CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses.

Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

Reduced specificity of Erns antibody ELISAs for samples from piglets with maternally derived antibodies induced by vaccination of sows with classical swine fever marker vaccine CP7_E2alf.

Successful implementation of marker vaccines against classical swine fever virus is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA) as well as the development of a testing scheme during emergency vaccination. In this context, special attention needs to be paid to breeding farms, because the offspring of marker vaccinated sows possess maternally derived antibodies (MDAs). So far, limited information is available on the influence of MDAs on serological testing in the context of a DIVA strategy. Therefore, two commercially available Erns antibody ELISAs were compared, using serum samples of piglets with a high-to-moderate titre of MDAs against marker vaccine CP7_E2alf. False-positive results were detected by both Erns antibody ELISAs for serum samples of piglets with an age of up to 4 weeks. Interestingly, most samples tested false-positive in the first Erns antibody ELISA were identified correctly by the other Erns antibody ELISA and vice versa. In conclusion, in case of emergency vaccination of sows, the specificity of both ELISAs in newborn piglets younger than 4 weeks may be relatively low. This could be addressed in a testing strategy by either not sampling piglets up to the age of 4 weeks or using both ELISAs in a screening-confirmation set-up.

Frequent infection of wild boar with atypical porcine pestivirus (APPV).

The recently identified atypical porcine pestivirus (APPV) was demonstrated to be the causative agent of the neurological disorder "congenital tremor" in newborn piglets. Despite its relevance and wide distribution in domestic pigs, so far nothing is known about the situation in wild boar, representing an important wild animal reservoir for the related classical swine fever virus. In this study, 456 wild boar serum samples obtained from northern Germany were investigated for the presence of APPV genomes and virus-specific antibodies. Results of real-time RT-PCR analyses revealed a genome detection rate of 19%. Subsequent genetic characterization of APPV (n = 12) from different hunting areas demonstrated close genetic relationship and, with exception of APPV from one location, displayed less than 3.3% differences in the analysed partial NS3 encoding region. Furthermore, indirect Erns ELISA revealed an antibody detection rate of approx. 52%, being in line with the high number of viremic wild boar. Analysis of fifteen wild boar samples from the Republic of Serbia by Erns antibody ELISA provided evidence that APPV is also abundant in wild boar populations outside Germany. High number of genome and seropositive animals suggest that wild boar may serve as an important virus reservoir for APPV.

The double-antigen ELISA concept for early detection of Erns -specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals.

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of Erns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel Erns -specific prototype ELISA (pigtype CSFV Erns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect Erns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other Erns -based antibody ELISAs were identified correctly by the novel prototype Erns ELISA and vice versa. In conclusion, the pigtype CSFV Erns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional Erns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.

A prognostic model for clinical stage I melanoma of the lower extremity. Location on foot as independent risk factor for recurrent disease.

Thirteen variables were studied to determine their usefulness in predicting recurrent disease in 158 patients with stage I melanoma of the lower extremity. A Cox proportional hazards analysis demonstrated that three variables were independent risk factors for recurrent disease in these patients: (1) thickness, in millimeters, of the primary tumor (P = 0.000009), (2) primary tumor location on the foot (P = 0.0003), and (3) the number of mitoses/mm2 (P = 0.0244). Life-table analyses of patient subgroups defined by different combinations of these three variables demonstrated that thick (greater than or equal to 3.0 mm) melanomas of the foot were associated with recurrent disease much more frequently than tumors of similar thickness located on the thigh or calf. These data provide guidelines that can be used to evaluate results of surgical and/or adjuvant therapy studies for patients with melanoma of the lower extremity.

A prognostic model for clinical stage I melanoma of the trunk. Location near the midline is not an independent risk factor for recurrent disease.

