The detection of long-lasting memory foot-and-mouth disease (FMD) virus serotype O-specific CD4+ T cells from FMD-vaccinated cattle by bovine major histocompatibility complex class II tetramer.

Foot-and-mouth disease (FMD) is a highly contagious, economically devastating disease of cloven-hooved animals. The development of long-lasting effective FMD vaccines would greatly benefit the global FMD control programme. Deep analysis of adaptive immunity in cattle vaccinated against FMD is technically challenging due to the lack of species-specific tools. In this study, we aimed to identify CD4+ T-cell epitopes in the FMD virus (FMDV) capsid and to phenotype the CD4+ T cells that recognize them using bovine major histocompatibility complex (BoLA) class II tetramer. A BoLA class II tetramer based on the DRA/DRB3*020:02 allele and FMDV antigen-stimulated PBMCs from bovine vaccinates were used to successfully identify four epitopes in the FMDV capsid, three of which have not been previously reported; two epitopes were identified in the structural protein VP1, one in VP3 and one in VP4. Specificity of the three novel epitopes was confirmed by proliferation assay. All epitope-expanded T-cell populations produced IFN-γ in vitro, indicating a long-lasting Th1 cell phenotype after FMD vaccination. VP3-specific CD4+ T cells exhibited the highest frequency amongst the identified epitopes, comprising >0·004% of the CD4+ T-cell population. CD45RO+ CCR7+ defined central memory CD4+ T-cell subpopulations were present in higher frequency in FMDV-specific CD4+ T-cell populations from FMD-vaccinated cattle ex vivo. This indicates an important role in maintaining cell adaptive immunity after FMD vaccination. Notably, FMDV epitope-loaded tetramers detected the presence of FMDV-specific CD4+ T cells in bovine PBMC more than four years after vaccination. This work contributes to our understanding of vaccine efficacy.

Association of intrauterine presence of Lactobacillus spp. with inflammation and pathogenic bacteria in the uterus in postpartum dairy cows.

The aim of this study was to clarify the influence of Lactobacillus spp. on the degree of endometrial inflammation in the postpartum period and the relationship between Lactobacillus spp. and pathogenic bacteria in the endometrium of postpartum dairy cows. Endometrial samples were collected from 41 Holstein-Friesian cows at 4 and 8 weeks postpartum using cytobrushes for polymorphonuclear neutrophil (PMN) count and bacterial culture to isolate Lactobacillus spp., Escherichia coli, and Trueperella pyogenes. The 4-week samples were divided into four groups (E+L+), (E+L-), (E-L+), (E-L-) according to whether endometritis was diagnosed (E+) and Lactobacillus spp. was isolated (L+). The diagnostic criterion for cytological endometritis was > 18% PMN. The average PMN% in the E+L+ group was lower than that in the E+L-group (P < 0.05) at 8 weeks postpartum. There were no significant correlations between the number of colonies of Lactobacillus spp. and E. coli or between that of Lactobacillus spp. and T. pyogenes. Lactobacillus spp. could reduce PMN% in dairy cows with endometritis during the puerperal period. In conclusion, the intrauterine presence of Lactobacillus spp. may have a positive effect on uterine involution in postpartum dairy cows.

Production of a correctly assembled fibrinogen using transgenic silkworms.

