Early weaning and biological sex shape long-term immune and metabolic responses in pigs.

During the early pre and postnatal life, host and environmental factors can impart a major influence on immune development, thus shaping lifelong disease resistance. Two major factors known to influence immune function and mortality in animals and people are early life stress and biological sex. How these two factors interact to shape long-term immune development and later life disease risk is poorly understood. Here we investigated how early weaning, a common early life stressor in pigs, and biological sex impacts long-term systemic inflammatory responses and hypothalamic-pituitary-adrenal axis (HPA axis) activation later in life. Ten-week-old female (F), intact-male (IM) and castrated-male (CM) pigs that were randomly assigned to early weaning (EW) and later weaning (LW) (at 15 or 28 days of age, respectively) were intramuscularly injected with either saline vehicle or lipopolysaccharide (LPS) to induce a systemic inflammatory response. Complete blood counts (CBC), proinflammatory cytokines, cortisol, testosterone, estradiol, and rectal temp were measured at 0 h, 2 h, and 4 h post-LPS challenge. At 4 h post-LPS, peritoneal fluid (PF) and white blood cells (WBC) were collected for differential analysis. LPS challenge significantly increased rectal temp and plasma cortisol level in all treatment groups. Together, the CBC results and immune cell counts in peritoneal cavity indicated that EW-F exhibited greater systemic immune response characterized by increased neutrophils to lymphocytes ratio (NLR) and enhanced neutrophil trafficking to the peritoneal cavity. Early weaning had an opposite effect on IM and CM pigs, which exhibited a suppressed LPS-induced neutrophil migration. Early weaning induced significantly greater cortisol responses only in IM pigs indicating a heightened HPA axis responses in EW-IM. how early weaning and biological sex affect immune and stress responses in pigs. Together, these results demonstrate that early weaning and biological sex and castration shape later life immune responses in pigs and provides insight into potential mechanisms driving sex differences in later life inflammatory disease risk and mortality.

ZFP36 ring finger protein like 1 significantly suppresses human coronavirus OC43 replication.

CCCH-type zinc figure proteins (ZFP) are small cellular proteins that are structurally maintained by zinc ions. Zinc ions coordinate the protein structure in a tetrahedral geometry by binding to cystine-cystine or cysteines-histidine amino acids. ZFP's unique structure enables it to interact with a wide variety of molecules including RNA; thus, ZFP modulates several cellular processes including the host immune response and virus replication. CCCH-type ZFPs have shown their antiviral efficacy against several DNA and RNA viruses. However, their role in the human coronavirus is little explored. We hypothesized that ZFP36L1 also suppresses the human coronavirus. To test our hypothesis, we used OC43 human coronavirus (HCoV) strain in our study. We overexpressed and knockdown ZFP36L1 in HCT-8 cells using lentivirus transduction. Wild type, ZFP36L1 overexpressed, and ZFP36L1 knockdown cells were each infected with HCoV-OC43, and the virus titer in each cell line was measured over 96 hours post-infection (p.i.). Our results show that HCoV-OC43 replication was significantly reduced with ZFP36L1 overexpression while ZFP36L1 knockdown significantly enhanced virus replication. ZFP36L1 knockdown HCT-8 cells started producing infectious virus at 48 hours p.i. which was an earlier timepoint as compared to wild -type and ZFP36L1 overexpressed cells. Wild-type and ZFP36L1 overexpressed HCT-8 cells started producing infectious virus at 72 hours p.i. Overall, the current study showed that overexpression of ZFP36L1 suppressed human coronavirus (OC43) production.

Determining the association between gut microbiota and its metabolites with higher intestinal Immunoglobulin A response.

