SARS-CoV-2 ORF8 accessory protein is a virulence factor.

IMPORTANCE: The relevance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF8 in the pathogenesis of COVID-19 is unclear. Virus natural isolates with deletions in ORF8 were associated with wild milder disease, suggesting that ORF8 might contribute to SARS-CoV-2 virulence. This manuscript shows that ORF8 is involved in inflammation and in the activation of macrophages in two experimental systems: humanized K18-hACE2 transgenic mice and organoid-derived human airway cells. These results identify ORF8 protein as a potential target for COVID-19 therapies.

Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures.

Human respiratory syncytial virus (HRSV) is the leading cause of bronchiolitis in infants. Two subgroups of HRSV (A and B) routinely cocirculate. Most research has been performed with HRSV-A strains because these are easier to culture than HRSV-B strains. In this study, we aimed to compare the replicative fitness and HRSV-induced innate cytokine responses of HRSV-A and HRSV-B strains in disease-relevant cell culture models. We used two recombinant (r) clinical isolate-based HRSV strains (A11 and B05) and one recombinant laboratory-adapted HRSV strain (A2) to infect commercially available nasal, bronchial, and small-airway cultures. Epithelial cells from all anatomical locations were susceptible to HRSV infection despite the induction of a dominant type III interferon response. Subgroup A viruses disseminated and replicated faster than the subgroup B virus. Additionally, we studied HRSV infection and innate responses in airway organoids (AOs) cultured at air-liquid interface (ALI). Results were similar to the commercially obtained bronchial cells. In summary, we show that HRSV replicates well in cells from both the upper and the lower airways, with a slight replicative advantage for subgroup A viruses. Lastly, we showed that AOs cultured at ALI are a valuable model for studying HRSV ex vivo and that they can be used in the future to study factors that influence HRSV disease severity.IMPORTANCE Human respiratory syncytial virus (HRSV) is the major cause of bronchiolitis and pneumonia in young infants and causes almost 200,000 deaths per year. Currently, there is no vaccine or treatment available, only a prophylactic monoclonal antibody (palivizumab). An important question in HRSV pathogenesis research is why only a fraction (1 to 3%) of infants develop severe disease. Model systems comprising disease-relevant HRSV isolates and accurate and reproducible cell culture models are indispensable to study infection, replication, and innate immune responses. Here, we used differentiated AOs cultured at ALI to model the human airways. Subgroup A viruses replicated better than subgroup B viruses, which we speculate fits with epidemiological findings that subgroup A viruses cause more severe disease in infants. By using AOs cultured at ALI, we present a highly relevant, robust, and reproducible model that allows for future studies into what drives severe HRSV disease.

Middle East respiratory syndrome coronavirus experimental transmission using a pig model.

Dromedary camels are the main reservoir of Middle East respiratory syndrome coronavirus (MERS-CoV), but other livestock species (i.e., alpacas, llamas, and pigs) are also susceptible to infection with MERS-CoV. Animal-to-animal transmission in alpacas was reported, but evidence for transmission in other species has not been proved. This study explored pig-to-pig MERS-CoV transmission experimentally. Virus was present in nasal swabs of infected animals, and limited amounts of viral RNA, but no infectious virus were detected in the direct contact pigs. No virus was detected in the indirect contact group. Furthermore, direct and indirect contact pigs did not develop specific antibodies against MERS-CoV. Therefore, the role of pigs as reservoir is probably negligible, although it deserves further confirmation.

A novel hepatitis B virus subgenotype D10 circulating in Ethiopia.

Hepatitis B virus (HBV) is genetically highly divergent and classified in ten genotypes and forty subgenotypes in distinct ethno-geographic populations worldwide. Ethiopia is a country with high HBV prevalence; however, little is known about the genetic variability of HBV strains that circulate. Here, we characterize the complete genome of 29 HBV strains originating from five Ethiopian regions, by 454 deep sequencing and Sanger sequencing. Phylogenetically, ten strains were classified as genotype A1 and nineteen as genotype D. Fifteen genotype D strains, provisionally named subgenotype D10, showed a novel distinct cluster supported by high bootstrap value and >4% nucleotide divergence from other known subgenotypes. In addition, the novel D10 strains harboured nine unique amino acid signatures in the surface, polymerase and X genes. Seventy-two per cent of the genotype D strains had the precore premature stop codon G1896A. In addition, 63% genotype A and 33% genotype D strains had the basal core promoter mutations, A1762T/G1764A. Furthermore, four pre-S deletion variants and two recombinants were identified in this study. In conclusion, we identified a novel HBV subgenotype D10 circulating in Ethiopia, underlining the high genetic variability of HBV strains in Africa.

