All five of the known PAstVs genotypes (1-5) prevailed in the pig population of Guangxi province and were distributed in all age groups of pigs, from suckling piglets to sows, with PAstV2 (47.7%), PAstV1 (26.2%) and PAstV5 (21.5%) seen predominantly. Phylogenetic analysis of partial ORF1b and partial capsid sequences from fecal and serum samples revealed that they were divided into the five lineages. Among these genotypes, based on partial ORF2 genes sequencing 23 strains were grouped as PAstV1, including 6 serum-derived strains, and were regarded as the causative agents of viremia in pigs. CONCLUSIONS: Due to the information regarding the types of PAstV in blood is limit. This is the first report for the presence of PAstV1 in blood and PAstV3 in the feces of nursery pigs of China. This study provides a reference for understanding the prevalence and genetic evolution of PAstVs in pigs in Guangxi province, China. It also provides a new perspective for understanding of the extra-intestinal infection of PAstVs in pigs.

Effect of an 88-amino-acid deletion in nsp2 of porcine reproductive and respiratory syndrome virus on virus replication and cytokine responses in vitro.

Previously, a spontaneous 88-amino-acid (aa) deletion in nsp2 was associated with cell-adaptation of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXM100, which arose during passaging of the highly pathogenic PRRSV (HP-PRRSV) strain JX143 in MARC-145 cells. Here, to elucidate the biological role of this deletion, we specifically deleted the region of a cDNA clone of HP-PRRSV strain JX143 (pJX143) corresponding to these 88 amino acids. The effect of the deletion on virus replication in cultured cells and transcriptional activation of inflammatory cytokines and chemokines in pulmonary alveolar macrophages (PAMs) was examined. Mutant virus with the 88-aa deletion in nsp2 (rJX143-D88) had faster growth kinetics and produced larger plaques in MARC-145 cells than the parental virus (rJX143), suggesting that the deletion enhanced virus replication in MARC-145 cells. In contrast, the overall yield of rJX143 was almost 1 log higher than that of rJX143-D88, suggesting that the 88-aa deletion in nsp2 decreased the production of infectious viruses in PAMs. Infection with the mutant virus with the 88-aa deletion resulted in increased mRNA expression of type I interferon (IFN-α and IFN-ß) and chemokines genes. In addition, the mRNA expression of antiviral genes (ISG15, ISG54 and PKR) regulated by the IFN response was upregulated in PAMs infected with the mutant virus rJX143-D88. Our results demonstrate that virus-specific host immunity can be enhanced by modifying certain nsp2 epitope regions. These findings provide important insights for understanding virus pathogenesis and development of future vaccines.

Construction of a reverse genetic system for porcine astrovirus.

In order to construct a full-length infectious cDNA clone of porcine astrovirus, three fragments covering the complete genome of PAstV1-GX1 strain were amplified by RT-PCR. All three PCR-amplified fragments were cloned into T-Vector pMD19 (Simple), and subsequently assembled into a full-length cDNA clone by subcloning. A silent nucleotide change creating a PstI site was engineered into the full-length cDNA clone to distinguish the rescued virus from the parental virus. Upon transfection of BHK-21 cells with the in vitro transcripts of both the original and constructed cDNAs, typical cytopathic effects were observed on PK-15 cells after serial passaging of the cell supernatant. The construction and recovery of the infectious cDNA clone of porcine astrovirus will provide a valuable experimental system to study the genome function and pathogenesis of astroviruses.

Mutations in a Highly Conserved Motif of nsp1ß Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (nsp1ß) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique -2/-1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ((123)GKYLQRRLQ(131)) reduced the ability of nsp1ß to suppress interferon beta (IFN-ß) activation and also impaired nsp1ß's function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1ß mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-α expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1ß mutants, IFN-α production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-γ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1ß function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCE: PRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1ß was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1ß function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines.

Detection and genetic characterization of canine astroviruses in pet dogs in Guangxi, China.

BACKGROUND: Astroviruses (AstVs) have been reported to infect and cause gastroenteritis in most animal species. Human AstVs were regarded the causative agent of viral diarrhea in children. In dogs, little is known about the epidemiology and clinical significance of AstV infection. FINDINGS: In this study, we collected and tested 253 rectal swabs from pet dogs; of which 64 samples (25.3%) tested positive for AstVs with diarrhea and 15 more samples (5.9%) also was identified as AstVs, however without any clinical signs. Phylogenetic analysis of 39 partial ORF1b sequences from these samples revealed that they are similar to AstVs, which can be subdivided into three lineages. Interestingly, out of the 39 isolates sequenced, 16 isolates are shown to be in the Mamastrovirus 5/canine astrovirus (CAstV) lineage and the remaining 23 isolates displayed higher similarities with known porcine astrovirus (PoAstV) 5 and 2. Further, analysis of 13 capsid sequences from these isolates showed that they are closely clustered with Chinese or Italy CAstV isolates. CONCLUSIONS: The findings indicate that CAstVs commonly circulate in pet dogs, and our sequencing results have shown the genomic diversity of CAstVs leading to increasing number of clusters.

Comparative analysis of routes of immunization of a live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in a heterologous virus challenge study.