Immunoglobulin A (IgA) is one of the important and most abundant immunoglobulins which neutralize invading pathogens at mucosal sites. Gut microbial community and their metabolites which are responsible for higher IgA are poorly known. The current study was carried out to determine those microbial community and their metabolites. Twenty-two healthy, 26 days wean piglets were used in the study. After 10 days of weaning, piglets were divided into two groups. Group 1 with significantly higher fecal IgA while group 2 with significantly lower IgA concentrations from each other. Both groups were analyzed for the fecal inflammatory cytokine, fecal microbial community using 16S ribosomal sequencing, and microbial metabolites using GC-MS. Results showed that Firmicutes and Bacteroidetes constituted 90.56% of the microbiome population in the fecal matter of pigs with higher IgA concentration while pigs with lower fecal IgA had Firmicutes and Bacteroidetes abundance as of 95.56%. Pigs with higher IgA had significantly higher Bacteroidota and Desulfobacterota populations, while significantly lower Firmicutes and Firmicutes/ Bacteroidota ratio (p <0.05). Roughly at the species level, animals with higher fecal IgA concentration had significantly higher bacteria which are associated with gut inflammation and infectious such Prevotella spp and Lachnospiraceae AC2044. Pigs with higher IgA had comparatively lower short-chain fatty acid (SCFA) such as acetic acid, butyric, formic acid, isovaleric acid, and propionic acid which has been associated with gut immune tolerance and immune homeostasis.

Developmental alterations of intestinal SGLT1 and GLUT2 induced by early weaning coincides with persistent low-grade metabolic inflammation in female pigs.

Early-life adversity (ELA) is linked with the increased risk for inflammatory and metabolic diseases in later life, but the mechanisms remain poorly understood. Intestinal epithelial glucose transporters sodium-glucose-linked transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) are the major route for intestinal glucose uptake but have also received increased attention as modulators of inflammatory and metabolic diseases. Here, we tested the hypothesis that early weaning (EW) in pigs, an established model of ELA, alters the development of epithelial glucose transporters and coincides with elevated markers of metabolic inflammation. The jejunum and ileum of 90-day-old pigs previously exposed to EW (16 days wean age), exhibited reduced SGLT1 activity (by ∼ 30%, P < 0.05) than late weaned (LW, 28 days wean age) controls. In contrast, GLUT2-mediated glucose transport was increased (P = 0.003) in EW pigs than in LW pigs. Reciprocal changes in SGLT1- and GLUT2-mediated transport coincided with transporter protein expression in the intestinal brush-border membranes (BBMs) that were observed at 90 days and 150 days of age. Ileal SGLT1-mediated glucose transport and BBM expression were inhibited by the ß-adrenergic receptor (ßAR) blocker propranolol in EW and LW pigs. In contrast, propranolol enhanced ileal GLUT2-mediated glucose transport (P = 0.015) and brush-border membrane vesicle (BBMV) abundance (P = 0.035) in LW pigs, but not in EW pigs. Early-weaned pigs exhibited chronically elevated blood glucose and C-reactive protein (CRP) levels, and adipocyte hypertrophy and upregulated adipogenesis-related gene expression in visceral adipose tissue. Altered development of intestinal glucose transporters by EW could underlie the increased risk for later life inflammatory and metabolic diseases.NEW & NOTEWORTHY These studies reveal that early-life adversity in the form of early weaning in pigs causes a developmental shift in intestinal glucose transport from SGLT1 toward GLUT2-mediated transport. Early weaning also induced markers of metabolic inflammation including persistent elevations in blood glucose and the inflammatory marker CRP, along with increased visceral adiposity. Altered intestinal glucose transport might contribute to increased risk for inflammatory and metabolic diseases associated with early-life adversity.

Bovine viral diarrhea virus compromises Neutrophil's functions in strain dependent manner.