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.

Middle East respiratory syndrome coronavirus (MERS-CoV) RNA and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar, April 2014.

Antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) were detected in serum and milk collected according to local customs from 33 camels in Qatar, April 2014. At one location, evidence for active virus shedding in nasal secretions and/or faeces was observed for 7/12 camels; viral RNA was detected in milk of five of these seven camels. The presence of MERS-CoV RNA in milk of camels actively shedding the virus warrants measures to prevent putative food-borne transmission of MERS-CoV.

The pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models.

Respiratory viruses that emerge in the human population may cause high morbidity and mortality, as well as concern about pandemic spread. Examples are severe acute respiratory syndrome coronavirus (SARS-CoV) and novel variants of influenza A virus, such as H5N1 and pandemic H1N1. Different animal models are used to develop therapeutic and preventive measures against such viruses, but it is not clear which are most suitable. Therefore, this review compares animal models of SARS and influenza, with an emphasis on non-human primates, ferrets and cats. Firstly, the pathology and pathogenesis of SARS and influenza are compared. Both diseases are similar in that they affect mainly the respiratory tract and cause inflammation and necrosis centred on the pulmonary alveoli and bronchioles. Important differences are the presence of multinucleated giant cells and intra-alveolar fibrosis in SARS and more fulminant necrotizing and haemorrhagic pneumonia in H5N1 influenza. Secondly, the pathology and pathogenesis of SARS and influenza in man and experimental animals are compared. Host species, host age, route of inoculation, location of sampling and timing of sampling are important to design an animal model that most closely mimics human disease. The design of appropriate animal models requires an accurate pathological description of human cases, as well as a good understanding of the effect of experimental variables on disease outcome.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) serology in major livestock species in an affected region in Jordan, June to September 2013.

Between June and September 2013, sera from 11 dromedary camels, 150 goats, 126 sheep and 91 cows were collected in Jordan, where the first human Middle-East respiratory syndrome (MERS) cluster appeared in 2012. All sera were tested for MERS-coronavirus (MERS-CoV) specific antibodies by protein microarray with confirmation by virus neutralisation. Neutralising antibodies were found in all camel sera while sera from goats and cattle tested negative. Although six sheep sera reacted with MERS-CoV antigen, neutralising antibodies were not detected.

Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in The Netherlands.

Arenaviruses are bi-segmented negative-stranded RNA viruses, which were until recently only detected in rodents and humans. Now highly divergent arenaviruses have been identified in boid snakes with inclusion body disease (IBD). Here, we describe the identification of a new species and variants of the highly divergent arenaviruses, which were detected in tissues of captive boid snakes with IBD in The Netherlands by next-generation sequencing. Phylogenetic analysis of the complete sequence of the open reading frames of the four predicted proteins of one of the detected viruses revealed that this virus was most closely related to the recently identified Golden Gate virus, while considerable sequence differences were observed between the highly divergent arenaviruses detected in this study. These findings add to the recent identification of the highly divergent arenaviruses in boid snakes with IBD in the United States and indicate that these viruses also circulate among boid snakes in Europe.

Performance evaluation of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, version 2.0, for detection and quantification of hepatitis C virus RNA.

To evaluate the analytical performance and explore the clinical applicability of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, v2.0 (CAP/CTM v2.0), a platform comparison was performed on panels and diagnostic samples with the Roche cobas AmpliPrep/cobas TaqMan HCV test (CAP/CTM v1.0), the Siemens Versant HCV RNA 3.0 branched DNA (bDNA) test, the Abbott m2000 RealTime HCV assay (Realtime assay), and the Siemens Versant HCV transcription-mediated amplification (TMA) test (TMA assay). The analytical performance of the CAP/CTM v2.0 on WHO and Acrometrix panels and clinical specimens of patients infected with HCV genotype 1, 2, 3, 4, 5, or 6 relative to that of the CAP/CTM v1.0 was significantly improved. In a qualitative comparison of the CAP/CTM v2.0 relative to the TMA assay on genotype 1 to 4 samples, the two tests proved to be almost equally sensitive. Response-guided therapy in one of five HCV genotype 4-infected patients previously tested with the CAP/CTM v1.0 would have significantly changed if tested with the CAP/CTM v2.0. In conclusion, the Roche CAP/CTM v2.0 has significantly better performance characteristics than the former CAP/CTM HCV v1.0 and the bDNA assay and performance characteristics comparable to those of the Realtime assay.

Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.

Pathology of experimental SARS coronavirus infection in cats and ferrets.

The pathology of severe acute respiratory syndrome-coronavirus (SARS-CoV) infection in cats and ferrets is poorly described, and the distribution of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV, in the respiratory tracts of these species is unknown. We observed SARS-CoV antigen expression and lesions in the respiratory tracts of 4 cats and 4 ferrets at 4 days postinoculation and ACE2 expression in the respiratory tracts of 3 cats and 3 ferrets without infection. All infected cats and ferrets had diffuse alveolar damage associated with SARS-CoV antigen expression. A novel SARS-CoV-associated lesion was tracheo-bronchoadenitis in cats. SARS-CoV antigen expression occurred mainly in type I and II pneumocytes and serous cells of tracheo-bronchial submucosal glands of cats and in type II pneumocytes of ferrets. ACE2 expression occurred mainly in type I and II pneumocytes, tracheo-bronchial goblet cells, serous epithelial cells of tracheo-bronchial submucosal glands in cats, and type II pneumocytes and serous epithelial cells of tracheo-bronchial submucosal glands in ferrets. In conclusion, the pathology of SARS-CoV infection in cats and ferrets resembles that in humans except that syncytia and hyaline membranes were not observed. The identification of tracheo-bronchoadenitis in cats has potential implications for SARS pathogenesis and SARS-CoV excretion. Finally, these results show the importance of ACE2 expression for SARS-CoV infection in vivo: whereas ACE2 expression in type I and II pneumocytes in cats corresponded to SARS-CoV antigen expression in both cell types, expression of both ACE2 and SARS-CoV antigen in ferrets was limited mainly to type II pneumocytes.

ALT and viral load decline during PEG-IFN alpha-2b treatment for HBeAg-positive chronic hepatitis B.

BACKGROUND: Alanine aminotransferase (ALT) is one of the main indicators for inflammatory activity in chronic hepatitis B. During interferon-based therapy, approximately 25%-40% of patients exhibit an ALT flare. OBJECTIVES AND STUDY DESIGN: To analyze the relation between ALT and HBV-DNA during pegylated interferon alpha-2b (PEG-IFN) treatment and compare different patterns of on-treatment viral load decline with the occurrence of ALT flares. RESULTS: Of the 123 patients included in this study 31 (25%) exhibited an ALT flare during treatment or follow-up. Six out of 8 (75%) host-induced flares, i.e. ALT flares which were followed by a HBV-DNA decrease associated with a favorable treatment outcome, occurred in patients with a delayed HBV-DNA decline pattern (delayed vs. non-delayed decline, p=.022); 5 of these 8 patients exhibited HBeAg loss and 4 even HBsAg loss at the end of follow-up. The prediction of ALT normalization was possible using on-treatment viral load. Based on the difference from baseline, the evolution of viral load and ALT level were strongly interrelated during treatment and follow-up. With a joint model we estimated a correlation coefficient of 0.38 (p<0.001) during the first 4 weeks of the treatment and of 0.72 (p<0.0001) thereafter. CONCLUSION: There was a strong relation between ALT and viral load in HBeAg-positive chronic hepatitis B patients treated with PEG-IFN alpha-2b, especially after 4 weeks of treatment. Patients with a delayed decline in viral load often exhibited a host-induced flare associated with a favorable outcome.

The emerging role of ACE2 in physiology and disease.