Fifteen variables were studied for their usefulness in predicting recurrent disease in 254 patients with clinical stage I melanoma of the trunk. Thickness of the primary tumor correctly predicted outcome with an accuracy of 90 percent or greater in 176 patients with melanoma primaries with a thickness of less than 1.70 mm or 5.5 mm or greater. No other variables significantly increased predictive accuracy over these ranges of thickness. A Cox proportional hazards analysis of the remaining 78 patients with primary tumors 1.70 to 5.49 mm thick demonstrated that the following four variables functioned as independent risk factors for recurrent disease: (1) thickness of the primary tumor (p = 0.0005), (2) mitoses/mm2 greater than 6 (p = 0.006), (3) a nearly absent or minimal lymphocyte response at the base of the tumor (p = 0.009), and (4) location on the upper trunk (p = 0.03). Trunk lesions located near the midline did not have a worse prognosis than more lateral melanomas of similar thickness.

Volume of malignant melanoma is superior to thickness as a prognostic indicator. Preliminary observation.

There are many clinical and histologic factors that are known to be valuable in predicting survival rates for patients with cutaneous malignant melanomas. Breslow thickness is considered to be the most reliable prognostic factor; however, thickness is a unidimensional measurement. A more accurate mensuration to predict biologic behavior might be one that takes into account the three-dimensional volume of the neoplasm. In a study of 35 primary malignant melanomas, the volumes of the dermal components of the tumors were calculated. Those patients with tumor volumes of 200 mm3 or less had a 91.4% 5-year disease-free survival rate, compared with survival rate of only 16.7% for those patients whose lesions had tumor volumes exceeding 200 mm3. On multivariate analysis, tumor volume exceeded thickness as a prognostic indicator. Thus, measurement of tumor volume proved to be of greater significance than thickness in predicting the outcome for patients with malignant melanomas.

Broad spectrum reactivity versus subtype specificity-trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA.

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl (n=793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity.

Acute monocytic leukaemia in a HIV-seropositive man.

Individuals infected with the human immunodeficiency virus (HIV) have been reported to develop a number of malignant neoplasms. We recently treated an HIV patient who had acute monocytic leukaemia which was first evident in the skin. To our knowledge, this is the first report of a case of acute monocytic leukaemia occurring in a HIV-infected person.

Dermatofibrosarcoma protuberans metastatic to a regional lymph node. Report of a case and review.

A case of dermatofibrosarcoma protuberans of the lower extremity with metastasis to an inguinal lymph node appearing 5 years after wide excision and skin grafting of the primary lesion is presented. The world literature is reviewed. This case is the 24th instance of metastasis and the 7th case of lymphatic metastasis. The other 17 cases were hematogenous metastases. The clinical and pathologic features of dermatofibrosarcoma protuberans are reviewed, and treatment is discussed, with the aim of emphasizing the need for long-term followup examination of the regional lymph nodes following wide and deep local excision.

The impact of intradermal lymphoscintigraphy on surgical management of clinical stage I truncal malignant melanoma.

Lymphoscintigraphy results in 35 stage I melanoma patients were compared with primary location and surgical management. Six of 25 primaries with potentially multidirectional lymphatic drainage had drainage to one regional node group. Three of 10 with expected unidirectional drainage showed multidirectional drainage. With unidirectional drainage, decision concerning lymphadenectomy was simplified.

Data quality and DRGs: an assessment of the reliability of federal beneficiary discharge data in selected Manhattan hospitals.

New York County Health Services Review Organization (NYCHSRO), the physicians' professional standards review organization of Manhattan, examined whether diagnostic coding errors identified in Manhattan hospitals would affect reimbursement under a diagnostic-related group (DRG) method of financing inpatient services. A sampling of 1,027 Medicare and Medicaid cases representing discharges from 18 Manhattan hospitals during 1982 and 1983 revealed incorrect DRG assignment for 17.5% of patient record abstracts, but these appear to have been unsystematic rather than deliberate errors. The difference between estimated reimbursement based on original and reabstracted records was not statistically significant either in the aggregate or for specific hospitals. It is emphasized that while New York State's Prospective Hospital Reimbursement Methodology (in effect during the study period) is not solely dependent upon DRG's case-mix is one of several factors used to make adjustments to existing per diem rates. A key recommendation is that hospitals conduct internal monitorings with all involved departments to improve the quality of the data abstracting process.