Fibrinogen from human blood is used as a main component of coagulants, including surgical tissue sealants. The development of a recombinant human fibrinogen (rFib) is anticipated to eliminate the risks of blood-borne infections. Here, we report the efficient production of rFib in a transgenic silkworm system. A silkworm line carrying cDNAs of the fibrinogen Aα and γ chains (Aα/γ-silkworm) produced Aα and γ chains in its cocoons, however, the Bß chains were not detected from cocoons of another silkworm line carrying the cDNA of fibrinogen Bß chains (Bß-silkworm). A silkworm line for all three fibrinogen chains was generated by crossing Aα/γ-silkworms with Bß-silkworms, which secreted Aα2Bß2γ2 fibrinogen (rFib) into cocoons at high contents. The N-terminal amino acid sequences of the three rFib chains were identical to those of the corresponding chains of native fibrinogen (nFib). The N-glycan profile of the rFib comprised oligomannose-type (53%), complex-type (34%), and paucimannose-type (13%); neither high-mannose-type (six or more mannose residues) nor core-fucosylated glycans were observed. The coagulation activity of the rFib was evaluated for the amount of thrombin-released fibrinopeptide A (FpA) and the kinetics for turbidity increase (non-covalent network formation) in the solution. FpA release rates were equivalent between rFib and nFib; by contrast, the kinetics of the turbidity increase for rFib were accelerated nearly two-fold, for both the rate and maximum value, compared to those of nFib. These results demonstrate that the rFib produced in the transgenic silkworm system is comparable to nFib in both physical and coagulative properties. This rFib is a promising candidate component for safe hemostatic pharmaceuticals.

A descriptive survey of porcine epidemic diarrhea in pig populations in northern Vietnam.

Porcine epidemic diarrhea (PED) virus (PEDV) is a globally emerging and re-emerging epizootic swine virus that causes massive economic losses in the swine industry, with high mortality in piglets. In Vietnam, PED first emerged in 2009 and has now developed to an endemic stage. This is the first cross-sectional survey performed to evaluate the proportion of PEDV-positive swine farms in Vietnam from January 2018 to February 2019. Fecal samples from 327 pig farms in northern Vietnam were collected and tested for PEDV infection by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method. The proportion of PEDV-positive farms was 30.9% and PEDV-positive farms were distributed throughout the study area. The highest proportion of PEDV-positive farms was 70% (7/10) among nucleus production type farms (P < 0.05). Higher proportions of PEDV-positive farms were found in the Northeast and Red River Delta areas, which are the major areas of pig production (P < 0.05). The proportion of PEDV-positive farms was higher among larger farms (P < 0.05). Our findings illustrate the high proportion of PEDV-positive farms in the Vietnamese pig population and will help to better understand the epidemiological dynamics of PED infection, to estimate impact, and establish and improve prevention and control measures.

Real-time decision-making during emergency disease outbreaks.

In the event of a new infectious disease outbreak, mathematical and simulation models are commonly used to inform policy by evaluating which control strategies will minimize the impact of the epidemic. In the early stages of such outbreaks, substantial parameter uncertainty may limit the ability of models to provide accurate predictions, and policymakers do not have the luxury of waiting for data to alleviate this state of uncertainty. For policymakers, however, it is the selection of the optimal control intervention in the face of uncertainty, rather than accuracy of model predictions, that is the measure of success that counts. We simulate the process of real-time decision-making by fitting an epidemic model to observed, spatially-explicit, infection data at weekly intervals throughout two historical outbreaks of foot-and-mouth disease, UK in 2001 and Miyazaki, Japan in 2010, and compare forward simulations of the impact of switching to an alternative control intervention at the time point in question. These are compared to policy recommendations generated in hindsight using data from the entire outbreak, thereby comparing the best we could have done at the time with the best we could have done in retrospect. Our results show that the control policy that would have been chosen using all the data is also identified from an early stage in an outbreak using only the available data, despite high variability in projections of epidemic size. Critically, we find that it is an improved understanding of the locations of infected farms, rather than improved estimates of transmission parameters, that drives improved prediction of the relative performance of control interventions. However, the ability to estimate undetected infectious premises is a function of uncertainty in the transmission parameters. Here, we demonstrate the need for both real-time model fitting and generating projections to evaluate alternative control interventions throughout an outbreak. Our results highlight the use of using models at outbreak onset to inform policy and the importance of state-dependent interventions that adapt in response to additional information throughout an outbreak.

