Spread rate of lumpy skin disease in the Balkans, 2015-2016.

After its introduction in Turkey in November 2013 and subsequent spread in this country, lumpy skin disease (LSD) was first reported in the western Turkey in May 2015. It was observed in cattle in Greece and reported to the World Organization for Animal Health (OIE) in August 2015. From May 2015 to August 2016, 1,092 outbreaks of lumpy skin disease were reported in cattle from western Turkey and eight Balkan countries: Greece, Bulgaria, The Former Yugoslav Republic of Macedonia, Serbia, Kosovo, and Albania. During this period, the median LSD spread rate was 7.3 km/week. The frequency of outbreaks was highly seasonal, with little or no transmission reported during the winter. Also, the skewed distribution of spread rates suggested two distinct underlying epidemiological processes, associating local and distant spread possibly related to vectors and cattle trade movements, respectively.

Sheep pox in Tunisia: Current status and perspectives.

Sheep pox, a well-known endemic capripox infection, has significant impacts on small ruminant populations in Tunisia. It is responsible for high economic losses throughout North Africa due to its enzootic nature and to the active animal transhumance existing in some governorates in Tunisia. The aim of this review was to analyse data gathered on annual vaccination campaigns designed to control its spread by reducing the level of endemicity and to describe diagnostic and management tools adapted to the Tunisian situation. Seasonal, temporal and spatial distributions of sheep pox outbreaks, as well as related clinical features, were found. It was concluded from this review that establishing strong herd immunization through individual animal immunization, creating adequate infrastructure, increasing awareness among breeders, setting up a field-based surveillance network and improving routine diagnostic methods need to be the major components of a programme to eradicate the disease. It was also felt that cost-benefit analyses of the surveillance and control strategies used would help in controlling its persistence.

Evaluation of indirect fluorescent antibody test (IFAT) for the diagnosis and screening of lumpy skin disease using Bayesian method.

The performance of indirect fluorescence antibody test (IFAT) for serological diagnosis and screening of lumpy skin disease (LSD) was evaluated using methods without gold standard. Virus neutralization test (VNT) was used as the second test and the study sites were selected from two different geographical places in Ethiopia to get different disease prevalence. The analysis of conditional dependent Bayesian model for the accuracy of IFAT showed that sensitivity, specificity, prevalence of the population Pi(1) and the population Pi(2) were 0.92 (0.89-0.95), 0.88 (0.85-0.91), 0.28 (0.25-0.32) and 0.06 (0.048-0.075), respectively. The posterior inferences obtained for VNT sensitivity, specificity and conditional correlation between the tests for sensitivity (rhoD) and specificity (rhoDc) were 0.78 (0.74-0.83), 0.97 (0.95-0.99), 0.052 (-0.03-0.15) and 0.019 (-0.01-0.06), respectively. The interval estimation of conditional correlation for both sensitivity and specificity clusters around zero and thus conditional dependence between the two tests was not significant. Although accuracy measure would not be the only basis for test selection, the result of our study demonstrated that IFAT has a reasonable high accuracy to be used for the diagnosis and sero-surveillance analysis of LSD in the target population.

Construction, characterization and immunogenicity of recombinant yellow fever 17D-dengue type 2 viruses.

Chimeric yellow fever (YF)-dengue type 2 (Den 2) viruses were constructed by replacing the premembrane (prM) and envelope (E) genes of YF 17D virus with those from Den 2 virus strains of south-east Asian genotype. Whereas viable chimeric viruses were successfully recovered when the YF 17D C gene and the Den 2 prM gene were fused at the signalase cleavage site, no virus could be rescued from the constructions fused at the viral protease cleavage site. Unlike YF virus that replicated in all the cell lines tested and similar to the Den 2 virus, the recombinant viruses did not replicate in vaccine-production certified CEF and MRC5 cells. Besides, chimeric 17D/Den 2 viruses and their parental viruses reached similar growth titers in Vero and C6/36 cell cultures. Analysis of mouse neurovirulence, performed by intracerebral inoculation, demonstrated that the 17D/Den 2 chimera is more attenuated in this system than the YF 17DD virus. Immunization of mice with this chimera induced a neutralizing antibody response associated with a partial protection against an otherwise lethal dose of mouse neurovirulent Den 2 NGC virus. Overall, these results provide further support for the use of chimeric viruses as an attractive methodology for the development of new live flavivirus vaccines.