"Small" melanomas: relation of prognostic variables to diameter of primary superficial spreading melanomas.

In a consecutive series of 648 superficial spreading melanomas a significantly better 5-year disease-free survival rate was observed for patients whose primary tumors were 14 mm or less in diameter when compared with those 15 mm or larger in diameter. Other distinguishing features of the group of "smaller" superficial spreading melanomas were that they occurred in younger patients; were of shorter durations; were more common in women; occurred disproportionately on the lower limbs; were less elevated; tended to be round in shape; were thinner (Breslow); penetrated less deeply (Clark levels); showed less histologic regression; and developed fewer metastases. Based on these findings it is recommended that educational programs be undertaken for the medical profession and for the public to promote early diagnosis and prompt treatment of superficial spreading melanomas when they are small in diameter and more often curable. A color atlas of "small" melanomas is presented.

A prognostic model for clinical stage I melanoma of the upper extremity. The importance of anatomic subsites in predicting recurrent disease.

Thirteen variables were studied for their relative usefulness in predicting recurrent disease in 107 patients with clinical Stage I melanoma of the upper extremity. After a mean follow-up period of 54 months, the only patents who have had recurrent disease to date are those who primary lesions were located either on the hand or posterior upper arm. The five-year disease-free survival role for 44 patients with melanoma at these sites was 68%. None of 63 patients with melanoma located on the forearm of anterior upper arm have had recurrent disease (i.e., the five-year, disease-free survival rate was 100% (p = 0.00004), compared with the hand or posterior arm group). A Cox proportional hazards (multivariate) analysis demonstrated that two primary tumor histologic variable, thickness in millimeters and ulceration, interacted to produce the best prognostic model for those 44 patients with melanoma of the hand or posterior upper arm. Twenty-one patients with primary lesions at these sites had primary tumors less than 2.25 mm in thickness and no evidence of ulceration histologically. Their five-year, disease-free survival role was 95%. For the remaining 23 patients with primary tumors on the hand or posterior upper arm who had either histologic evidence of ulceration or primary tumors greater than or equal to 2.25 mm, the five-year disease-free survival rate was 37% (p = 0.002, compared with group nonulcerated, thin lesions). The excellent survival rate for patients with melanomas on the forearm or anterior upper arm was not completely explained by pathologic stage, by primary tumor thickness, or by histologic ulceration of the primary tumor.

Malignant melanoma patients with positive nodes and relatively good prognoses: microstaging retains prognostic significance in clinical stage I melanoma patients with metastases to regional nodes.

Fifteen variables were tested for their value in predicting recurrent disease in 46 clinical Stage I melanoma patients with metastases to regional nodes. A stepwise proportional hazards general linear model (Cox multivariate analysis) separated these melanoma patients with regional node metastases into at least two risk groups. Twenty patients in the relatively low-risk group had a five-year disease-free survival of 80% (in spite of having nodal metastases). This compares to a five-year disease-free survival of 17.5% for 26 patients in the high-risk group (P less than 0.001, Lee-Desu Statistic). Criteria for the high-risk group required that a patient have only one of the following two values: (1) The number of regional lymph nodes that contained tumor divided by the total number of nodes removed x 100% (percentage of positive nodes) greater than or equal to 20%; or (2) a primary tumor thickness of greater than 3.5 mm (regardless of node percentage). Conversely, patients in the low-risk group had neither of the above features. The high-risk group could further be stratified by the lymphocytic response at the base of the tumor. These findings have direct immediate application to the elective regional node dissection controversy and to adjuvant therapy studies containing these patients.

Prognostic factors for melanoma patients with lesions 0.76 - 1.69 mm in thickness. An appraisal of "thin" level IV lesions.

