Evaluation of the diagnostic sensitivity and specificity of two pen-side tests for detecting African swine fever virus in experimentally infected pigs.

African swine fever virus (ASFV) has spread through many countries and regions worldwide, causing significant losses. Timely detection of ASFV-infected pigs is crucial for disease control. In this study, we assessed the performance of two pen-side tests: a portable real-time PCR (qPCR) test for detecting viral genomic DNA and a lateral flow immunoassay (LFIA) for detecting viral antigens. To determine the time from infection to the earliest detection, 10 ASFV-seronegative pigs were inoculated intramuscularly with 104.0 hemadsorption dose 50 of a highly virulent ASFV strain. Whole blood and oral swab samples were alternately collected from each group of five pigs daily until all succumbed to the infection. Samples were promptly subjected to the two pen-side tests upon collection, and a subset was transported to a veterinary diagnostic laboratory for analysis using a reference qPCR assay. Viral genomic DNA was consistently detected by the reference qPCR assay in all blood samples from 2 days postinfection (dpi), preceding the onset of clinical signs, and in oral swabs from 4 dpi onwards. The portable qPCR test demonstrated comparable performance to the reference qPCR assay for both whole blood and oral swab samples. The LFIA exhibited 100% specificity when testing with whole blood samples but showed reduced sensitivity, particularly for blood samples collected early or late after infection. The antigen test did not perform well with oral swabs.

Comparative Analysis of Swine Antibody Responses following Vaccination with Live-Attenuated and Killed African Swine Fever Virus Vaccines.

African swine fever virus (ASFV) is circulating in many swine-producing countries, causing significant economic losses. It is observed that pigs experimentally vaccinated with a live-attenuated virus (LAV) but not a killed virus (KV) vaccine develop solid homologous protective immunity. The objective of this study was to comparatively analyze antibody profiles between pigs vaccinated with an LAV vaccine and those vaccinated with a KV vaccine to identify potential markers of vaccine-induced protection. Thirty ASFV seronegative pigs were divided into three groups: Group 1 received a single dose of an experimental LAV, Group 2 received two doses of an experimental KV vaccine, and Group 3 was kept as a non-vaccinated (NV) control. At 42 days post-vaccination, all pigs were challenged with the parental virulent ASFV strain and monitored for 21 days. All pigs vaccinated with the LAV vaccine survived the challenge. In contrast, eight pigs from the KV group and seven pigs from the NV group died within 14 days post-challenge. Serum samples collected on 41 days post-vaccination were analyzed for their reactivity against a panel of 29 viral structural proteins. The sera of pigs from the LAV group exhibited a strong antibody reactivity against various viral structural proteins, while the sera of pigs in the KV group only displayed weak antibody reactivity against the inner envelope (p32, p54, p12). There was a negative correlation between the intensity of antibody reactivity against five ASFV antigens, namely p12, p14, p15, p32, and pD205R, and the viral DNA titers in the blood of animals after the challenge infection. Thus, antibody reactivities against these five antigens warrant further evaluation as potential indicators of vaccine-induced protection.

Nanocellulose and Graphene Oxide Aerogels for Adsorption and Removal Methylene Blue from an Aqueous Environment.

The characteristics of aerogel materials such as the low density and large surface area enable them to adsorb large amounts of substances, so they show great potential for application in industrial wastewater treatment. Herein, using a combination of completely environmentally friendly materials such as cellulose nanofibers (CNFs) extracted from the petioles of the nipa palm tree and graphene oxide (GO) fabricated by simple solvent evaporation, a composite aerogel was prepared by a freeze-drying method. The obtained aerogel possessed a light density of 0.0264 g/cm3 and a porosity of more than 98.2%. It was able to withstand a weight as much as 2500 times with the maximum force (1479.5 N) to break up 0.2 g of an aerogel by compression strength testing and was stable in the aquatic environment, enabling it to be reused five times with an adsorption capacity over 90%. The CNF/GO aerogel can recover higher than 85% after 30 consecutive compression recovery cycles, which is convenient for the reusability of this material in wastewater treatments. The obtained aerogel also showed a good interaction between the component phases, a high thermal stability, a 3D network structure combined with thin walls and pores with a large specific surface area. In addition, the aerogel also exhibited a fast adsorption rate for methylene blue (MB) adsorption, a type of waste from the textile industry that pollutes water sources, and it can adsorb more than 99% MB in water in less than 20 min. The excellent adsorption of MB onto the CNF/GO aerogel was driven by electrostatic interactions, which agreed with the pseudo-second-order kinetic model with a correlation coefficient R 2 = 0.9978. The initial results show that the CNF/GO aerogel is a highly durable "green" light material that might be applied in the treatment of domestic organic waste water and is completely recoverable and reusable.