Longitudinal analysis of CD8(+) T-cell phenotype and IL-7, IL-15 and IL-16 mRNA expression in different tissues during primary simian immunodeficiency virus infection.

Infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) represents a useful model of HIV infection that offers the unique opportunity to investigate the very early modifications that affect CD8(+) T-lymphocyte subsets and related cytokines during lentiviral infection. Herein, three cynomolgus macaques were inoculated intravenously with a pathogenic isolate of SIVmac 251. In fresh isolated mononuclear cells from blood, lymph node and bronchoalveolar lavage, we analyzed changes in the phenotype of CD8(+) T cells and we used reverse transcription-PCR to monitor the expression of IL-7, IL-15 and IL-16 mRNA. We demonstrated that an expansion of CD8(+)CD28(-) T cells occurs from the third week of infection on in the peripheral blood and in the lung, whereas CD8(+)CD28(+) T cells expand in the lymph nodes. Concomitantly, we evidenced mRNA modulations in IL-16, IL-15 and IL-7 expression in the three compartments studied. The containment of systemic viral replication was associated with an overexpression of IL-16 mRNA in the lung and in the peripheral blood. Given the immunomodulatory properties of IL-15 and IL-7 and the potential antiviral ability of IL-16, these perturbations could have important implications in early viral dissemination and HIV immunopathogenesis.

The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines.

The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF), dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain.

Secretion of beta-chemokines by bronchoalveolar lavage cells during primary infection of macaques inoculated with attenuated nef-deleted or pathogenic simian immunodeficiency virus strain mac251.

Primary infection of macaques with simian immunodeficiency virus (SIV) as a model of human immunodeficiency virus (HIV) infection represents a unique opportunity to investigate early lentivirus-host interactions. In order to gain insight into immunopathogenic events taking place in the lung during lentiviral infection, we analysed lymphocyte expansion in the lung and chemokine secretion by mononuclear cells obtained by bronchoalveolar lavage (BALMCs) during primary infection by a pathogenic and a non-pathogenic SIV. Two groups of cynomolgus macaques were inoculated intravenously with a fully pathogenic isolate of SIVmac251 or with an attenuated, nef-deleted, molecular clone of SIVmac251. Spontaneous MIP-1alpha, MIP-1beta and RANTES production was assessed by ELISA in supernatants of short-term cultured BALMCs. Kinetics of haematological, virological and immunological parameters were investigated simultaneously. All 11 inoculated animals became infected. Monkeys inoculated with the nef-deleted SIV clone exhibited a significantly reduced plasma virus load and a less pronounced accumulation of lymphocytes in the lung compared to monkeys infected with the pathogenic SIVmac251 isolate. Compared to pre-infection levels, we observed an increase in the levels of RANTES, MIP1-alpha and MIP1-beta production in the two groups of monkeys, by the time of peak viraemia. Strikingly, a greater enhancement of RANTES and MIP-1alpha production was detected in monkeys infected with the attenuated virus. Given the potential influence of beta-chemokines on the immune response and virus replication, such results suggest that RANTES, MIP1-alpha and MIP1-beta could contribute to the singular features of the immune response elicited during infection of macaques with an attenuated SIV.

RANTES, IFN-gamma, CCR1, and CCR5 mRNA expression in peripheral blood, lymph node, and bronchoalveolar lavage mononuclear cells during primary simian immunodeficiency virus infection of macaques.

Primary infection of macaques with pathogenic isolates of simian immunodeficiency virus (SIV) (as a model of HIV infection in humans) represents a unique opportunity to study early lentivirus/host interactions. We sought to determine whether there is a temporal relationship linking SIV replication and dissemination and the expression of the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) and the SIV/HIV coreceptor CCR5 in different tissues during acute SIV infection of macaques. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate of SIVmac251. RT-PCR was used to monitor the expression of RANTES and CCR5 mRNA in fresh isolated mononuclear cells from blood, lymph node, and bronchoalveolar lavages. These expressions were compared to those of IFN-gamma as an indicator of the development of the immune response and to another receptor for RANTES, CCR1, which is not described as a coreceptor for SIV/HIV-1 entry. An enhancement of CCR1/CCR5 mRNA expression was noticed during primary SIVmac251 infection of macaques, mainly in tissue. In the three different compartments investigated, IFN-gamma and RANTES overexpression was noticed by the time of systemic viral replication containment. Our results put CCR5 and RANTES mRNA expression back in the context of inflammatory and immune responses to SIV primary infection.

