A longitudinal and cross-sectional study ofEpstein-Barr virus DNA load: a possible predictor of AIDS-related lymphoma in HIV-infected patients.

INTRODUCTION: HIV-infected patients are more than 100-fold greater at risk for developing malignant AIDS-related lymphoma (ARL) compared to the general population. Most ARLs are EBV related. The main purpose of this study was to investigate whether a high peak EBV DNA load in HIV-infected patients is predictive of ARL, including classical Hodgkin lymphoma. METHODS: From an ongoing prospective HIV positive cohort study, we conducted a case-control study between 2004 and 2016 among patients from whom at least one EBV DNA load in serum or plasma was available. We compared peak EBV DNA load between patients with (49 cases) and without ARL (156 controls). RESULTS: The geometric mean of the peak EBV DNA load measured before diagnosis of malignant lymphoma was 52,565 IU/mL in EBER-positive lymphoma patients vs. 127 IU/mL in controls (p < .001). Patients with EBV DNA loads >100,000 IU/mL have an increased risk for diagnosis of malignant lymphoma compared to patients with EBV DNA loads ≤100,000 IU/mL (adjusted OR 12.53; 95%CI: 4.08; 38.42). In the longitudinal study, including 13 patients with at least three left-over plasma samples available for retesting, measurements of EBV-DNA during the preceding 12 months proved to be of poor value for predicting subsequent lymphoma diagnosis. CONCLUSIONS: A EBV DNA load >100,000 IU/mL can be useful in clinical setting to accelerate time to diagnosis and treatment. EBV-DNA loads in samples taken during the preceding year of ARL diagnosis showed to be of poor predictive value.

The sample of choice for detecting Middle East respiratory syndrome coronavirus in asymptomatic dromedary camels using real-time reversetranscription polymerase chain reaction.

The newly identified Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease, particularly in people with comorbidities, requires further investigation. Studies in Qatar and elsewhere have provided evidence that dromedary camels are a reservoir for the virus, but the exact modes of transmission of MERS-CoV to humans remain unclear. In February 2014, an assessment was made of the suitability and sensitivity of different types of sample for the detection of MERSCoV by real-time reverse-transcription polymerase chain reaction (RT-PCR) for three gene targets: UpE (upstream of the E gene), the N (nucleocapsid) gene and open reading frame (ORF) 1a. Fifty-three animals presented for slaughter were sampled. A high percentage of the sampled camels (79% [95% confidence interval 66.9-91.5%, standard error 0.0625]; 42 out of 53) were shown to be shedding MERS-CoV at the time of slaughter, yet all the animals were apparently healthy. Among the virus-positive animals, nasal swabs were most often positive (97.6%). Oral swabs were the second most frequently positive (35.7%), followed by rectal swabs (28.5%). In addition, the highest viral load, expressed as a cycle threshold (Ct) value of 11.27, was obtained from a nasal swab. These findings lead to the conclusion that nasal swabs are the candidate sample of choice for detecting MERS-CoV using RT-PCR technology in apparently healthy camels.

Des travaux de recherche approfondis sont encore nécessaires concernant le coronavirus responsable du syndrome respiratoire du Moyen-Orient (MERSCoV), un virus identifié récemment et qui provoque des troubles respiratoires sévères en particulier chez les individus atteints de pathologies multiples. Les études effectuées au Qatar et ailleurs ont démontré que les dromadaires font office de réservoirs du virus ; toutefois, les modalités précises de la transmission du MERS-CoV à l'être humain demeurent obscures. En février 2014, une équipe de chercheurs a évalué l'adéquation et la sensibilité de plusieurs types d'échantillons pour détecter le MERS-CoV en utilisant l'amplification en chaîne par polymérase couplée à une transcription inverse en temps réel (RT-PCR) spécifique pour trois cibles génétiques, à savoir la séquence UpE (en amont du gène E), le gène N (nucléocapside) et le cadre de lecture ORF1a. Pour ce faire, divers prélèvements ont été effectués sur 53 dromadaires destinés à l'abattage. Un fort pourcentage de ces dromadaires (79 % [intervalle de confiance à 95 % compris entre 66,9 et 91,5 %, erreur standard : 0,0625], soit 42 sur 53) excrétaient le MERSCoV au moment de l'abattage, mais aucun ne présentait le moindre signe clinique. Les échantillons dans lesquels le plus de cas positifs ont été détectés étaient les écouvillons nasaux (97,6 %). Venaient ensuite les écouvillons oraux, qui ont détecté 35,7 % de cas positifs, puis les écouvillons rectaux (28,5 % de cas positifs détectés). Par ailleurs, ce sont les écouvillons nasaux qui ont permis d'obtenir l'intensité la plus élevée de la réponse de la RT-PCR, exprimée en une valeur du seuil de cycles de 11,27. Ces résultats permettent de conclure que les écouvillons nasaux sont les échantillons à privilégier pour la détection du MERS-CoV par RTPCR chez les dromadaires asymptomatiques.