Infertility-related stress, social support, and coping of women experiencing infertility in Vietnam.

BACKGROUND: Women with an infertility problem living in traditional and developing countries face extensive social pressure, infertility-related stress, and distress, which possibly affect their choices of coping strategies. The present study aims to investigate the impact of infertility-related stress and social support on coping of Vietnamese women who live with an infertility diagnosis. PARTICIPANTS AND PROCEDURE: A cross-sectional study was conducted with 192 women diagnosed with infertility at two hospitals in the north and central regions of Vietnam. Participants completed a questionnaire consisting of the Multidimensional Scale of Perceived Social Support, the Copenhagen Multi-centre Psychosocial Infertility coping scales and the Fertility Problem Inventory, and questions about their sociodemographic characteristics, infertility-related history, and key social relationships. Four linear regression analyses were performed on four coping strategies: active-avoidance coping (AAC), active-confronting coping (ACC), passive-avoidance coping (PAC), and meaning-based coping (MBC). RESULTS: The findings show that high infertility-related stress significantly predicted the use of avoidance coping strategies (AAC and PAC) among these women, while those with a high level of perceived social support tended to use ACC and MBC. None of the four linear regression models support the moderating role of social support in the relationship between infertility-related stress and coping styles. CONCLUSIONS: The study findings show that levels of infertility-related stress and perceived social support have a direct effect on the choice of coping strategies among Vietnamese women diagnosed with infertility. The study results have practical implications in the Vietnamese context, including: (i) the development and adaptation of evidence-based and culturally appropriate interventions and counselling strategies; and (ii) social policy advocacy to better support women diagnosed with infertility, their husbands, and both as couples.

Diabetes and higher HbA1c levels are independently associated with adverse renal outcomes in inpatients following multiple hospital admissions.

OBJECTIVE: To assess the association between glycaemic status prior to the first hospital presentation with developing adverse renal outcomes overtime in patients with multiple hospital re-admissions. DESIGN: A prospective observational cohort study. PARTICIPANTS: All inpatients aged ≥54 years admitted between 2013 and 16 to a tertiary hospital. MAIN OUTCOMES: We prospectively measured HbA1c levels in all inpatients aged ≥54 years admitted between 2013 and 16. Diabetes was defined as prior documented diagnosis of diabetes and/or HbA1c ≥6.5% (47·5 mmol/L). Included patients had ≥ two admissions (at least 90 days apart), baseline estimated glomerular filtration rate (eGFR) >30 ml/min/1·73m2 and no history of renal replacement therapy. We assessed several renal outcomes: (a) 50% decline in eGFR; (b) rapid decline in renal function (eGFR decline >5 mL/min/1·73m2/year) and (c) final eGFR<30 ml/min/1·73m2. RESULTS: Of 4126 inpatients with a median follow-up of 465 days (254, 740), 26% had diabetes. The presence of diabetes was associated with higher odds of (a) 50% decline in eGFR (OR = 1·42;95% CI:1·18-1·70;p < 0·001); (b) rapid decline in renal function (OR = 1·40;95%CI:1·20-1·63;p < 0·001), and (c) reaching eGFR<30 ml/min/1.73m2 (OR = 1·25;95%CI:1·03-1·53;p < 0·05). Every 1% (11 mmol/L) increase in baseline HbA1c was associated with significantly greater odds of (a) >50% decline in eGFR (OR = 1·07;95% CI:1·01-1·4;p < 0·05) and (b) rapid decline in renal function (OR = 1·11;95% CI:1·05-1·18;p < 0·001). CONCLUSIONS: In patients with ≥two admissions, the presence of diabetes and higher HbA1c levels were strongly and independently associated with adverse renal outcomes at follow up. Such patients are at high risk of relatively rapid deterioration in renal function and a logical target for structured preventive interventions.

