Serological and genomic evidence of Rift Valley fever virus during inter-epidemic periods in Mauritania.

Rift Valley fever virus (RVFV) is an emerging pathogen of major concern throughout Africa and the Arabian Peninsula, affecting both livestock and humans. In the past recurrent epidemics were reported in Mauritania and studies focused on the analysis of samples from affected populations during acute outbreaks. To verify characteristics and presence of RVFV during non-epidemic periods we implemented a multi-stage serological and molecular analysis. Serum samples of small ruminants, cattle and camels were obtained from Mauritania during an inter-epidemic period in 2012-2013. This paper presents a comparative analysis of potential variations and shifts of antibody presence and the capability of inter-epidemic infections in Mauritanian livestock. We observed distinct serological differences between tested species (seroprevalence: small ruminants 3·8%, cattle 15·4%, camels 32·0%). In one single bovine from Nouakchott, a recent RVF infection could be identified by the simultaneous detection of IgM antibodies and viral RNA. This study indicates the occurrence of a low-level enzootic RVFV circulation in livestock in Mauritania. Moreover, results indicate that small ruminants can preferably act as sentinels for RVF surveillance.

Prostaglandin profiling reveals a role for haematopoietic prostaglandin D synthase in adipose tissue macrophage polarisation in mice and humans.

BACKGROUND/OBJECTIVES: Obesity has been associated with both changes in adipose tissue lipid metabolism and inflammation. A key class of lipid-derived signalling molecules involved in inflammation are the prostaglandins. In this study, we aimed to determine how obesity affects the levels of prostaglandins within white adipose tissue (WAT) and determine which cells within adipose tissue produce them. To avoid the effects of cellular stress on prostaglandin levels, we developed a multivariate statistical approach in which metabolite concentrations and transcriptomic data were integrated, allowing the assignment of metabolites to cell types. SUBJECTS/METHODS: Eicosanoids were measured by liquid chromatography-tandem mass spectrometry and mRNA levels using real-time PCR. Eicosanoid levels and transcriptomic data were combined using principal component analysis and hierarchical clustering in order to associate metabolites with cell types. Samples were obtained from C57Bl/6 mice aged 16 weeks. We studied the ob/ob genetically obese mouse model and diet-induced obesity model. We extended our results in mice to a cohort of morbidly obese humans undergoing bariatric surgery. RESULTS: Using our modelling approach, we determined that prostglandin D2 (PGD2) in adipose tissue was predominantly produced in macrophages by the haematopoietic isoform of prostaglandin D synthase (H-Pgds). Analysis of sub-fractionated WAT confirmed that H-Pgds was expressed in adipose tissue macrophages (ATMs). Furthermore, H-Pgds expression in ATMs isolated from lean and obese mice was consistent with it affecting macrophage polarisation. Functionally, we demonstrated that H-PGDS-produced PGD2 polarised macrophages toward an M2, anti-inflammatory state. In line with a potential anti-inflammatory role, we found that H-PGDS expression in ATMs was positively correlated with both peripheral insulin and adipose tissue insulin sensitivity in humans. CONCLUSIONS: In this study, we have developed a method to determine the cellular source of metabolites within an organ and used it to identify a new role for PGD2 in the control of ATM polarisation.

Anti-HEV seroprevalence and rate of viremia in a German cohort of dogs, cats, and horses.

Hepatitis E virus (HEV) genotype 3 infections in Germany are mainly transmitted zoonotically through the consumption of swine meat. Furthermore, there is evidence that pets might come into contact with HEV, but the relevance of companion animals as possible sources of HEV transmission in Germany still needs to be defined. A monitoring study was therefore carried out on dogs, cats, and horses from Germany. In total 365 serum samples from pets (124 dogs, 119 cats, and 122 horses) were tested for HEV by PCR and for anti-HEV antibodies by a commercial ELISA. The HEV seroprevalence determined by the sero-assay varied significantly between dogs (10%), cats (6%), and horses (2%). Liver injury-related enzymes, alanine transaminase (ALT), and aspartate transaminase (AST) showed no differences between HEV-positive or negative animals. None of the pet serum samples tested positive for PCR. This serological study suggests that dogs and cats are significantly exposed to HEV in Germany, while horses are of minor relevance.

Detection of Crimean-Congo hemorrhagic fever virus in blood-fed Hyalomma ticks collected from Mauritanian livestock.