Nitric oxide synthesis during acute SIV mac251 infection of macaques.

During HIV1 infection, nitric oxide (NO) could significantly contribute to immune dysregulation by its multiple effects on the modulation of the host immune response. The in vivo regulation of NO production is attributable to several nitric oxide synthases, one of which is a cytokine-inducible enzyme (iNOS). In vitro experiments suggest that iNOS expression in macrophages may be directly modulated by HIV infection. Acute infection of macaques with a pathogenic strain of the simian immunodeficiency virus (SIV) represents a relevant animal model for the in vivo study of the relationships between iNOS expression and lentiviral replication. Indeed, acute infection in this model is characterized by high rates of viral replication associated with early cytokine dysregulations, in the absence of opportunistic infection. In our experiment, two cynomolgus macaques were inoculated intravenously with a pathogenic isolate of SIVmac251, and iNOS gene expression was investigated ex vivo during acute infection in mononuclear cells obtained from bronchoalveolar lavage (BALMCs). An enhancement of this gene expression was observed as early as the second week of infection, at the time of peak of systemic viraemia, and increased until day 31 p.i. This overexpression was concomitant with a marked linear increase in IFN gamma expression in BALMCs. At the time of systemic viral load peak, the production of NO in plasma of these two monkeys was evidenced by the detection of large amounts of nitrate.

Selective quasispecies transmission after systemic or mucosal exposure of macaques to simian immunodeficiency virus.

Sexual transmission is the major cause of the AIDS epidemic. For the development of new antiviral and vaccine strategies, we therefore need to understand the mechanisms by which lentiviruses cross the mucosal barrier and the subsequent pathogenic consequences. For this purpose, experimental approaches are greatly facilitated by the development of relevant animal models. In this study, macaques were inoculated intravenously, intrarectally, or intravaginally with a pathogenic cell-free isolate of SIVmac251. Patterns of virological and immunological events significantly differed between vaginally (transient viremia, late seroconversion) and intravenously or intrarectally inoculated monkeys (persistent viremia and early seroconversion). Two weeks after infection, analysis of the env gene nucleotide sequences of proviruses recovered from PBMCs demonstrated that most of the differences were observed in the V1 loop. Three viral variants were specifically associated with vaginal transmission, whereas no such selection was evidenced after intravenous or intrarectal transmissions. These results are in favor of specific mechanisms associated with vaginal transmission, implicating viral envelope structure.

Variation in virological parameters and antibody responses in macaques after atraumatic vaginal exposure to a pathogenic primary isolate of SIVmac251.

We developed an animal model for the male-to-female transmission of human immunodeficiency virus, consisting of an atraumatic vaginal application of simian immunodeficiency virus onto the intact vaginal mucosa of cynomolgus macaques. Different doses of a pathogenic isolate of SIVmac251, with or without seminal plasma, were infused into the vaginas of female macaques. Infection of macaques could be achieved after a single exposure to the virus. Two patterns of infection were underscored with no relation to the virus dose inoculated: in 50% of the monkeys, SIV was persistently recovered and a strong antibody response to SIV was evidenced in blood and vaginal secretions. In the other infected animals, SIV infection was only transiently evidenced and a weak systemic antibody response was detected. It appeared that the presence of seminal plasma may be implicated in this variability only when low doses of virus are inoculated. Sequence analysis of the env gene of SIV revealed that most of the persistently viraemic animals were infected with a viral variant different from that of transiently viraemic macaques.

Consequences of ddI-induced reduction of acute SIVmac251 virus load on cytokine profiles in cynomolgus macaques.