Routine use of HbA1c amongst inpatients hospitalised with decompensated heart failure and the association of dysglycaemia with outcomes.

Diabetes is an independent risk factor for development of heart failure and has been associated with poor outcomes in these patients. The prevalence of diabetes continues to rise. Using routine HbA1c measurements on inpatients at a tertiary hospital, we aimed to investigate the prevalence of diabetes amongst patients hospitalised with decompensated heart failure and the association of dysglycaemia with hospital outcomes and mortality. 1191 heart failure admissions were identified and of these, 49% had diabetes (HbA1c ≥ 6.5%) and 34% had pre-diabetes (HbA1c 5.7-6.4%). Using a multivariable analysis adjusting for age, Charlson comorbidity score (excluding diabetes and age) and estimated glomerular filtration rate, diabetes was not associated with length of stay (LOS), Intensive Care Unit (ICU) admission or 28-day readmission. However, diabetes was associated with a lower risk of 6-month mortality. This finding was also supported using HbA1c as a continuous variable. The diabetes group were more likely to have diastolic dysfunction and to be on evidence-based cardiac medications. These observational data are hypothesis generating and possible explanations include that more diabetic patients were on medications that have proven mortality benefit or prevent cardiac remodelling, such as renin-angiotensin system antagonists, which may modulate the severity of heart failure and its consequences.

Critical Risk Results - An Update on International Initiatives.

Direct communication of significant (often life-threatening) results is a universally acknowledged role of the pathology laboratory, and an important contributor to patient safety. Amongst the findings of a recent survey of 871 laboratories from 30 countries by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM), only 3 tests were noted to be common to 90% of alert lists, and only 48% of laboratories consulted clinicians in developing these alert lists despite ISO15189 recommendations to do so. These findings are similar to previous national surveys demonstrating significant variation worldwide in how critical risk results are managed and also in how these protocols are developed. In order to promote "best practice" and harmonization of critical risk results management, guidelines and recommendations have been published, most recently by Clinical and Laboratory Standards Institute (CLSI) and Australasian Association of Clinical Biochemists (AACB). These statements in particular have placed strong emphasis on patient risk and risk assessment in the management of critical risk results. This focus has resulted in recommendations to adopt new terminology, the consideration of risk assessment when compiling alert tables, consultative involvement of laboratory users in setting up protocols, and the need for outcome-based evidence to support our practices. With time it is expected that emerging evidence and technological improvements will facilitate the advancement of laboratories down this path to harmonization, best practice, and improve patient safety.

Splenic CD11c+ cells derived from semi-immune mice protect naïve mice against experimental cerebral malaria.

BACKGROUND: Immunity to malaria requires innate, adaptive immune responses and Plasmodium-specific memory cells. Previously, mice semi-immune to malaria was developed. Three cycles of infection and cure ('three-cure') were required to protect mice against Plasmodium berghei (ANKA strain) infection. METHODS: C57BL/6 J mice underwent three cycles of P. berghei infection and drug-cure to become semi-immune. The spleens of infected semi-immune mice were collected for flow cytometry analysis. CD11c(+) cells of semi-immune mice were isolated and transferred into naïve mice which were subsequently challenged and followed up by survival and parasitaemia. RESULTS: The percentages of splenic CD4(+) and CD11c(+) cells were increased in semi-immune mice on day 7 post-infection. The proportion and number of B220(+)CD11c(+)low cells (plasmacytoid dendritic cells, DCs) was higher in semi-immune, three-cure mice than in their naïve littermates on day 7 post-infection (2.6 vs 1.1% and 491,031 vs 149,699, respectively). In adoptive transfer experiment, three months after the third cured P. berghei infection, splenic CD11c(+) DCs of non-infected, semi-immune, three-cure mice slowed Plasmodium proliferation and decreased the death rate due to neurological pathology in recipient mice. In addition, anti-P. berghei IgG1 level was higher in mice transferred with CD11c(+) cells of semi-immune, three-cure mice than mice transferred with CD11c(+) cells of naïve counterparts. CONCLUSION: CD11c(+) cells of semi-immune mice protect against experimental cerebral malaria three months after the third cured malaria, potentially through protective plasmacytoid DCs and enhanced production of malaria-specific antibody.