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), which infects primarily the respiratory tract of pigs. Thus intranasal (IN) delivery of a potent vaccine-adjuvant formulation is promising. In this study, PRRS-MLV (VR2332) was coadministered ± an adjuvant Mycobacterium vaccae whole cell lysate or CpG ODN through intramuscular (IM) or IN route as a mist, and challenged with a heterologous PRRSV 1-4-4 IN at 42 days post-vaccination (dpv). At 14 and 26 dpv, vaccine viral RNA copies were one log greater in the plasma of PRRS-MLV IM compared to IN vaccinated pigs, and the infectious replicating vaccine virus was detected only in the IM group. In PRRS-MLV ± adjuvant IM vaccinated pigs, reduced viral RNA load and absence of the replicating challenged virus was observed at 7, 10 and 14 days post-challenge (dpc). At 14 dpc, in BAL fluid ≥ 5 log viral RNA copies were detected in all the pig groups, but the replicating challenged virus was undetectable only in IM groups. Immunologically, virus neutralizing antibody titers in the plasma of IM (but not IN) vaccine groups was ≥ 8 against the vaccine and challenged viruses. At 26 dpv, PRRS-MLV IM (without adjuvant) received pigs had significantly increased population of CD4 and CD8 T cells in PBMC. At 14 dpc, relatively increased population of IFN-γ(+) total lymphocytes, NK, CD4, CD8 and γδ T cells were observed in the MLV-IM group. In conclusion, PRRS-MLV IM vaccination induced the virus specific T cell response in pigs, but still it is required to improve its cross-protective efficacy.

Development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is a leading cause of economic burden to the pork industry worldwide. The routinely used modified live PRRS virus vaccine (PRRS-MLV) induces clinical protection, but it has safety concerns. Therefore, in an attempt to develop a safe and protective inactivated PRRSV vaccine, we generated PRRS-virus-like-particles (PRRS-VLPs) containing the viral surface proteins GP5-GP4-GP3-GP2a-M or GP5-M using a novel baculovirus expression system. Our in vitro results indicated that the desired PRRSV proteins were incorporated in both the VLPs preparations based on their reactivity in immunogold electron microscopy and ELISA. To boost their immunogenicity in pigs, we entrapped the PRRS-VLPs in PLGA nanoparticles and coadministered them intranasally with a potent adjuvant. We then evaluated their efficacy in pigs against a viral challenge using a virulent heterologous field isolate. Our results indicated that PRRS-VLPs induced an anamnestic immune response, since we observed boosted IgG and IFN-γ production in vaccinated and virus-challenged animals, but not during the pre-challenge period. Importantly, a two-log reduction in the lung viral load was detected in PRRS-VLP-vaccinated animals. In conclusion, we generated PRRS-VLPs containing up to five viral surface proteins and demonstrated their immunogenicity in pigs, but further studies are required to improve its immunogenicity and efficacy as a vaccine candidate.

Evaluation of humoral immune status in porcine epidemic diarrhea virus (PEDV) infected sows under field conditions.

Porcine epidemic diarrhea virus (PEDV) is an economically devastating enteric disease in the swine industry. The virus infects pigs of all ages, but it cause severe clinical disease in neonatal suckling pigs with up to 100% mortality. Currently, available vaccines are not completely effective and feedback methods utilizing PEDV infected material has variable success in preventing reinfection. Comprehensive information on the levels and duration of effector/memory IgA and IgG antibody secreting B cell response in the intestines and lymphoid organs of PEDV-infected sows, and their association with specific antibody levels in clinical samples such as plasma, oral fluid, and feces is important. Therefore, our goal in this study was to quantify PEDV specific IgA and IgG B cell responses in sows at approximately 1 and 6 months post-infection in commercial swine herds, including parity one and higher sows. Our data indicated that evaluation of both PEDV specific IgA and IgG antibody levels in the plasma and oral fluid (but not feces) samples is beneficial in disease diagnosis. PEDV specific B cell response in the intestine and spleen of infected sows decline by 6 months, and this associates with specific antibody levels in the plasma and oral fluid samples; but the virus neutralization titers in plasma remains high beyond 6 months post-infection. In conclusion, in sows infected with PEDV the presence of effector/memory B cell response and strong virus neutralization titers in plasma up to 6 months post-infection, suggests their potential to protect sows from reinfection and provide maternal immunity to neonates, but challenge studies are required to confirm such responses.

The impact of ammonia and microcystin-LR on neurobehavior and glutamate/gamma-aminobutyric acid balance in female zebrafish (Danio rerio): ROS and inflammation as key pathways.

Ammonia and microcystin-LR (MC-LR) are both toxins that can be in eutrophic waters during cyanobacterial blooms. While previous studies have focused on the effects of ammonia exposure on fish neurobehavioral toxicity, little attention has been given to the effects of MC-LR and combined exposures to both. This study exposed adult female zebrafish to ammonia (30 mg/L) and MC-LR (10 µg/L) alone and in combination for 30 days to investigate their neurotoxic effects and underlying mechanisms. Behavioral results showed that exposure to ammonia and MC-LR, both alone and in combination, led to decreased locomotor activity and increased anxiety in fish. Histomorphological analysis revealed the formation of thrombi and vacuolization in the brain across all exposure groups. Exposure to ammonia and MC-LR resulted in significant increases in MDA contents, decreases in Mn-SOD activities, and alterations in GSH contents compared to the control. Single and combined exposure to ammonia and MC-LR also induced the release of inflammatory factors (IL-1ß and TNF-α) by activating the NOD/NF-κB signaling pathway. Furthermore, both ammonia and MC-LR significantly changed the expression of genes related to the glutamatergic and GABAergic systems, elevated Glu and GABA contents, as well as increased the Glu/GABA ratio, indicating that a shift towards increased Glu levels. Overall, these findings suggested that exposure to MC-LR and ammonia, individually and in combination, could decrease locomotor activity and increase anxiety of female zebrafish. This was likely due to brain damage from over-activated ROS and the release of pro-inflammatory cytokines, which led to a disruption in the balance of glutamatergic and GABAergic systems. However, there was no significant interaction between MC-LR and ammonia in fish neurobehavioral toxicity.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus.

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.