Es preciso investigar más a fondo el coronavirus del síndrome respiratorio de Oriente Medio (MERS-CoV), recién identificado, que provoca una grave enfermedad respiratoria, sobre todo en personas con afecciones concomitantes. Estudios realizados en Qatar y otros lugares han deparado pruebas de que los dromedarios son un reservorio del virus, pero aún no están del todo claros los modelos exactos de transmisión del MERS-CoV al ser humano. Los autores describen un análisis realizado en febrero de 2014 de la idoneidad y sensibilidad de distintos tipos de muestra para detectar el MERS-CoV mediante una reacción en cadena de la polimerasa acoplada a transcripción inversa en tiempo real (RTPCR) dirigida contra tres genes: el gen UpE (upstream of the E gene: en dirección 5' desde el gen E); el gen N (nucleocápside) y el marco de lectura abierto (ORF) 1a. Para ello se tomaron muestras de 53 animales enviados al sacrificio. Se comprobó que un elevado porcentaje de los dromedarios analizados (un 79% [intervalo de confianza al 95%: 66,9­91,5%; error estándar: 0,0625], esto es, 42 de 53) excretaban virus en el momento del sacrificio, pese a que todos los animales parecían estar sanos. Entre los ejemplares positivos para el MERS-CoV, las muestras que con más frecuencia arrojaban resultado positivo eran los frotis nasales (97,6%). Las segundas, por orden de frecuencia, eran los frotis bucales (35,7%), seguidos de los frotis rectales (28,5%). Además, la carga viral más alta, expresada por un valor de ciclo umbral (Ct) (o punto de cruce) de 11,27, se obtuvo a partir de un frotis nasal. Estos resultados llevan a la conclusión de que los frotis nasales son el tipo de muestra más adaptado para detectar el MERS-CoV en dromedarios aparentemente sanos mediante la técnica de RT-PCR.

[Chronotherapy for affective disorders]. / Chronotherapie bij stemmingsstoornissen.

BACKGROUND: In the literature there is increasing interest in the chronobiology of affective disorders and in the most important chronotherapies for treating these disorders. AIM: To discuss the background to and the main features of the most important therapies for affective disorders. METHOD: Using PubMed, we performed a concise review of the literature on the use of chronotherapeutics in affective disorders and we also studied the standard textbooks on the subject. RESULTS: Light therapy is the type of chronotherapy that has been studied most. Chronotherapies show interesting and promising results in open label studies, but so far there have been no randomized double-blind placebo controlled trials. CONCLUSION: Chronotherapeutics provides a neurobiological model and a series of promising, possibly effective non-pharmacological therapies, particularly for affective disorders. Light therapy deserves to be included in the multidisciplinary treatment guidelines relating to affective disorders. However, more, better and longer trials are needed in order to evaluate the various types of chronotherapies.

[Is there adequate discussion between psychiatrists and their patients regarding patients' sexual problems?]. / Het bespreken van seksuele problemen door psychiaters en aiossen.