Bovine viral diarrhea virus (BVDV) infection is a major problem that results in economically important diseases of the cattle industry worldwide. The two major consequences of this disease are persistent infection and immune dysfunction. A number of studies have been done to determine the underline mechanisms of BVDV-induced immune dysfunction, in particular targeting antigen-presenting cells, T- and B- cells and cytokine gene expression. However, little research has focused Eon the effect of BVDV on neutrophils. Neutrophils are one of the predominant leukocytes circulating in blood and are considered the first line of defense in the innate immune system along with macrophages. Neutrophils not only eliminate the invading bacteria but also activate innate as well as adaptive immune responses. Therefore, compromised neutrophil function would affect both arms of immune system and caused immune suppression. In the current study, we used virus strains from both BVDV-1 and BVDV-2 species. Including a highly virulent non-cytopathic type 2a BVDV (ncp BVDV2a-1373), moderately virulent non-cytopathic type 2a (ncp BVDV2a 28508-5), and a pair of non-cytopathic type 1b BVDV (ncp BVDV1b TGAN) and cytopathic type 1b BVDV (cp BVDV1b TGAC) strain isolated from a case of mucosal disease. The highly virulent ncp BVDV2a-1373 significantly increased neutrophil apoptosis. However, none of the other BVDV strains affected neutrophil viability. All BVDV strains used significantly reduced CD18 and L-selectin expression on neutrophils as well as their oxidative burst and neutrophil extracellular traps (NET) activity. Cp BVDV significantly reduced neutrophil's phagocytic activity but ncp BVDV did not have any effect on it. On the other hand, ncp BVDV significantly increased neutrophil's CD14 expression and chemotactic activity while cp BVDV did not show any effect either on neutrophil's CD14 expression or on chemotactic activity. In conclusion, BVDV affected neutrophils variability and functional activity in strain dependent manner. Results of the current study will further help in understanding the pathophysiology of different BVDV strains.

The Effect of Bovine Viral Diarrhea Virus (BVDV) Strains and the Corresponding Infected-Macrophages' Supernatant on Macrophage Inflammatory Function and Lymphocyte Apoptosis.

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study, we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis, bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells, lymphocytes or BL-3 cells. By protein electrophoresis, several protein bands were unique for high-virulence, 1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta, IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages.

Comparative humoral immune response against cytopathic or non-cytopathic bovine viral diarrhea virus infection.

Bovine viral diarrhea virus (BVDV) infection causes immune dysfunction. The current study investigated the effect of cytopathic (cp) or noncytopathic (ncp) strains of BVDV on immunomodulation by the levels of total serum immunoglobulin G (IgG), the IgG1, IgG2, BVDV neutralizing antibodies and total white blood cell (WBC) count. Twenty (20) BVDV seronegative dairy calves (5-6 months old) were divided in two groups of ten. The animals were infected with either a cp or ncp virus isolated from the same animal (ncp BVDV1b-TGAN or cp BVDV1b-TGAC). One group of 10 was infected with ncp TGAN while the other group of 10 was infected with cp TGAC. Calves infected with cp BVDV had a significant decrease in total IgG as well as IgG1 concentration at 7 days post infection (DPI) that recovered by 21 DPI (total IgG) and 35 DPI (IgG1), respectively. There was no effect of ncp BVDV infection on total IgG concentration in the first 7 days of infection (DOI); however, IgG1 concentration was significantly reduced and IgG2 concentration was significantly increased at 7 DOI. At 35 DPI, ncp TGAN-infected calves had significantly higher total IgG, IgG1 as well as IgG2 compared to cp TGAC-infected calves. Ncp BVDV induced higher BVDV homologous and heterologous neutralizing antibodies compared to the cp BVDV strain. Calves infected with ncp BVDV had significantly reduced WBC counts at 7 DPI that recovered by 14 DPI. Overall, these findings indicate that humoral immunosuppression occurs early following BVDV infection with the largest effect on IgG1 levels.

Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability.