A longitudinal study of thyroid autoantibodies in pregnancy: the importance of test timing.

OBJECTIVE: Thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TGAb) are frequently measured to investigate thyroid dysfunction in pregnancy. Despite the recognized fall of these autoantibodies in pregnancy, there is limited guidance on the timing of such testing. We assessed optimal test timing of TPOAb/TGAb for the detection of Hashimoto's thyroiditis and post-partum thyroid dysfunction (PPTD). DESIGN: Prospective longitudinal study with recruitment in Trimester 1. PATIENTS: Healthy women ≤13 weeks' gestation from Mercy Hospital for Women, a tertiary obstetric hospital in Melbourne. MEASUREMENTS: Serum TPOAb, TGAb, TSH and fT4 were measured at Trimester 1 (T1), Trimester 2(T2), Trimester 3(T3) and postpartum (PP) in each participant. Post-partum thyroid dysfunction (PPTD) was defined if TSH deviated from the assay's nonpregnant reference interval. Longitudinal random-effect logistic regression was used to investigate the association between time and positive/negative thyroid autoantibody status. RESULTS: Samples from 140 women at T1 (12·0: 10·3-13·0) (median: IQR weeks' gestation); 95 at T2 (24·3: 23·0-25·9), 79 at T3 (35·9: 34·8-36·7) and 83 at PP (12·4: 10·8-14·6 weeks post-partum) were attained. At T1, 13 (9%) and 15 (11%) women had positive TPOAb and TGAb, respectively. The odds of having a positive TPOAb were 96% lower at T2 [OR = 0·04 (95% CI: 0·02-0·8; P = 0·03)] and 97% lower at T3 [OR = 0·03 (95% CI: 0·001-0·6; P = 0·02)] than at T1. Similarly, the odds of having a positive TGAb were 99·4% lower [OR = 0·006 (95% CI: 0-0·3; P = 0·01)] at T2, and 99·5% lower [OR = 0·005 (95% CI: 0-0·4; P = 0·02)] at T3 than at T1. The ROC analysis diagnostic ORs for a positive TPOAb and/or TGAb to predict PPTD were 7·8 (95% CI: 2·2-27·6), 1·2 (95% CI: 0-8·9), 2·0 (95% CI: 0-16·8), and 12·2 (95% CI: 3·3-44·9) at T1, T2, T3 and post-partum, respectively. CONCLUSIONS: A significant proportion of pregnant women lose their thyroid autoantibody positivity after T1. The gestation-dependent loss of TPOAb/TGAb positivity and reduction in diagnostic accuracy for predicting PPTD limits the value of testing at T2 and T3.

Ontogenic changes in lung cholesterol metabolism, lipid content, and histology in mice with Niemann-Pick type C disease.

Niemann-Pick Type C (NPC) disease is caused by a deficiency of either NPC1 or NPC2. Loss of function of either protein results in the progressive accumulation of unesterified cholesterol in every tissue leading to cell death and organ damage. Most literature on NPC disease focuses on neurological and liver manifestations. Pulmonary dysfunction is less well described. The present studies investigated how Npc1 deficiency impacts the absolute weight, lipid composition and histology of the lungs of Npc1(-/-) mice (Npc1(nih)) at different stages of the disease, and also quantitated changes in the rates of cholesterol and fatty acid synthesis in the lung over this same time span (8 to 70days of age). Similar measurements were made in Npc2(-/-) mice at 70days. All mice were of the BALB/c strain and were fed a basal rodent chow diet. Well before weaning, the lung weight, cholesterol and phospholipid (PL) content, and cholesterol synthesis rate were all elevated in the Npc1(-/-) mice and remained so at 70days of age. In contrast, lung triacylglycerol content was reduced while there was no change in lung fatty acid synthesis. Despite the elevated PL content, the composition of PL in the lungs of the Npc1(-/-) mice was unchanged. H&E staining revealed an age-related increase in the presence of lipid-laden macrophages in the alveoli of the lungs of the Npc1(-/-) mice starting as early as 28days. Similar metabolic and histologic changes were evident in the lungs of the Npc2(-/-) mice. Together these findings demonstrate an intrinsic lung pathology in NPC disease that is of early onset and worsens over time.

Working memory training is associated with lower prefrontal cortex activation in a divergent thinking task.