Fourteen variables were tested for their prognostic usefulness in 203 patients with clinical Stage I melanoma and primary tumor 0.76-169 mm thick. Only two variables, primary tumor location and level of invasion, were useful in predicting death from melanoma for these patients. Of the 12 deaths from melanoma, 11 occurred in patients with primary tumors located on the upper back, posterior arm, posterior neck, and posterior scalp (=BANS). There has been only one death from melanoma in 136 patients with melanoma located at other sites (11/67 vs 1/136, p less than 0.0001 Fisher's Exact Test). Of the 67 BANS patients, 51 had level II or level III lesions and five (10%0 died of melanoma. This compared with six deaths from melanoma in 16 patients (37.5%) with level IV BANS lesions (5/51 vs 6/16, p = 0.01 Fisher's Exact Test). The relatively high incidence of both melanoma deaths and regional node metastases for the BANS group merits consideration for testing the efficacy of elective regional node dissection for these patients.

A multivariate analysis of prognostic factors for melanoma patients with lesions greater than or equal to 3.65 mm in thickness. The importance of revealing alternative Cox models.

Fourteen prognostic factors were examined in 79 patients with clinical Stage I melanoma greater than or equal to 3.65 mm in thickness. All nine patients with melanoma of the hands or feet died of melanoma. A Cox proportional hazards (multivariate) analysis of the remaining 70 patients showed that a combination of the following four variables best predicted bony or visceral metastases: 1) a nearly absent or minimal lymphocyte response at the base of the tumor, 2) histologic type other than superficial spreading melanoma, 3) location on the trunk, and 4) positive nodes or no initial node dissection. Ulceration and/or ulceration width were not useful in predicting outcome either singly or in combination with other variables. Patients with negative lymph nodes and primary tumors of the trunk, hands, and feet did not do better than patients with positive nodes at those sites. Conversely, non of 16 patients with negative lymph nodes and extremity melanomas (excluding the hands and feet) or head and neck melanomas developed visceral or bony metastases (i.e., five-year disease-free survival rate 100%).

Prognostic factors for patients with clinical stage I melanoma of intermediate thickness (1.51 - 3.39 mm). A conceptual model for tumor growth and metastasis.

Fourteen variables were tested for their ability to predict visceral or bony metastases in 177 patients with clinical Stage I melanoma of intermediate thickness (1.51 - 3.39 mm). A Cox multivariate analysis yielded a combination of four variables that best predicted bony or visceral metastases for these patients: 1) mitoses greater than 6/min 2 (p = 0.0007), 2) location other than the forearm of leg) p = 0.009, 3) ulceration width greater than 3 mm (p = 0.04), 4) microscopic satellites (p = 0.05). The overall prognostic model chi square was 32.40 with 4 degrees of freedom (p less than 10 (-5). Combinations of the above variables were used to separate these patients into at least two risk groups. The high risk patients had at least a 35% or greater chance of developing visceral metastases within five years, while the low risk group had greater than an 85% chance of being disease free at five years. Criteria for the high risk group were as follows: 1) mitoses greater than 6/mm 2 in at least one area of the tumor, irrespective of primary tumor location, or 2) a melanoma located at some site other than the forearm or leg and histologic evidence in the primary tumor of either ulceration greater than 3 mm wide or microscopic satellites. The low risk group was defined as follows: 1) mitoses less than or equal to 6/mm 2 and a location on the leg or forearm, or 2) mitoses less than or equal to 6/mm 2 and the absence in histologic sections of the primary tumor of both microscopic satellites and ulceration greater then 3 mm wide. The number of patients in this series who did not undergo elective regional node dissection (N = 47) was probably too small to detect any benefit from this procedure. Based on survival rates from this and other studies, it is estimated that approximately 1500 patients with clinical Stage I melanoma of intermediate thickness in each arm of a randomized clinical trial would be needed to detect an increase in survival rates from elective regional node dissection.