BACKGROUND: Sexual problems arise frequently among psychiatric patients, either as a symptom of psychiatric disorders or as a side effect of psychotropic medication. However, it is questionable whether in daily practice psychiatrists and trainee psychiatrists give enough attention to patients' sexual problems. GOAL: To investigate how much attention psychiatrists and trainees give to patients' sexual problems and to discover what factors influence the amount of attention they give. METHODS: All psychiatrists and trainees working at two academic psychiatric centres and three mental health institutes in the province of North Holland were asked to complete an online questionnaire about the discussion of sexual problems. RESULTS: 164 psychiatrists and trainee psychiatrists completed the questionnaire (response rate 44%). About 50% of the respondents stated that they spent less than five minutes per week discussing sexual problems with their patients. When prescribing antidepressants and antipsychotics, psychiatrists and trainees often failed to inform patients about sexual side effects (33% in the case of antidepressants and 50% for antipsychotics). CONCLUSIONS: The investigation reveals that psychiatrists and trainees give little attention to sexuality problems of patients. The main reason for this seems to be feelings of shame and incompetence. Lack of time was not identified as a significant factor. We believe that the situation will improve considerably if psychiatrists and trainees involved in training programmes and supervisory activities give more attention to sexual problems.

Development and implementation of real-time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens.

Several real-time PCR and nucleic acid sequence-based amplification (NASBA) primer pairs and a modified real-time PCR primer pair for the detection of enteroviruses were compared. The modified real-time PCR primer pair was evaluated on clinical samples in comparison with cell culture using the MagnaPure LC Isolation instrument for nucleic acid extraction. Six hundred forty samples could be examined both by cell culture and real-time PCR. Faecal specimens (n = 285), cerebrospinal fluid (n = 210), throat swabs (n = 113), biopsies (n = 1--, vesicular fluid (n = 11), and pleural fluid specimens (n = 9) were included. By culture, 26/640 (4%) samples were positive for enterovirus. By real-time PCR, the number of positive specimens was 50 (7.8%). Of the 210 cerebrospinal fluid samples, three were positive by culture and nine by real-time PCR. Seventeen and 33 of a total of 285 faecal specimens were positive by culture and real-time PCR, respectively. In case of discrepant results, the clinical symptoms were in accordance with an infection due to enteroviruses. Genotyping using the VP1 gene correlated with serotyping by neutralization. In contrast, six of the 19 specimens that could be typed both by neutralization and by sequencing using the VP4 domain yielded a different genotype, yet within the same species. Real-time PCR turned out to be suitable for the detection of enteroviruses in the daily routine setting. In comparison to rapid culture, it offers a rapid, more sensitive, and reliable assay; especially in cerebrospinal fluid, the yield of enteroviruses is much higher.

Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union.

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Oral transmission of porcine reproductive and respiratory syndrome virus by muscle of experimentally infected pigs.

The current study was performed to determine if porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted to pigs by feeding muscle tissue obtained from recently infected pigs. Muscle obtained from pigs infected with either a European strain (EU donor pigs) or American strain (US donor pigs) of PRRSV was fed to PRRSV-free receiver pigs. The donor pigs were slaughtered 11 days post-infection (dpi). PRRSV was detected by conventional virus isolation in muscle at 11 dpi from 7 of 12 EU donor pigs and 5 of 12 US donor pigs. In contrast to conventional virus isolation, all muscle samples from infected pigs were positive for viral nucleic acid by PCR, except for muscle from one animal infected with the American strain of PRRSV. Five hundred grams of raw semimembranosus muscle from each of the donor pigs was fed over a 2 days period (250 g per day) to each of two receiver pigs (48 receiver pigs). The receiver pigs were housed separately in five groups. One of the five groups was fed muscle obtained from US donor pigs that was also spiked with the American strain of PRRSV. Sentinel pigs were placed in-contact with the group of receiver pigs fed spiked muscle. All receiver pigs became viraemic by 6 days post-feeding (dpf). There was evidence of horizontal transmission with sentinel pigs, in-contact with receiver pigs, becoming viraemic. The study demonstrates that PRRSV could be infectious through the oral route via the feeding of meat obtained from recently infected pigs.

Virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages.