BACKGROUND: Psychological stress and heightened mast cell (MC) activation are linked with important immunologic disorders, including allergy, anaphylaxis, asthma, and functional bowel diseases, but the mechanisms remain poorly defined. We have previously demonstrated that activation of the corticotropin-releasing factor (CRF) system potentiates MC degranulation responses during IgE-mediated anaphylaxis and psychological stress through corticotropin-releasing factor receptor subtype 1 (CRF1) expressed on MCs. OBJECTIVE: In this study we investigated the role of corticotropin-releasing factor receptor subtype 2 (CRF2) as a modulator of stress-induced MC degranulation and associated disease pathophysiology. METHODS: In vitro MC degranulation assays were performed with bone marrow-derived mast cells (BMMCs) derived from wild-type (WT) and CRF2-deficient (CRF2-/-) mice and RBL-2H3 MCs transfected with CRF2-overexpressing plasmid or CRF2 small interfering RNA. In vivo MC responses and associated pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF2-/-, and MC-deficient KitW-sh/W-sh knock-in mice. RESULTS: Compared with WT mice, CRF2-/- mice exhibited greater serum histamine levels and exacerbated IgE-mediated anaphylaxis and colonic permeability. In addition, CRF2-/- mice exhibited increased serum histamine levels and colonic permeability after acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF2 expressed on MCs suppresses store-operated Ca2+ entry signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient KitW-sh/W-sh mice systemically engrafted with WT and CRF2-/- BMMCs demonstrated the functional importance of MC CRF2 in modulating stress-induced pathophysiology. CONCLUSIONS: MC CRF2 is a negative global modulator of stimuli-induced MC degranulation and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis.

Site-directed non-covalent polymer-drug complexes for inflammatory bowel disease (IBD): Formulation development, characterization and pharmacological evaluation.

Inflammatory Bowel Diseases (IBD) is a debilitating condition that affects ~70,000 new people every year and has been described as "an expensive disease with no known cure". In addition, IBD increases the risk of developing colon cancer. The current therapeutics for IBD focus on the established disease where the immune dysfunction and bowel damage have already occurred but do not prevent or delay the progression. The current work describes a polymer-based anti-inflammatory technology (Ora-Curcumin-S) specifically targeted to the luminal side of the colon for preventing and/or treating IBD. Ora-Curcumin-S was prepared by molecular complexation of curcumin with a hydrophilic polymer Eudragit® S100 using co-precipitation method. Curcumin interacted with the polymer non-covalently and existed in an amorphous state as demonstrated by various physicochemical techniques. Ora-Curcumin-S is a polymer-drug complex, which is different than solid dispersions in that the interactions are retained even after dissolving in aqueous buffers. Ora-Curcumin-S was >1000 times water soluble than curcumin and importantly, the enhanced solubility was pH-dependent, which was observed only at pHs above 6.8. In addition, around 90% of Ora-Curcumin-S was stable in phosphate buffer, pH 7.4 and simulated intestinal fluid after 24 h, in contrast to 10-20% unformulated curcumin. Ora-Curcumin-S inhibited Monophosphoryl Lipid-A and E. coli induced inflammatory responses in dendritic cells and cells over expressing Toll-Like Receptor-4 (TLR-4) suggesting that Ora-Curcumin-S is a novel polymer-based TLR-4 antagonist. Preliminary pharmacokinetics in mice showed targeted delivery of soluble curcumin to the colon lumen without exposing to the systemic circulation. Furthermore, Ora-Curcumin-S significantly prevented colitis and associated injury in a mouse model of ulcerative colitis estimated using multiple preclinical parameters: colonoscopy pictures, body weight, colon length, colon edema, spleen weight, pro-inflammatory signaling and independent pathological scoring. Overall, the outcome of this innovative proof-of-concept study provides an exciting and locally-targeted pathway for a dietary therapeutic option for IBD patients to help limit colonic inflammation and thus susceptibility to colitis-associated colorectal cancer.

Dendritic Cell-Targeted Nanovaccine Delivery System Prepared with an Immune-Active Polymer.