BACKGROUND: Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Orthonairovirus (Nairovididae) and is a (re)emerging tick-borne pathogen. It is endemic in most parts of Africa, Asia and southern Europe, and can cause severe hemorrhagic symptoms in humans, with high fatality rates (5-30%). METHODS: Hyalomma ticks were collected from four different livestock herds (cattle and camels) in Mauritania in 2018. The tick species were determined morphologically and confirmed molecularly by using the cytochrome oxidase 1 gene marker. For the detection of CCHFV, ticks were tested individually by one-step multiplex real-time reverse-transcriptase quantitative polymerase chain reaction. The small segment of all positive samples was sequenced to determine the CCHFV genotype. RESULTS: In total, 39 of the 1523 ticks (2.56%) collected from 63 cattles and 28 camels tested positive for CCHFV. Three Hyalomma species were identified. Hyalomma rufipes had the largest proportion of positivity (5.67%; 16/282), followed by Hyalomma dromedarii (1.89%; 23/1214). No Hyalomma impeltatum tested positive (0%; 0/21). Positive ticks were found in only six out of 91 host animals. Viral sequence analysis revealed the presence of two different CCHFV lineages (Africa I and Africa III). CONCLUSIONS: In this study, 2.56% of Hyalomma ticks collected from camels and cattle in Mauritania tested positive for CCHFV. However, the true prevalence of CCHFV in unfed ticks may be lower, as a considerable number of ticks may have been passively infected during blood-feeding by co-feeding ticks or due to viremia of the host. The results indicate the need to track the actual area of circulation of this virus.

Rift Valley fever virus detection in susceptible hosts with special emphasis in insects.

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes.

Antibody response against koala retrovirus (KoRV) in koalas harboring KoRV-A in the presence or absence of KoRV-B.

Koala retrovirus (KoRV) is in the process of endogenization into the koala (Phascolarctos cinereus) genome and is currently spreading through the Australian koala population. Understanding how the koala's immune system responds to KoRV infection is critical for developing an efficacious vaccine to protect koalas. To this end, we analyzed the antibody response of 235 wild koalas, sampled longitudinally over a four-year period, that harbored KoRV-A, and with or without KoRV-B. We found that the majority of the sampled koalas were able to make anti-KoRV antibodies, and that there was a linear increase in anti-KoRV IgG levels in koalas up to approximately seven years of age and then a gradual decrease thereafter. Koalas infected with both KoRV-A and KoRV-B were found to have slightly higher anti-KoRV IgG titers than koalas with KoRV-A alone and there was an inverse relationship between anti-KoRV IgG levels and circulating KoRV viral load. Finally, we identified distinct epitopes on the KoRV envelope protein that were recognized by antibodies. Together, these findings provide insight into the koala's immune response to KoRV and may be useful in the development of a therapeutic KoRV vaccine.

Seroprevalence and Associated Risk Factors of Rift Valley Fever in Domestic Small Ruminants in the North Region of Cameroon.

Rift Valley fever (RVF) is a zoonotic vector borne infectious disease of major medical and veterinary importance particularly in sub-Saharan Africa. However, there is dearth of epidemiological knowledge of the disease in Cameroon. We conducted a cross-sectional study (January 2016-January 2017) to investigate the seroprevalence and potential risk factors of Rift Valley fever virus (RVFV) in sheep and goats in the North region of Cameroon. Stratified sampling approach was used to select herds where sera were collected from 680 randomly selected small ruminants (355 goats and 325 sheep) in eight localities (Kismatari, Lagdo, Pitoa, Garoua, Bocklé, Dembo, Poli and Touboro) within three administrative divisions (Bénoué, Mayo-Rey and Faro) in the North region. Anti-RVFV antibodies were detected using a competitive Enzyme-Linked Immunosorbent Assay (ELISA), while a capture ELISA was used for the detection of specific RVFV-Immunoglobulin M (Ig-M) antibodies. We evaluated the associated potential risk factors of RVF in small ruminants based on observations of animal-related intrinsic and extrinsic factors in combination with serological results. The results revealed that 3.4% (95% confidence interval (CI): 2.2-5.1%) of sampled animals and 24.6% (95% CI: 15.1-37.1%) of 65 sampled herds were seropositive for anti-RVFV antibodies and no difference in seropositivity between sheep and goats at individual animal as well as at herd levels was observed. Localities along hydrographic or large water banks such as Kismatari (OR: 14.333, (95% CI: 1.436-145.088)) and Pitoa (OR = 11.467 (95% CI: 1.249-50.306)) were significantly associated to RVFV antibody seroprevalence in a simple logistic regression. In addition, the multiple regression analysis showed that age and access to water points significantly influenced RVFV antibody seroprevalence in small ruminants. This study revealed that anti-RVFV antibodies are present in sheep and goats in the North region of Cameroon. It highlights the likely endemic circulation of RVFV in the considered localities despite the absence of clinical cases reported in animals or humans. Under these conditions, it is necessary to set up an early warning, surveillance and control strategy based on epizootic risk.