Working memory (WM) training has been shown to lead to improvements in WM capacity and fluid intelligence. Given that divergent thinking loads on WM and fluid intelligence, we tested the hypothesis that WM training would improve performance and moderate neural function in the Alternate Uses Task (AUT)-a classic test of divergent thinking. We tested this hypothesis by administering the AUT in the functional magnetic resonance imaging scanner following a short regimen of WM training (experimental condition), or engagement in a choice reaction time task not expected to engage WM (active control condition). Participants in the experimental group exhibited significant improvement in performance in the WM task as a function of training, as well as a significant gain in fluid intelligence. Although the two groups did not differ in their performance on the AUT, activation was significantly lower in the experimental group in ventrolateral prefrontal and dorsolateral prefrontal cortices-two brain regions known to play dissociable and critical roles in divergent thinking. Furthermore, gain in fluid intelligence mediated the effect of training on brain activation in ventrolateral prefrontal cortex. These results indicate that a short regimen of WM training is associated with lower prefrontal activation-a marker of neural efficiency-in divergent thinking.

The impact of agricultural Best Management Practices on water quality in a North German lowland catchment.

Research on water quality degradation caused by point and diffuse source pollution plays an important role in protecting the environment sustainably. Implementation of Best Management Practices (BMPs) is a conventional approach for controlling and mitigating pollution from diffuse sources. The objectives of this study were to assess the long-term impact of point and diffuse source pollution on sediment and nutrient load in a lowland catchment using the ecohydrological model Soil and Water Assessment Tool (SWAT) and to evaluate the cost and effectiveness of BMPs for water quality improvement in the entire catchment. The study area, Kielstau catchment, is located in the North German lowlands. The water quality is not only influenced by the predominating agricultural land use in the catchment as cropland and pasture, but also by six municipal wastewater treatment plants. Diffuse entries as well as punctual entries from the wastewater treatment plants are implemented in the model set-up. Results from model simulations indicated that the SWAT model performed satisfactorily in simulating flow, sediment, and nutrient load in a daily time step. Two approaches to structural and nonstructural BMPs have been recommended in relation to cost and effectiveness of BMPs in this study. These BMPs include extensive land use management, grazing management practice, field buffer strip, and nutrient management plan. The results showed that BMPs would reduce fairly the average annual load for nitrate and total nitrogen by 8.6% to 20.5%. However, the implementation of BMPs does not have much impact on reduction in the average annual load of sediment and total phosphorus at the main catchment outlet. The results obtained by implementing those BMPs ranged from 0.8% to 4.9% and from 1.1% to 5.3% for sediment and total phosphorus load reduction, respectively. This study also reveals that reduction only in one type of BMP did not achieve the target value for water quality according to the European Water Framework Directive. The combination of BMPs improved considerably water quality in the Kielstau catchment, achieving a 53.9% and a 46.7% load reduction in nitrate and total nitrogen load, respectively, with annual implementation cost of 93,000 Euro.

Impact of troponin 1 on long-term mortality after emergency orthopaedic surgery in older patients.

OBJECTIVES: To determine the association between post-operative troponin rises and longer term (2-year) mortality after emergency orthopaedic surgery in patients over 60 years of age. METHODS: One hundred and two patients were recruited in 2006 and had inpatient troponin 1 measurements. These patients were followed up by a telephone call annually for complications. RESULTS: At 2 years, 29.4% (30/102) of patients had died. Twenty-five patients (25/54 or 49.3%) with a troponin rise were dead at 2 years compared with five patients without a troponin rise (5/48 or 10.4%), which was significantly different P < 0.0001. Patients with a higher troponin level (>0.1 µg/L) were more likely to be dead at 2 years compared with those with a lower level of troponin. However, when adjusted for other comorbidities the association between troponin elevation and death at 2 years did not persist. Using Cox regression multivariate analysis, only one factor, sustaining an in-hospital cardiac event odds ratio 4.3 (95% confidence interval 1.8-10.3, P = 0.001), was associated with 2 years all-cause mortality . Furthermore, patients who sustained a symptomatic troponin rise (P < 0.0001) or asymptomatic troponin rise (P = 0.004) were more likely to have died at 2 years compared with those with no troponin rise. Three factors were significantly associated with a cardiac event during the second year: (i) post-operative troponin rise (P = 0.05); (ii) pre-morbid atrial fibrillation (P = 0.04); and (iii) post-operative renal failure (P < 0.001). CONCLUSION: Elevated post-operative troponin levels are predictive of 1-year but not 2-year mortality in older patients undergoing emergency orthopaedic surgery.