Targeting dendritic cells (DCs), either ex vivo (Ex. Sipuleucel-T) or in vivo, for stimulating cellular immunity has been a leading approach for cancer vaccines. We have rationally engineered a nanoparticle (NP)-based delivery system for vaccines (InAc-NPs) using inulin acetate (InAc) as the polymer to target DCs. The material and the antigen-encapsulated InAc-NPs (∼190 nm in diameter) were characterized for their physicochemical properties. As a potent vaccine adjuvant, InAc-NPs activated TLR4 on multiple immune cells, including DCs and primary swine and human cells, to secrete various cytokines as detected by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. In addition, InAc-NPs promoted the maturation of DCs as observed by a decreased phagocytic ability and enhanced capability to activate various maturation markers (MHC-I, MHC-II, CD40, and CD80) quantified using flow cytometry. In mice, the InAc-NPs produced strong serum antibody titers (total IgG, IgG1, and IgG2a) against the encapsulated antigen (ovalbumin) similar to complete Freund's adjuvant. Additionally, as a dose-sparing delivery system, antigen delivered through InAc-NPs generated higher antibody titers (IgG1, 1.57 times; IgG-total, 1.66 times; and IgG2a, 29.8 times) even at 100 times less antigen dose. High amounts of cytokines representing both humoral (IL4 and IL10) and cell-mediated (IL2 and IFN-γ) immunities were secreted from splenocytes of mice immunized with InAc-NPs. Importantly, InAc-NPs provided complete protection in 100% of the vaccinated mice from metastasis of intravenously injected melanoma cells (B16-F10) to lungs. In addition, the InAc-NPs were cleared from the injection site within 30 h of injection (in vivo imaging) and displayed no toxicity at the injection site (histology). The current study demonstrates that the multifunctional InAc-based nanovaccine delivery system has potential applications in cancer immunotherapy and delivering vaccines against various infectious diseases.

BVDV Npro protein mediates the BVDV induced immunosuppression through interaction with cellular S100A9 protein.

The innate immune response is a vital part of the body's antiviral defense system. The innate immune response is initiated by various receptor interactions, including danger associated molecular patterns (DAMPs). The S100A9 is a member of the DAMPs protein family and, is released by activated phagocytic cells such as neutrophils, monocytes, macrophages or endothelial cells, and S100A9 induces its effect through TLR4/MyD88 pathway. Bovine viral diarrhea virus (BVDV) is one of the major devastating disease in the cattle industry worldwide. It shows its effect through immunosuppression and develops persistent infection in calves born from infected cows. The current study revealed that BVDV potentially induced immunosuppression by the interaction of BVDV Npro protein with cellular S100A9 protein. The Inhibition of S100A9 protein expression by small interfering RNA (siRNA) enhanced the virus replication in infected cells. Overexpression of bovine S100A9 enhanced the ncpBVDV2a 1373 mediated Type-I interferon production. A co-immunoprecipitation experiment demonstrated a strong interaction between ncp BVDV2a 1373 Npro protein and cellular S100A9 protein. This suggested that BVDV Npro reduced the S100A9 protein availability/activity in infected cells, resulting in reduced Type-I interferon production. A further study of S100A9-BVDV interaction will be need for better understanding of BVDV pathophysiology.

Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate.

Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. RESULTS: showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p<0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.

Weaning stress and gastrointestinal barrier development: Implications for lifelong gut health in pigs.

The gastrointestinal (GI) barrier serves a critical role in survival and overall health of animals and humans. Several layers of barrier defense mechanisms are provided by the epithelial, immune and enteric nervous systems. Together they act in concert to control normal gut functions (e.g., digestion, absorption, secretion, immunity, etc.) whereas at the same time provide a barrier from the hostile conditions in the luminal environment. Breakdown of these critical GI functions is a central pathophysiological mechanism in the most serious GI disorders in pigs. This review will focus on the development and functional properties of the GI barrier in pigs and how common early life production stressors, such as weaning, can alter immediate and long-term barrier function and disease susceptibility. Specific stress-related pathophysiological mechanisms responsible for driving GI barrier dysfunction induced by weaning and the implications to animal health and performance will be discussed.

Discovery of inulin acetate as a novel immune-active polymer and vaccine adjuvant: synthesis, material characterization, and biological evaluation as a toll-like receptor-4 agonist.