The renin-angiotensin-aldosterone system (RAAS) is a key regulator of systemic blood pressure and renal function and a key player in renal and cardiovascular disease. However, its (patho)physiological roles and its architecture are more complex than initially anticipated. Novel RAAS components that may add to our understanding have been discovered in recent years. In particular, the human homologue of ACE (ACE2) has added a higher level of complexity to the RAAS. In a short period of time, ACE2 has been cloned, purified, knocked-out, knocked-in; inhibitors have been developed; its 3D structure determined; and new functions have been identified. ACE2 is now implicated in cardiovascular and renal (patho)physiology, diabetes, pregnancy, lung disease and, remarkably, ACE2 serves as a receptor for SARS and NL63 coronaviruses. This review covers available information on the genetic, structural and functional properties of ACE2. Its role in a variety of (patho)physiological conditions and therapeutic options of modulation are discussed.

Virus specific immune responses after human neoadjuvant adenovirus-mediated suicide gene therapy for prostate cancer.

PURPOSE: Neoadjuvant gene therapy potentially improves the outcome of primary treatment of prostate cancer by radical prostatectomy in patients with high risk of recurrence. We conducted a Phase I escalating dose study with a replication-defective adenovirus expressing the herpes simplex virus-thymidine kinase gene (Adv-HSV-tk vector). The primary end point was toxicity, while the evaluation of the patients' cellular and humoral immune responses served as a secondary endpoint. MATERIAL AND METHODS: The Adv-HSV-tk vector was injected into the prostate in two doses (2x10(10) to 2x10(11) viral particles), followed by ganciclovir twice daily for 14 days and retropubic radical prostatectomy on day 21. Adenovirus-specific neutralizing, IgG and IgA antibodies were evaluated. Peripheral blood mononuclear cells (PBMC) were stimulated by Adv-HSV-tk and analysed for IFN-gamma production and 3H-thymidine incorporation. Prostate specimens were immunostained for B (CD20+) and for T (CD3+) lymphocytes. RESULTS: Toxicity was minor in all 8 patients treated. In the prostate, no virus related cytopathic effect could be observed. Dose-dependent infiltration of T and B lymphocytes in the whole prostate and in tumor areas was observed. Boosting of adenovirus-specific antibody responses was observed in 7 patients, and an increased adenovirus-specific PBMC proliferation and IFN-gamma production was seen after Adv-HSV-tk stimulation. CONCLUSION: Neo-adjuvant adenovirus-mediated cytotoxic gene therapy prior to prostatectomy for prostate cancer is feasible and safe in an outpatient setting for intraprostatic vector doses up to 2x10(11) viral particles. Activation of the immune system was observed. Application of higher vector doses may be considered.

Monitoring intrahepatic CD8+ T cells by fine-needle aspiration cytology in chronic hepatitis C infection.

Infection of the liver with hepatitis C virus (HCV) causes compartmentalization of CD8+ cytotoxic T cells to the site of disease. These cells are thought to be involved in viral clearance during interferon therapy. The repetitive analysis of the intrahepatic immune response is hampered by the difficulty to obtain the intrahepatic T cells. The fine-needle aspiration biopsy (FNAB) technique was evaluated for its use to obtain liver-derived CD8+ T cells in a minimally invasive way. In 26 chronic HCV patients who were evaluated for Peg-interferon and ribavirin combination therapy, pre-treatment FNABs and peripheral blood specimens were obtained simultaneously with liver tissue biopsies, and CD3+ and CD8+ T cells were quantified by immunocytochemistry. The CD8+/CD3+ ratio was significantly higher in the FNABs than in peripheral blood (P < 0.01), and similar to those in portal areas in the tissue biopsies. A significant correlation was observed between numbers of CD3+CD8+ T lymphocytes in the FNABs and the numbers of CD8+ cells in the lobular fields or in the portal tracts of the liver tissue biopsies, but not with CD3+CD8+ T lymphocytes in peripheral blood. Finally, the ratio of CD8+/CD3+ T lymphocytes in FNABs was significantly higher in those patients who responded rapidly to therapy when compared with slow responders at 4 weeks of treatment (P = 0.02). These findings demonstrate that the intrahepatic T-cell composition is reflected in FNABs, and that the FNAB technique can be used for predicting early virological response to therapy of patients chronically infected with HCV.

A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection.