Development of indirect enzyme-linked immunosorbent assay for detection of porcine epidemic diarrhea virus specific antibodies (IgG) in serum of naturally infected pigs.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED. RESULTS: One hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80-100%, respectively. CONCLUSIONS: The developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

Total serum protein reference value as a clinical diagnostic index of equine proliferative enteropathy.

Equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis is characterized by hypoproteinemia. There are currently no reliable reports that provide a reference value for the total serum protein (TP) concentration to clinically diagnose EPE. The objective of this study was to statistically determine the reference value. Feces and sera of 99 foals with EPE-like clinical signs and of 35 healthy foals were obtained. The samples were used for specific-gene detection of L. intracellularis, TP measurement, and specific-antibody detection against L. intracellularis. Based on these results, the optimal reference value for the TP concentration as a clinical diagnostic index of EPE was found to be ≤ 4.8 g/dl. This clinical diagnostic index will provide an effective approach for diagnosing EPE.

Novel recombinant feline interferon carrying N-glycans with reduced allergy risk produced by a transgenic silkworm system.

BACKGROUND: The generation of recombinant proteins for commercialisation must be cost-effective. Despite the cost-effective production of recombinant feline interferon (rFeIFN) by a baculovirus expression system, this rFeIFN carries insect-type N-glycans, with core α 1,3 fucosyl residues that act as potential allergens. An alternative method of production may yield recombinant glycoproteins with reduced antigenicity. RESULTS: A cDNA clone encoding the fifteenth subtype of FeIFN-α (FeIFN-α15) was isolated from a Japanese domestic cat. This clone encoded a protein of 189 amino acids with a molecular mass of 21.1 kDa. The rFeIFN-α15 was expressed using a transgenic silkworm system, which was expected to yield an N-glycan structure with reduced antigenicity compared with the protein produced by the baculovirus system. The resulting rFeIFN-α15 accumulated in the sericin layer of silk fibres and was easily extracted and purified by column chromatography. The N-terminal amino acid sequence of purified rFeIFN-α15 was identical to the mature form of natural sequence. Moreover, its N-glycans did not include detectable core α 1,3 fucosyl residues. Its anti-vesicular stomatitis virus activity (2.6 × 108 units/mg protein) was comparable to that of the baculovirus-expressed rFeIFN. CONCLUSIONS: The lower allergy risk of rFeIFN produced by the transgenic silkworm system than by the baculovirus expression system is due to the former lacking core α 1,3 fucosyl residues in its N-glycans. The rFeIFN-α15 produced by the transgenic silkworm system may be a prospective candidate for the next generation of rFeIFN in veterinary medicine.

Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay.

BACKGROUND: Porcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd. RESULTS: In this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed. CONCLUSIONS: The developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.

Soluble Thrombomodulin Ameliorates Ischemia-Reperfusion Injury of Liver Grafts by Modulating the Proinflammatory Role of High-Mobility Group Box 1.

Transplantation using grafts obtained after cardiac death (CD) is considered a promising solution for graft shortages. However, no standard criteria for organ preservation have been established for CD donors. High-mobility group box 1 (HMGB1) is a DNA-binding protein that is released from dying hepatocytes as an early mediator of inflammation and organ tissue damage. HMGB1 stimulates immunocytes to produce inflammatory cytokines, thereby amplifying the inflammatory response. Thrombomodulin is an integral membrane protein that functions as an endothelial anticoagulant cofactor, and it binds HMGB1 through the extracellular domain. We investigated the effects of ART-123, recombinant human soluble thrombomodulin, on warm ischemia-reperfusion injury in liver grafts. Male Wistar rats were divided into four ex vivo groups: heart-beating (HB) group, in which livers were isolated from HB donors; CD group, in which livers were isolated from CD donors exposed to apnea-induced conditions and warm ischemic conditions for 30 min after cardiac arrest; and two CD groups pretreated with ART-123 (1 or 5 mg/kg). Each isolated liver was reperfused for 1 h after cold preservation for 6 h. The perfusate levels of HMGB1, LDH, TNF-α, and IL-6 were significantly lower in the CD group pretreated with ART-123 (5 mg/kg) than in the CD group. Bile production was significantly higher in the CD group pretreated with ART-123 (5 mg/kg) than in the CD group. The sinusoidal spaces were significantly narrower in the CD group than in the other groups. We propose that ART-123 maintains sinusoidal microcirculation by reducing endothelial cell damage during warm ischemia-reperfusion injury.