The objective of this study was to measure the effect of two variables (pig age and virus strain) on selected responses (clinical signs, viraemia, virus excretion and seroconversion) of pigs following exposure to porcine reproductive and respiratory syndrome (PRRS) virus. Therefore, young (6 till 8 weeks old) and old (6 months old) pigs were infected with 3 different PRRSV strains, i.e. LV ter Huurne (LVTH), LV4.2.1 and SDSU#73. Regardless of the strain used for exposure, young pigs were more susceptible to infection as shown by a higher number of viraemic and virus excreting pigs. Strain differences were also evident. LV ter Huurne induced virus excretion in a higher number of pigs and with a higher virus titre, whereas SDSU#73 induced most severe clinical signs. LV4.2.1 induced viraemia and virus excretion in a low number of pigs. The kinetics of the antibody response differed per virus strain. The results presented here are useful in developing a less expensive standardised infection model, consisting of young pigs intranasally infected with a virulent PRRSV strain, to study the efficacy of new vaccine strains.

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus.

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.

Discrimination of different subsets of cytolytic cells in pseudorabies virus-immune and naive pigs.

We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6(+) CD8(bright+) CD4(-) (CTL phenotype), CD6(+) CD8(dull+) CD4(+) (the fraction containing memory T helper cells), CD6(+) CD8(-) CD4(+) (T helper cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(+) ( gammadelta-T cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(-) (NK phenotype) and CD6(-) CD8(-) CD4(-) gammadelta-T(-) or gammadelta-T(+). Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6(+) CD8(+) and CD4(+) CD8(+) and fewer CD6(-) CD8(+) and gammadelta-T(+) CD8(+) lymphocytes than IL-2 stimulation of naive PBMC (P<0.05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6(+) CD8(+) T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6(-) CD8(+) T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.

Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for CD8(+) cells.

Porcine reproductive and respiratory virus (PRRSV) primarily infects and destroys alveolar macrophages of the pig. The aim of the present study was to characterize the changes of leukocyte populations in the broncho-alveolar lavage fluid (BALF) of PRRSV-infected pigs. Piglets were inoculated intranasally with PRRSV strain LV ter Huurne. On various days post-infection the piglets were sacrificed and the lungs removed, washed semi-quantitatively and analysed by flow cytometry. The total number of recovered BALF cells increased approximately 10 times between day 10 and day 21 of infection and decreased thereafter. The number of small low-autofluorescent cells (SLAC), i.e. lymphocytic and monocytic cells, increased very strongly from day 2 until day 21 of infection; in contrast, the number of large highly autofluorescent cells (LHAC), i.e. mostly macrophages, remained constant until day 14 of infection, increased slightly on day 21 and then decreased. On day 21 of infection in specific-pathogen-free piglets approximately 60% of the SLAC consisted of CD2(+)CD8(+)CD4(-)gammadeltaTCR(-) cells, which were partly CD8(+)CD6(+) and partly CD8(+)CD6(-). These phenotypes correspond to that of cytotoxic T-cells and natural killer cells respectively. From these results we can conclude that during a PRRSV infection the total number of BALF cells increases mainly due to an influx of lymphocytic cells with a cytolytic phenotype.

Establishment and characterization of porcine cytolytic cell lines and clones.