Airway iron and iron-regulatory cytokines in cystic fibrosis.

Iron availability is critical to Pseudomonas aeruginosa. The current authors determined sputum iron, ferritin, microalbumin levels and total cell counts (TCC) in 19 adult patients with cystic fibrosis (CF) during an acute exacerbation and repeated analyses following a median of 12 days antibiotic treatment. The current authors also determined sputum interleukin (IL)-1beta and tumour necrosis factor (TNF)-alpha levels because of their putative role in intracellular iron homeostasis. Additional data were obtained from 17 stable CF patients, eight patients with stable chronic obstructive pulmonary disease (COPD) and six normal subjects. Overall, sputum iron, ferritin, microalbumin, IL-1beta and TNF-alpha concentrations and TCCs were significantly elevated in the CF patients compared to those with COPD and normal controls. Sputum ferritin levels were significantly elevated in acute versus stable CF patients and there was a trend for sputum TCC to be higher, but all other inflammatory indices were similar. In the CF patients, sputum iron was positively and strongly related to IL-1beta, TNF-alpha, ferritin and microalbumin levels, but negatively related to forced expiratory volume in one second % predicted. In those acute patients who clinically improved with antibiotics (n=14), there were significant decreases in sputum TCC, iron, ferritin and IL-1beta content, but not TNF-alpha or albumin levels. However, changes in sputum TNF-alpha in acute patients were still closely related to changes in iron, ferritin and albumin content, and changes in IL-1beta were related to changes in sputum ferritin content. Iron and iron-regulatory cytokines may play a role in cystic fibrosis lung disease and the increased iron content may even facilitate Pseudomonas aeruginosa infection.

Expansion techniques after Mohs' surgery on the face.

Mohs' surgery in the face has established itself as the optimal technique for a high cure rate of basal cell and squamous cell carcinoma occurring in the skin of the face. However, after the resection in Mohs' surgery, the defects, when extensive, require careful, planned reconstruction in order to produce a good cosmetic result. Although flap reconstruction is available for smaller lesions, larger defects can be covered often by expansion techniques. The expansion technique involves placing a silastic expander of various size and designs underneath the adjacent skin and, over a period of weeks, injecting saline into the expander in order to increase the amount of skin available for future flap reconstruction. This usually represents a two-stage procedure consisting of the insertion of the expander and, some weeks later, removal of the filled expander when there is sufficient tissue, and using this tissue to reconstruct the initial Mohs' defect by a flap. When defects are greater than half the aesthetic unit of the face, or greater than one-third in the forehead, or over 6 cm in the scalp, expansion techniques should be considered for reconstruction.

The role of components of recombination signal sequences in immunoglobulin gene segment usage: a V81x model.

It has long been appreciated that some immunoglobulin (and T-cell receptor) gene segments are used much more frequently than others. The VHsegment V81x is a particularly striking case of overusage. Its usage varies with the stage of B-cell development and with the strain of mice, but it is always high in B cell progenitors. We have found that the coding sequence and the recombination signal sequences (RSS) are identical in five mouse strains, including CAST/Ei, a strain derived from the species Mus castaneus. Thus, the strain differences cannot be attributed to sequences within V81x itself. V81x RSS mediated recombination at rates significantly higher than another VHRSS. Although the V81x nonamer differs at one base pair from the consensus sequence, an RSS with this nonamer and a consensus heptamer recombines as well as the consensus RSS. When the V81x spacer is replaced by that of VA1, the frequency of recombination decreases by approximately 5-fold; thus, the contribution of variation in natural spacers to variability in VHusage in vivo is likely to be more than has been previously appreciated. Furthermore, the contribution of the heptamer and nonamer to differential VHusage in our assay is correlated inversely with their conservation throughout the VHlocus.

A mouse with a monoclonal primary immunoglobulin repertoire not further diversified by V-gene replacement.

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a lambda1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a "knocked-in" V(H)DJ(H) rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB's primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 lambda light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.

Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function.

Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.