Identification of Core Alpha 1,3-Fucosyltransferase Gene From Silkworm: An Insect Popularly Used to Express Mammalian Proteins.

Silkworm has great potential as production system of recombinant mammalian proteins. When the protein products are used for medical purpose, it is required to reduce the risk of an allergy, the content of core alpha 1,3-fucosyl residue attached to the N-glycan of proteins, for example. We isolated the gene of an enzyme responsible for the transfer of core alpha 1,3-fucosyl residue, core alpha 1,3-fucosyltransferase (Fuc-T C3), from silkworm. A candidate cDNA for silkworm Fuc-T C3 was isolated as a homolog of the fruit fly enzyme gene fucTA. The gene was located on chromosome 7 of the silkworm genome and was composed of seven exons, which spanned approximately 10 kb on the genome. The coding region of the gene was 1,350 bp and encoded a 450-amino acid protein with a molecular mass of 52.2 kDa. Deduced amino acid sequence of the coding region showed one transmembrane domain in its N-terminal and typical motifs common to fucosyltransferases including Fuc-T C3s of other organisms in its C-terminal. The extract of CHO cells transfected with the cDNA showed Fuc-T C3 activity using GDP-fucose and DABS-GnGn peptide as substrates. These results showed this cDNA clone actually encodes silkworm Fuc-T C3.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Hepatic venous reconstruction using the superficial femoral vein in a right-lobe living donor liver transplant patient with interrupted inferior vena cava.

Anatomical abnormalities in patients with BA often include polysplenia, preduodenal portal vein, interrupted retrohepatic IVC, cardiac abnormalities, and situs inversus. In LDLT patients who had congenital vascular anomalies, additional surgical modifications for the reconstruction of hepatic venous branches are sometimes necessary to prevent venous parenchymal congestion. We report a 12-yr-old female with post-Kasai BA with interrupted retrohepatic IVC who underwent right-lobe LDLT because the left liver graft volume was insufficient. The donor right liver graft had three major hepatic branches, including the RHV, IRHV, and MHV tributary (V8). We performed hepatic venous reconstruction by creating a large, wide triple orifice consisting of the RHV and two SFVs, which were anastomosed to the V8 and IRHV using the donor's SFV as an interposition graft. In conclusion, the reconstruction of venous orifices for right-lobe LDLT patients with the absent retrohepatic IVC is can be carried out using an SFV graft derived from the living donor or the recipient.

Evaluating swine disease occurrence on farms using the state-space model based on meat inspection data: a time-series analysis.

BACKGROUND: Data on abnormal health conditions in animals obtained from slaughter inspection are important for identifying problems in fattening management. However, methods to objectively evaluate diseases on farms using inspection data has not yet been well established. It is important to assess fattening management on farms using data obtained from slaughter inspection. In this study, we developed the state-space model to evaluate swine morbidity using slaughter inspection data. RESULTS: The most appropriate model for each disease was constructed using the state-space model. Data on 11 diseases in slaughterhouses over the past 4 years were used to build the model. The model was validated using data from 14 farms. The local-level model (the simplest model) was the best model for all diseases. We found that the analysis of slaughter data using the state-space model could construct a model with greater accuracy and flexibility than the ARIMA model. In this study, no seasonality or trend model was selected for any disease. It is thought that models with seasonality were not selected because diseases in swine shipped to slaughterhouses were the result of illness at some point during the 6-month fattening period between birth and shipment. CONCLUSION: Evaluation of previous diseases helps with the objective understanding of problems in fattening management. We believe that clarifying how farms manage fattening of their pigs will lead to improved farm profits. In that respect, it is important to use slaughterhouse data for fattening evaluation, and it is extremely useful to use mathematical models for slaughterhouse data. However, in this research, the model was constructed on the assumption of normality and linearity. In the future, we believe that we can build a more accurate model by considering models that assume non-normality and non-linearity.