Glycoproteins B (gB), gC and gD of pseudorabies virus (PRV) have been implicated as important antigens in protective immunity against PRV infection. As cell-mediated immunity plays a major role in this protective immunity, we determined the significance of these glycoproteins in the actual induction of cell-mediated immunity. We vaccinated pigs with plasmid DNA constructs coding for gB, gC or gD and challenged them with the virulent NIA-3 strain of pseudorabies virus. Vaccination with plasmid DNA coding for gB induced the strongest cell-mediated immune responses including cytotoxic T cell responses, whereas plasmid DNA coding for gD induced the strongest virus neutralising antibody responses. Interestingly, vaccination with gB-DNA reduced virus excretion early after challenge infection while vaccination with gC-DNA or gD-DNA did not.This is the first study to demonstrate that DNA vaccination induces cytotoxic T cell responses in pigs and that cell-mediated immunity induced by vaccination with gB-DNA is important for the reduction of virus excretion early after challenge infection.

Foreign-body reaction to dermal sheep collagen in interferon-gamma-receptor knock-out mice.

This study was performed to gain more insight into the role of interferon-gamma (IFN-gamma), a potent macrophage activator, in the foreign-body reaction to hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC). Because the results of earlier studies aimed at modulating the foreign-body reaction in AO rats by local or systemic treatment with anti-IFN-gamma were not completely unambiguous, we extended our investigations to IFN-gamma-receptor knock-out (KO) mice. Several parameters (i.e., macrophages, giant cells, T-cells, B-cells, granulocytes, expression of MHC class II, stroma formation, and degradation and calcification of the biomaterial) were compared between wild-type (WT) and KO mice. Remarkably, the foreign-body reaction was very similar in WT and KO mice. In both, giant cells were formed, but in contrast to previous results in AO rats, phagocytosis of HDSC bundles occurred hardly at all up to 9 weeks, and MHC class II expression was minimal. Stroma formation and vascularization were high and calcification occurred. T-cells comprised less than 1%; a few plasma cells were present in the KO mice at later time points. Granulocytes, mainly eosinophils, were present at all explantation time points. Because of the similar results in WT and KO mice, we question whether IFN-gamma plays a role at all in the foreign-body reaction in mice.

Vaccination of pigs against pseudorabies virus with plasmid DNA encoding glycoprotein D.

We analysed the ability of a plasmid carrying the gene encoding glycoprotein D (gD) of pseudorabies virus (PRV) to induce humoral and cell-mediated immune responses and assessed the protection provided by PRV-gD DNA vaccination against challenge infection with PRV. Immunization with plasmid PRV-gD induced neutralizing antibodies and lymphocyte proliferative responses both in mice and pigs. Moreover, when challenged with virulent PRV six weeks following the last immunization, PRV-gD DNA vaccinated pigs excreted virus for a significantly shorter period and showed less clinical symptoms than pigs vaccinated with a control plasmid. Thus, in the target animal, DNA vaccination with PRV-gD DNA induces protective immunity against challenge infection.

Effect of vaccination route and composition of DNA vaccine on the induction of protective immunity against pseudorabies infection in pigs.

Vaccination with naked DNA may be an alternative to conventional vaccines because it combines the efficacy of attenuated vaccines with the biological safety of inactivated vaccines. We recently showed that the vaccination with naked DNA coding for the immunorelevant glycoprotein D (gD) of pseudorabies virus (PRV) induced both antibody and cell-mediated immunity in pigs and provided protection against challenge infection. To determine whether the efficacy of the naked DNA vaccination against PRV could be improved, we compared three sets of variables. First, the efficacy of the naked DNA vaccine coding only for the immunorelevant gD was compared with a cocktail vaccine containing additional plasmids coding for two other immunorelevant glycoproteins, gB and gC. Second, the intramuscular route of vaccination was compared with the intradermal route. Third, the commonly used needle method of inoculation was compared with the needleless Pigjet injector method. Five groups of five pigs were vaccinated three times at 4-weeks intervals and challenged with the virulent NIA-3 strain of PRV 6 weeks after the last vaccination. Results showed that although the cocktail vaccine induced stronger cell-mediated immune responses than the vaccine containing only gD plasmid, both vaccines protected pigs equally well against challenge infection. Intradermal inoculation with a needle induced significantly stronger antibody and cell-mediated immune responses and better protection against challenge infection than intramuscular inoculation. Our data show that the route of administering DNA vaccines in pigs is important for an optimal induction of protective immunity.