Vaccine adjuvants are an essential part of modern vaccine design, especially against intracellular pathogens such as M. tuberculosis, malarial parasite, HIV, influenza virus and Ebola. The present work offers a unique approach to designing novel vaccine adjuvants by identifying polymers that mimic "pathogen associated molecular patterns" (PAMPS) and engineering an immune-active particulate vaccine delivery system that uses the polymer. By using this strategy, we report the discovery of the first plant polymer based toll-like receptor-4 (TLR-4) agonist, inulin acetate (InAc). InAc was synthesised from the plant polysaccharide inulin. Inulin acetate as a polymer and particles prepared using InAc were characterised using various physicochemical techniques. The TLR-4 agonistic activity of InAc was established in multiple immune, microglial, dendritic, peripheral blood mononuclear (human and swine) and genetically modified epithelial cells (HEK293) that exclusively express TLR-4 on their surface. InAc activated all the above-mentioned cells to release proliferative cytokines; however, InAc failed to activate when the were cells either pre-incubated with a TLR-4 specific antagonist or isolated from mice deficient in adapter proteins involved in TLR signalling (Mal/MyD88). Antigen encapsulated microparticles prepared with TLR-4 agonist InAc mimicked pathogens to offer improved antigen delivery to dendritic cells compared to soluble antigen (47 times) or antigen encapsulated poly(lactic-co-glycolic acid) (PLGA) particles (1.57 times). In conclusion, InAc represents a novel polymer-based modern vaccine adjuvant targeting specific signalling pathways of the innate immune system, which could be formulated into a platform vaccine delivery system against cancer and viral diseases.

Immune response to bovine viral diarrhea virus--looking at newly defined targets.

Bovine viral diarrhea virus (BVDV) has long been associated with a wide variety of clinical syndromes and immune dysregulation, many which result in secondary bacterial infections. Current understanding of immune cell interactions that result in activation and tolerance are explored in light of BVDV infection including: depletion of lymphocytes, effects on neutrophils, natural killer cells, and the role of receptors and cytokines. In addition, we review some new information on the effect of BVDV on immune development in the fetal liver, the role of resident macrophages, and greater implications for persistent infection.

Characterization of the cytopathic BVDV strains isolated from 13 mucosal disease cases arising in a cattle herd.

Bovine viral diarrhea virus (BVDV) is a positive single stranded RNA virus belonging to the Pestivirus genus of the Flaviviridae family. BVDV has a wide host range that includes most ruminants. Noncytopathic (ncp) BVDV may establish lifelong persistent infections in calves following infection of the fetus between 40 and 120 days of gestation. Cytopathic (cp) BVDV strains arise from ncp strains via mutations. The most common cp mutations are insertions of RNA derived from either host or a duplication of viral sequences into the region of the genome coding for the NS2/3 protein. Superinfection of a persistently infected animal with a cp virus can give rise to mucosal disease, a condition that is invariably fatal. A herd of 136 bred 3-year old cows was studied. These cows gave birth to 41 PI animals of which 23 succumbed to mucosal disease. In this study, we characterized the ncp and cp viruses isolated from 13 of these animals. All viruses belonged to the BVDV type 2a genotype and were highly similar. All the cp viruses contained an insertion in the NS2/3 coding region consisting of the sequences derived from the transcript encoding a DnaJ protein named Jiv90. Comparison of the inserted DnaJ regions along with the flanking viral sequences in the insertion 3' end of the 13 cp isolates revealed sequence identities ranging from 96% to 99% with common borders. This suggested that one animal likely developed a cp virus that then progressively spread to the other 12 animals. Interestingly, when the inserted mammalian gene replicated within viral genome, it showed conservation of the same conserved motifs between the different species, which may indicate a role for these motifs in the insertion function within the virus genome. This is the first characterization of multiple cp bovine viral diarrhea virus isolates that spread in a herd under natural conditions.

Cleavage site stability of Egyptian highly pathogenic avian influenza viruses in backyard chickens during 2009-2011.