Although non-major-histocompatibility-complex-restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, the phenotype and functional characteristics of these cells are not well defined. To allow a detailed analysis of these subsets, we established and characterized cell lines and clones of interleukin-2-activated (IL-2) cytolytic cells. Cell lines and clones were obtained from peripheral blood mononuclear cells of minipigs of the swine-leucocyte-antigen-complex (SLA) d/d haplotype. Cells were cultured in the presence of human recombinant IL-2 and cloned by double limiting dilution in the presence of gamma-irradiated L14 cells (a retrovirus immortalized B-lymphoblastoid cell line of the haplotype SLAd/d) or gamma-irradiated autologous peripheral blood mononuclear cells as feeder cells. Cytolytic cell lines and clones were characterized for their ability to kill different target cells and for their cell surface phenotype. All obtained clones expressed CD2 and CD8 and were negative for CD4. The following three subsets of cytolytic cells were identified: Subset 1) CD3- CD5- cells that killed K562 cells (a natural killer cell susceptible target cell line), as well as the pseudorabies virus (PRV)-infected or uninfected porcine kidney cells. These cells were considered to be typical natural killer cells. Subset 2) CD3 gamma/delta + CD5- T-cells that killed K562 cells and PRV virus-infected or uninfected porcine kidney cells, infected or uninfected L14 cells, and L14 cells constitutively expressing the PRV viral glycoprotein gB or gC. These cells were considered to be gamma/delta T-cells with natural killer activity. Subset 3) CD3 alpha/beta + CD5+ T-cells that killed L14 cells, PRV-infected L14 cells, and PRV gB- and gC-transfected L14 cells. These cells were possibly induced by the L14 feeder cells, used in the in vitro culture system. None of the cytolytic effector cells killed only MHC-matched viral infected cells. In conclusion, we describe a method to isolate, clone, and culture cytolytic cells from pigs. The clones could be cultured for 5 months, which allowed appropriate phenotypic and functional characterization of the various clones. Two of the subsets, CD3 gamma/delta T- and the natural killer cell subset may be involved in antiviral immunity in this species.

Cell-mediated immunity to pseudorabies virus: cytolytic effector cells with characteristics of lymphokine-activated killer cells lyse virus-infected and glycoprotein gB- and gC-transfected L14 cells.

We examined cytolytic cells that lyse pseudorabies virus (PRV)-infected cells in pigs. In vitro stimulation of peripheral blood mononuclear cells from PRV-immune pigs with live PRV generated cells that lysed PRV-infected immortalized B cells. Several lines of evidence indicated a major contribution of non-major histocompatibility complex (MHC)-restricted cytolytic cells, which displayed characteristics of natural killer (NK) or lymphokine-activated killer cells: cytolysis was non-MHC-restricted, depended on CD2+CD4-CD8bright- (or CD2+CD4-CD8dull+) cells, was strongly augmented by in vitro antigenic stimulation and was not limited to virus-infected cells, i.e. the NK cell-susceptible target cell line K562 was also lysed. Cytolytic cells were also generated by in vitro antigenic stimulation with UV-inactivated PRV. Target cells transfected with and stably expressing PRV gB or gC were lysed to the same degree as PRV-infected target cells.

Development and antigen specificity of the lymphoproliferation responses of pigs to pseudorabies virus: dichotomy between secondary B- and T-cell responses.

To better understand the contribution of T cells to the immunity of pigs to pseudorabies virus (PRV), we examined the lymphoproliferation response to this virus. Depletion studies demonstrated that both CD2+CD8+ and CD2+CD4+ cells contributed to lymphoproliferation, but to varying degrees upon stimulation with live and ultraviolet (UV) light-inactivated PRV. Flow cytometric analysis revealed the emergence of both CD2+CD8+ and CD2+CD4+ lymphoblastoid cells. To examine the contribution of specific viral proteins, we prepared immortalized porcine B cells of haplotype d/d that stably expressed a single PRV protein, and used these cells for in vitro stimulation of lymphocytes from PRV-immune miniature pigs of the same haplotype. Cells expressing PRV gB or gC induced proliferation. An immunization/challenge experiment showed that the lymphoproliferation response was stronger upon immunization with the virulent NIA-3 strain than with the attenuated Bartha strain. Upon challenge inoculation, the NIA-3-immunized pigs were almost completely immune, in contrast to the Bartha-immunized pigs. Such poorly protected pigs showed secondary B- and T-cell immune responses upon challenge. In contrast, the better protected NIA-3-immunized pigs did not show a secondary B-cell response. However, they developed a secondary lymphoproliferation response, which was quicker and stronger than in the Bartha-immunized pigs. This dichotomy between secondary B- and T-cell responses indicates that an effective T-cell memory response is able to quickly eliminate challenge virus in immune pigs, so preventing a secondary B-cell response.

Interaction of pseudorabies virus with immortalized porcine B cells: influence on surface class I and II major histocompatibility complex and immunoglobulin M expression.