This study evaluates the consequences of antiretroviral treatment of the acute simian immunodeficiency virus (SIV) primary infection on virus load and cytokine responses. Four cynomolgus macaques were inoculated intravenously with a pathogenic primary isolate (SIVmac251). Animals were pretreated with 10.8 mg/kg/day of dideoxyinosine (ddI) from 4 days before inoculation, and treatment was continued for 28 days. Proinflammatory (IL6, IL1 beta and TNF alpha) and antiinflammatory (IL10) cytokine and lymphokine (IL2, IL4 and IFN gamma) polymerase chain reaction (PCR) ratios were monitored in unmanipulated peripheral blood mononuclear cells (PBMCs) during acute infection by using a semiquantitative reverse transcription (RT)-PCR method. PBMC-associated virus loads were dramatically reduced compared to those of placebo-treated macaques. Nevertheless, a transient rise in IL6, IL1 beta, TNF alpha and IL10 mRNA expression was observed in PBMCs. IL2, IL4 and IFN gamma mRNAs were either undetectable or weakly detectable throughout the study, with no major changes. Despite a dramatic reduction in the acute viral loads in ddI-treated monkeys, early cytokine mRNA profiles were comparable to those of untreated SIVmac251-infected monkeys. Contrary to what was previously evidenced during primary infection with an attenuated SIV clone, no increase in IL2 and IL4 mRNA was detected in PBMCs of the ddI-treated monkeys, although these monkeys exhibited virus loads similar to those evidenced in macaques infected by attenuated SIV. These data indicate that differential lymphokine expression patterns found in pathogenic and Nef-truncated SIV-infected monkeys may not be strictly dependent on virus load levels.

Chemoattractant factors (IP-10, MIP-1alpha, IL-16) mRNA expression in mononuclear cells from different tissues during acute SIVmac251 infection of macaques.

We have used semiquantitative RT-PCR to monitor the expression of mRNA encoding chemoattractant factors IP-10, MIP-1alpha, and IL-16 in freshly isolated peripheral blood mononuclear cells (PBMCs), lymph node mononuclear cells (LNMCs), and mononuclear cells obtained after bronchoalveolar lavages (BALMCs) of two cynomolgus macaques inoculated intravenously with a pathogenic isolate of simian immunodeficiency virus, SIVmac251. Concomitant with the peak of systemic viral replication (two weeks after experimental inoculation) and proinflammatory cytokine IL-6 mRNA expression, high levels of MIP-1alpha and IP-10 mRNA were produced in LNMCs and BALMCs. In BALMCs, in which we have reported a marked progressive overexpression of IFN-gamma mRNA coinciding with an increase in the CD8+ lymphocyte percentages, we noticed a progressive overexpression of IL-16 mRNA. Our results suggest the role of chemokines IP-10, MIP-1alpha, and IL-16 in the development of inflammatory and immune responses during the early stages of lentiviral infection.

Cytokine mRNA expression in mononuclear cells from different tissues during acute SIVmac251 infection of macaques.

We used semiquantitative RT-PCR to monitor the expression of mRNA encoding cytokines (IL-1 beta, IL-6, TNF-alpha, and IL-10) and IFN-gamma in fresh isolated peripheral blood mononuclear cells (PBMCs), lymph node mononuclear cells (LNMCs), and mononuclear cells obtained after bronchoalveolar lavages (BALMCs), of four cynomolgus macaques inoculated intravenously with a pathogenic isolate of simian immunodeficiency virus (SIVmac251). To investigate the effects of the viral load on the expression of the cytokines, two monkeys received 30 mg kg-1 day-1 of didanosine (ddI). The two nontreated monkeys became infected and seroconverted, whereas the ddI-treated monkeys were completely protected as demonstrated by all criteria of diagnosis of SIV infection. Concomitant with the peak of viral replication (2 weeks after the experimental inoculation), high levels of IL-6 mRNA were produced in PBMCs, LNMCs, and BALMCs of the two placebotreated infected monkeys. Overexpression of TNF-alpha and IL-10 mRNAs was sometimes observed in LNMCs and BALMCs. A progressive overexpression of IFN-gamma mRNA, starting 2 weeks after experimental inoculation, was observed in BALMCs from infected animals. Concurrently, a marked increase in the CD8+ lymphocyte percentage in the BAL fluids was detected by FACS analysis. Thus, our results emphasize the importance of a comparative study of the expression of cytokines in different tissues. They suggest the interactions of monocyte/macrophage monokine production with viral replication, as well as the role of IFN-gamma in the development of lung cellular immunity to SIV infection.