PURPOSE: Two distinguishable subclades of H5N1 (classic and variant strains) are cocirculating among the poultry populations in Egypt despite the intensive vaccination programs. A study to investigate the genetic relationship between avian influenza virus (AIV) isolates from backyard chickens in Sharkia (2009-2011), subclades, and commercially available vaccines was carried out. METHODS: Forty-eight suspected AIV infected birds were clinically examined and used for virus isolation followed by reverse transcription-polymerase chain reaction. Four H5N1 virus isolates were sequenced and analyzed. The intravenous pathogenicity index (IVPI) of three AIV isolates was determined. RESULTS: Thirty-four hemagglutinating viral agents (30 AIV subtype H5N1 and 4 Newcastle disease virus) were detected. Both the nucleotide and amino acid sequence identities of four H5N1 virus isolates (SHZA-0412/2009, SHZA-0801/2010, SHMK-1903/2010, and SHAH-1403/2011) were high--98.4-99.7% and 100%, respectively--indicative of their genetic homogeneity. The hemagglutinin cleavage site characterization revealed the presence of multiple basic amino acids (-PQRERRRKKR/GL-) of the highly pathogenic phenotype. These results were supported by IVPI in chickens of 2.69-2.90. The similarity of our isolates with H5N1 AIV vaccine strains (93.9-95.1%) was higher than that with H5N2 strains (77.8-91.9%). The divergence of four sequences with classic and variant lineages is 2-2.7% and 2.3-3%, respectively, with two amino acid substitutions (A249P and N251Y). CONCLUSION: Genetic characterization and IVPI data of backyard H5N1 isolates are indicative of a highly pathogenic avian influenza virus with hemagglutinin cleavage site constancy and two amino acids substitutions with Egyptian classic and variant lineages, suggesting a beginning of antigenic drift.

The effect of bovine viral diarrhea virus (BVDV) strains on bovine monocyte-derived dendritic cells (Mo-DC) phenotype and capacity to produce BVDV.

BACKGROUND: Dendritic cells (DC) are important antigen presentation cells that monitor, process, and present antigen to T cells. Viruses that infect DC can have a devastating impact on the immune system. In this study, the ability of bovine viral diarrhea virus (BVDV) to replicate and produce infectious virus in monocyte-derived dendritic cells (Mo-DC) and monocytes was studied. The study also examined the effect of BVDV infection on Mo-DC expression of cell surface markers, including MHCI, MHCII, and CD86, which are critical for DC function in immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from bovine blood through gradient centrifugation. The adherent monocytes were isolated from PBMCs and differentiated into Mo-DC using bovine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GMCSF). To determine the effect of BVDV on Mo-DC, four strains of BVDV were used including the severe acute non-cytopathic (ncp) BVDV2a-1373; moderate acute ncp BVDV2a 28508-5; and a homologous virus pair [i.e., cytopathic (cp) BVDV1b TGAC and ncp BVDV1b TGAN]. The Cooper strain of bovine herpesvirus 1 (BHV1) was used as the control virus. Mo-DC were infected with one of the BVDV strains or BHV-1 and were subsequently examined for virus replication, virus production, and the effect on MHCI, MHCII, and CD86 expression. RESULTS: The ability of monocytes to produce infectious virus reduced as monocytes differentiated to Mo-DC, and was completely lost at 120 hours of maturation. Interestingly, viral RNA increased throughout the course of infection in Mo-DC, and the viral non-structural (NS5A) and envelope (E2) proteins were expressed. The ncp strains of BVDV down-regulated while cp strain up-regulated the expression of the MHCI, MHCII, and CD86 on Mo-DC. CONCLUSIONS: The study revealed that the ability of Mo-DC to produce infectious virus was reduced with its differentiation from monocytes to Mo-DC. The inability to produce infectious virus may be due to a hindrance of virus packaging or release mechanisms. Additionally, the study demonstrated that ncp BVDV down-regulated and cp BVDV up-regulated the expression of Mo-DC cell surface markers MHCI, MHCII, and CD86, which are important in the mounting of immune responses.