Receptors and entry cofactors for retroviruses include single and multiple transmembrane-spanning proteins as well as newly described glycophosphatidylinositol-anchored and secreted proteins.

In the past few years, many retrovirus receptors, coreceptors, and cofactors have been identified. These molecules are important for some aspects of viral entry, although in some cases it remains to be determined whether they are required for binding or postbinding stages in entry, such as fusion. There are certain common features to the molecules that many retroviruses use to gain entry into the cell. For example, the receptors for most mammalian oncoretroviruses are multiple membrane-spanning transport proteins. However, avian retroviruses use single-pass membrane proteins, and a sheep retrovirus uses a glycosylphosphatidylinositol-anchored molecule as its receptor. For some retroviruses, particularly the lentiviruses, two cell surface molecules are required for efficient entry. More recently, a soluble protein that is required for viral entry has been identified for a feline oncoretrovirus. In this review, we will focus on the various strategies used by mammalian retroviruses to gain entry into the cell. The choice of receptors will also be discussed in light of pressures that drive viral evolution and persistence.

Molecular and serological surveillance of Hepatitis E virus in wild and domestic carnivores in Brandenburg, Germany.

Hepatitis E virus (HEV) is a zoonotic virus which circulates in pigs and wild boars as main reservoir species. To reveal the infection rate in carnivores, we have carried out a monitoring study of raccoons, raccoon dogs, dogs and cats sampled in Brandenburg, Germany. In summary, 53.8% (43 of 80) of the raccoons, 34.3% (25 of 73) of the raccoon dogs, 56.6% (47 of 83) of dogs and 32.3% (21 of 65) of cats were tested positive for HEV-specific antibodies. No viral RNA could be detected. This first description of anti-HEV antibodies in raccoons and raccoon dogs worldwide and in dogs and cats in Germany highlights the natural host range expansion of HEV.

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.

Type 1 human T-cell leukemia virus small envelope protein expressed in mouse cells by using a bovine papilloma virus-derived shuttle vector.

In an attempt to express the small (transmembrane) envelope protein p21e of type 1 human T-cell leukemia (lymphotrophic) virus (HTLV-1) exclusive of other viral gene products, we have constructed a recombinant plasmid clone (pMBE-1) in a bovine papillomavirus-derived mammalian expression vector. Mouse C127 cells transfected with the pMBE-1 plasmid expressed the introduced HTLV-1 viral gene(s) as demonstrated by Northern blot and indirect immunofluorescence with natural human antisera. The transfected mouse cells were injected into BALB/c mice, and a monoclonal antibody was recovered which specifically recognizes a 21-kilodalton protein present in HTLV-1 virions, indicating that the pMBE-1 plasmid encodes the small envelope protein.

DNA methylation and expression of HLA-DR alpha.

B-cell lines established from two individuals with T-cell acute lymphocytic leukemia (T-ALL) express HLA-DR antigens, whereas the isogenic T-cells do not. The lack of expression correlates with a lack of detectable HLA-DR mRNA. All of the DR alpha DNA sequences detected by a cloned DR alpha cDNA probe are contained in a BglII fragment which varies slightly in size (4.0 to 4.8 kilobases) from one individual to another. In DNA from the T-cells not expressing DR alpha mRNA, all of the potential HpaII sites within the BglII fragment appeared to be methylated. In contrast, at least some of these sites were not methylated in DNA from the B-cells expressing high levels of DR alpha mRNA. Treatment of these T-cells with 5-azacytidine resulted in the induction of DR surface antigen expression, the appearance of DR alpha mRNA, and the partial demethylation of the DR alpha DNA sequences. T-cell lines established from human T-cell leukemia-lymphoma virus associated T-cell neoplasias, in contrast to the T-cell acute lymphocytic leukemia cell lines, expressed both DR antigens and DR alpha mRNA; the HpaII sites within the BglII fragment of DR alpha DNA of these human T-cell leukemia-lymphoma virus-positive T-cell lines were in all cases at least partially unmethylated. Uncultured peripheral blood T-cells from human T-cell leukemia-lymphoma virus-infected individuals expressed DR antigens at a low level, and the DR alpha locus was partially unmethylated. After 48 h in culture, DR antigen expression was substantially increased, but no significant changes were observed in methylation of the DR alpha locus or in the amount of DR mRNA which was present. This suggests that expression of DR antigens also can be modulated post-transcriptionally.