A stochastic modeling study of quarantine strategies against foot-and-mouth disease risks through cattle trades across the Thailand-Myanmar border.

Foot-and-mouth disease (FMD) is an important endemic disease in livestock in Southeast Asia. Transboundary movement of animals may result in the transnational disease spread. A major cattle market is located at the Thailand-Myanmar border, where most cattle imported from Myanmar are traded. In this study, we built a stochastic susceptible-exposed-infectious-recovered (SEIR) model to investigate the effectiveness of a private animal quarantine service center in preventing FMDV from entering the major cattle market. We computed with different parameters and found that, with 50 % vaccine effectiveness, the risk of releasing infected cattle to the market per batch was generally low during the quarantine period of 21 and 28 days, with the risk ranging from 0.071 to 0.078 and 0.032 to 0.036, respectively. Despite the best scenario, the zero-risk state is difficult to attain. The sensitivity analysis highlights that the percentage of immune animals before entering the quarantine centers and the vaccine effectiveness are important factors. In conclusion, the 21-day quarantine period mitigates the risk of FMDV introduction into the cattle market. This control measure should be rigorously maintained to sustainably prevent FMDV outbreaks through transboundary animal movements, especially among countries in FMD-endemic regions.

Identifying Pathogen and Allele Type Simultaneously in a Single Well Using Droplet Digital PCR.

In the transmission control of chronic and untreatable livestock diseases such as bovine leukemia virus (BLV) infection, the removal of viral superspreaders is a fundamental approach. On the other hand, selective breeding of cattle with BLV-resistant capacity is also critical for reducing the viral damage to productivity by keeping infected cattle. To provide a way of measuring BLV proviral load (PVL) and identifying susceptible/resistant cattle simply and rapidly, we developed a fourplex droplet digital PCR method targeting the BLV pol gene, BLV-susceptible bovine major histocompatibility complex (BoLA)-DRB3*016:01 allele, resistant DRB3*009:02 allele, and housekeeping RPP30 gene (IPATS-BLV). IPATS-BLV successfully measured the percentage of BLV-infected cells and determined allele types precisely. Furthermore, it discriminated homozygous from heterozygous carriers. Using this method to determine the impact of carrying these alleles on the BLV PVL, we found DRB3*009:02-carrying cattle could suppress the PVL to a low or undetectable level, even with the presence of a susceptible heterozygous allele. Although the population of DRB3*016:01-carrying cattle showed significantly higher PVLs compared with cattle carrying other alleles, their individual PVLs were highly variable. Because of the simplicity and speed of this single-well assay, our method has the potential of being a suitable platform for the combined diagnosis of pathogen level and host biomarkers in other infectious diseases satisfying the two following characteristics of disease outcomes: (i) pathogen level acts as a critical maker of disease progression; and (ii) impactful disease-related host genetic biomarkers are already identified. IMPORTANCE While pathogen-level quantification is an important diagnostic of disease severity and transmissibility, disease-related host biomarkers are also useful in predicting outcomes in infectious diseases. In this study, we demonstrate that combined proviral load (PVL) and host biomarker diagnostics can be used to detect bovine leukemia virus (BLV) infection, which has a negative economic impact on the cattle industry. We developed a fourplex droplet digital PCR assay for PVL of BLV and susceptible and resistant host genes named IPATS-BLV. IPATS-BLV has inherent merits in measuring PVL and identifying susceptible and resistant cattle with superior simplicity and speed because of a single-well assay. Our new laboratory technique contributes to strengthening risk-based herd management used to control within-herd BLV transmission. Furthermore, this assay design potentially improves the diagnostics of other infectious diseases by combining the pathogen level and disease-related host genetic biomarker to predict disease outcomes.

Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA.

Bovine leukemia virus (BLV) is the causative agent of a B-cell tumor called enzootic bovine leukosis. Preventing BLV spreading is required to reduce economic loss related to BLV infection of livestock. To quantify proviral load (PVL) more easily and rapidly, we developed a quantification system of PVL using droplet digital PCR (ddPCR). This method uses a multiplex TaqMan assay of the BLV provirus and housekeeping gene RPP30 for the quantification of BLV in BLV-infected cells. Furthermore, we combined ddPCR with DNA purification-free sample preparation (unpurified genomic DNA). The percentage of BLV-infected cells based on unpurified genomic DNA was highly correlated with that based on purified genomic DNA (correlation coefficient: 0.906). Thus, this new technique is a suitable method to quantify PVL of BLV-infected cattle in a large sample number.

Development of a highly sensitive point-of-care test for African swine fever that combines EZ-Fast DNA extraction with LAMP detection: Evaluation using naturally infected swine whole blood samples from Vietnam.

BACKGROUND: While early detection and early containment are key to controlling the African swine fever (ASF) pandemic, the lack of practical testing methods for use in the field are a major barrier to achieving this feat. OBJECTIVES: To describe the development of a rapid and sensitive point-of-care test (POCT) for ASF, and its evaluation using swine whole blood samples for field settings. METHODS: In total, 89 swine whole blood samples were collected from Vietnamese swine farms and were performed the POCT using a combination of crude DNA extraction and LAMP (loop-mediated isothermal amplification) amplification. RESULTS: The POCT enabled crude DNA to be extracted from swine whole blood samples within 10 min at extremely low cost and with relative ease. The entire POCT required a maximum of 50 min from the beginning of DNA extraction to final judgment. Compared to a conventional real-time PCR detection, the POCT showed a 1 log reduction in detection sensitivity, but comparable diagnostic sensitivity of 100% (56/56) and diagnostic specificity of 100% (33/33). The POCT was quicker and easier to perform and did not require special equipment. CONCLUSIONS: This POCT is expected to facilitate early diagnosis and containment of ASF invasion into both regions in which it is endemic and eradicated.

Persistent expression of the full genome of hepatitis C virus in B cells induces spontaneous development of B-cell lymphomas in vivo.

Extrahepatic manifestations of hepatitis C virus (HCV) infection occur in 40%-70% of HCV-infected patients. B-cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection. The mechanism by which HCV infection of B cells leads to lymphoma remains unclear. Here we established HCV transgenic mice that express the full HCV genome in B cells (RzCD19Cre mice) and observed a 25.0% incidence of diffuse large B-cell non-Hodgkin lymphomas (22.2% in males and 29.6% in females) within 600 days after birth. Expression levels of aspartate aminotransferase and alanine aminotransferase, as well as 32 different cytokines, chemokines and growth factors, were examined. The incidence of B-cell lymphoma was significantly correlated with only the level of soluble interleukin-2 receptor α subunit (sIL-2Rα) in RzCD19Cre mouse serum. All RzCD19Cre mice with substantially elevated serum sIL-2Rα levels (> 1000 pg/mL) developed B-cell lymphomas. Moreover, compared with tissues from control animals, the B-cell lymphoma tissues of RzCD19Cre mice expressed significantly higher levels of IL-2Rα. We show that the expression of HCV in B cells promotes non-Hodgkin-type diffuse B-cell lymphoma, and therefore, the RzCD19Cre mouse is a powerful model to study the mechanisms related to the development of HCV-associated B-cell lymphoma.

Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova.

Maternal proteins are rapidly degraded by the ubiquitin-proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficient gad. Furthermore, we assessed morphological features in gad mouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the 'maternal antigen that embryos require' (NLRP5 (MATER)) protein level increased significantly in gad mouse ova compared with that in wild-type mice. In an ultrastructural study, gad mouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.