We examined whether the L14 cell line, an immortalized B cell line originating from inbred miniature pigs of the MHC haplotype d/d, could be useful to study T cell responses of pigs to pseudorabies virus (PRV). Compared with porcine kidney cells, the replication of PRV in L14 cells was slower and yielded lower quantities of infectious virus, which agrees with the reported poor replication of PRV in peripheral blood lymphocytes of swine. The virus yield and the number of L14 cells expressing the viral glycoprotein gE were both maximal at 48 h postinfection, when approximately 90% of all viable L14 cells expressed gE. Morphologically detectable effects of PRV replication in L14 cells were not obvious, but the number of viable cells at 72 h postinfection was lower in infected cultures than in uninfected cultures. Major histocompatibility complex (MHC) class I and II antigen expression was significantly higher at different time points postinfection on infected than on uninfected L14 cells. In contrast, expression of IgM appeared very slightly reduced on infected L14 cells, indicating a selective influence of PRV on cellular protein expression. PRV-infected L14 cells were lysed by lymphocytes from PRV-immune minipigs of MHC haplotype d/d, indicating their usefulness in in vitro cytolytic assays.

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity.

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.

Vaccine properties of pseudorabies virus strain 783 are not affected by a deletion of 71 base pairs in the promoter/enhancer region of the viral immediate early gene.

Strain 783 of pseudorabies virus (PRV) is a genetically engineered vaccine which contains three deletions. The purpose of this study was to examine the effect of one of the deletions, which until now has not been characterized. The deletion occurs within the inverted repeats. Seventy-one base pairs (bp) were deleted, including one of the repeat sequence elements related to the TAATGARATTC boxes detected within the promoter and enhancer region of the immediate early (IE) genes of herpes simplex virus. The deletion affected neither the transcription of the IE gene nor viral growth in vitro. In our animal experiments, one group of pigs was inoculated with the original strain 783 and another with strain 783 which had had the repeat sequences restored. These two groups were then compared to determine the protective efficacy of the two vaccine strains against PRV infection. The deletion in the inverted repeats does not affect the vaccine properties of PRV strain 783: strain 783, with and without the 71 bp deletion in the repeats, protected pigs equally well.

Longitudinal analysis of hepatitis C virus infection and genetic drift of the hypervariable region.

Hepatitis C virus (HCV) infections in a cohort of chimpanzees were studied retrospectively. All animals had been inoculated intravenously with materials derived from a single-source chimpanzee plasma implicated in non-A, non-B hepatitis, prepared by extensive ultracentrifugation. Anti-HCV and HCV RNA were monitored by the confirmatory line immunoassay and by an RNA-capture polymerase chain reaction method, respectively. In a chronically infected chimpanzee, HCV RNA was detectable after 32 days and throughout the acute phase, dropped transiently below detection level, and became detectable again. In 3 other chimpanzees with acute resolving infections, HCV RNA was detected 7-11 days after inoculation and became permanently undetectable after alanine aminotransferase normalization. Various anti-HCV profiles were detected among the chimpanzees. Analysis of the hypervariable region in E2/NS1 in 7 chimpanzees suggested genome stability on transmission, revealed different mutation frequencies during chronic infection, and suggested the importance of immune selection during chronic HCV infection.

Rapid detection of hepatitis C virus RNA by direct capture from blood.

A new diagnostic assay for hepatitis C virus RNA detection is described. HCV genomic RNA is captured onto streptavidin-coated magnetic beads by solution hybridization with biotinylated complementary oligonucleotides. The specificity of the capture assay is confirmed using different capture oligonucleotides as well as sera representing different types of HCV. Sensitivity was determined by testing serial dilutions of a HCV infected plasma. A panel of 50 sera was tested for anti-HCV by a Line Immunoassay and for HCV-RNA by both a conventional guanidinium extraction method and the new capture assay. The specificity of the capture assay was 95.8% and the sensitivity was 92.3% compared to the standard protocol. This method provides a rapid and simple alternative for HCV-RNA detection in blood samples.