Rift Valley fever virus infections in Egyptian cattle and their prevention.

Rift Valley fever virus (RVFV) causes consistently severe outbreaks with high public health impacts and economic losses in livestock in many African countries and has also been introduced to Saudi Arabia and Yemen. Egypt with its four large outbreaks in the last 40 years represents the northernmost endemic area of RVFV. The purpose of this study was to provide an insight into the current anti-RVFV antibody status in immunized as well as non-immunized dairy cattle from the Nile Delta of Egypt. During 2013-2015, a total of 4,167 dairy cattle from four governorates including Dakahlia, Damietta, Gharbia and Port Said were investigated. All cattle were born after 2007 and therewith after the last reported Egyptian RVFV outbreak in 2003. The samples derived from vaccinated animals from 26 different dairy farms as well as non-immunized cattle from 27 different smallholding flocks. All samples were examined following a three-part analysis including a commercially available competition ELISA, an in-house immunofluorescence assay and a virus neutralization test. Additionally, a subset of samples was analysed for acute infections using IgM ELISA and real-time reverse transcriptase PCR. The results indicated that the RVFV is still circulating in Egypt as about 10% of the non-immunized animals exhibited RVFV-specific antibodies. Surprisingly, the antibody prevalence in immunized animals was not significantly higher than that in non-vaccinated animals which points out the need for further evaluation of the vaccination programme. Due to the substantial role of livestock in the amplification and transmission of RVFV, further recurrent monitoring of the antibody prevalence in susceptible species is highly warranted.

Vaccination of alpacas against Rift Valley fever virus: Safety, immunogenicity and pathogenicity of MP-12 vaccine.

Rift Valley fever (RVF) is an emerging zoonosis of major public health concern in Africa and Arabia. Previous outbreaks attributed camelids a significant role in the epidemiology of Rift Valley fever virus (RVFV), making them an important target species for vaccination. Using three alpacas as model-organisms for dromedary camels, the safety, immunogenicity and pathogenicity of the MP-12 vaccine were evaluated in this study. To compare both acute and subacute effects, animals were euthanized at 3 and 31days post infection (dpi). Clinical monitoring, analysis of liver enzymes and hematological parameters demonstrated the tolerability of the vaccine, as no significant adverse effects were observed. Comprehensive analysis of serological parameters illustrated the immunogenicity of the vaccine, eliciting high neutralizing antibody titers and antibodies targeting different viral antigens. RVFV was detected in serum and liver of the alpaca euthanized 3dpi, whereas no virus was detectable at 31dpi. Viral replication was confirmed by detection of various RVFV-antigens in hepatocytes by immunohistochemistry and the presence of mild multifocal necrotizing hepatitis. In conclusion, results indicate that MP-12 is a promising vaccine candidate but still has a residual pathogenicity, which requires further investigation.

Hepatitis E virus in wild rabbits and European brown hares in Germany.

Recently, a change of hepatitis E from being a typical travel-associated disease to an autochthonous zoonosis in Germany was observed. An increasing number of autochthonous infections with the hepatitis E Virus (HEV) have been recognized in developed countries. Venison from wild boar is already known to be a potential source of infection, if not prepared properly by the consumer. In Germany, certain wild animals are known to be a reservoir for HEV. However, current information is missing about European brown hares (Lepus europaeus) and wild rabbits (Oryctolagus cuniculus). Thus, a total of 833 hunting-harvested animals (European brown hares n = 669; wild rabbits n = 164) were tested for the occurrence of HEV RNA and HEV antibodies. For this, liver and blood specimens were taken after hunts in six German federal states. HEV antibodies were found by ELISA in 2.2% (624/14) of European brown hares, but no HEV RNA was detectable by nested real-time RT-PCR. In contrast, a seroprevalence of 37.3% (126/47) was observed for wild rabbits, and 17.1% (164/28) of the samples were HEV RNA positive. Genomic analysis revealed that these partial sequences clustered within the rabbit clade of HEV-3 genotype. In addition, one rabbit sequence segregated into subtype 3g of HEV-3. Highest seroprevalences for hares and rabbits were detected in the federal states of Bavaria and of Schleswig-Holstein, respectively. Comparing urban, rural and insular areas, the highest seroprevalence was shown for wild rabbits in rural areas and for European brown hares on the northern island Fehmarn. This study provides evidence that European brown hares and wild rabbits from Germany can be infected with HEV. The different prevalences indicate that wild rabbits are a potential reservoir for HEV in Germany, whereas European brown hares seem to be only of minor importance for the epidemiology of HEV.

Evidence for enzootic circulation of Rift Valley fever virus among livestock in Cameroon.

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen, causing serious epidemics in Africa and the Arabian Peninsula. In Cameroon serological data indicate the presence of RVFV, but active circulation of RVFV, causing clinical infections has not been proven yet. For this purpose we carried out a serological and molecular study on a total of 1953 randomly selected serum samples of small ruminants and cattle, which were collected in years 2013 and 2014 in Cameroon. In a first step, sera were screened serologically using a variety of assay formats to reveal RVFV specific antibodies. At the second stage, seropositive specimen were assessed for acute RVFV infections via IgM-specific ELISA and quantitative real-time RT-PCR. Our data show a significant difference in the antibody prevalence in cattle (13.5% [95% confidence interval: 11.4-15.7]) and small ruminants (3.4% [95% confidence interval: 2.3-4.7]), with indications for annual fluctuations and significant regional differences of seropositivity. One small ruminant and three bovines were eventually found to be positive in IgM ELISA and indications for viremia were found in one bovine by RVFV genome detection using quantitative real-time RT-PCR. The results of this study therefore corroborate the presence of acute RVFV-infection and its circulation in Cameroon.

Experimental Evaluation of Faecal Escherichia coli and Hepatitis E Virus as Biological Indicators of Contacts Between Domestic Pigs and Eurasian Wild Boar.

Domestic pigs and Eurasian wild boar (Sus scrofa) share several important viral and bacterial pathogens. Therefore, direct and indirect contacts between domestic pigs and wild boar present a risk of pathogen spillover and can lead to long-term perpetuation of infection. Biological indicators could be a powerful tool to understand and characterize contacts between wild boar and domestic pigs. Here, faecal Escherichia coli and Hepatitis E virus (HEV) were explored as potential biological indicators under experimental conditions. The data gained in our pilot study suggest that faecal E. coli can be used as biological indicator of contact between wild boar and domestic pig. For HEV, faecal transmission was also confirmed. However, molecular studies on full-genome basis did not reveal markers that would allow tracing of transmission direction. Based on these promising results, future field studies will especially target the practicability of E. coli microbiome molecular typing as surrogate of contacts at the wildlife-livestock interface.

Atypical BSE in Germany--proof of transmissibility and biochemical characterization.

Intensive active surveillance has uncovered two atypical German BSE cases in older cattle which resemble the two different atypical BSE phenotypes that have recently been described in France (designated H-type) and Italy (designated L-type or BASE). The H-type is characterized by a significantly higher molecular size, but a conventional glycopattern of the proteinase K treated abnormal prion protein (PrP(Sc)), while the L-type PrP(Sc) has only a slightly lower molecular size and a distinctly different glycopattern. In this paper we describe the successful transmission of both German atypical BSE cases to transgenic mice overexpressing bovine PrP(C). Upon challenge with the L-type, these mice developed BSE after a substantially shorter incubation period than any classical BSE transmission using these mice to date. In contrast, the incubation period was distinctly prolonged when these mice were challenged with the H-type. PrP(Sc) accumulated in the brains of these mice were of the same atypical BSE type that had been used for the transmission. These atypical cases suggest the possible existence of sporadic BSE cases in bovines. It is thus feasible that the BSE epidemic in the UK could have also been initiated by an intraspecies transmission from a sporadic BSE case.

Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas.

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.