Spatial-Temporal Dynamics of Hepatitis E Virus Infection in Foxes (Vulpes vulpes) in Federal State of Brandenburg, Germany, 1993-2012.

Hepatitis E virus (HEV) is the main course for acute hepatitis in humans throughout the world. Human associated genotypes 1 and 2 as well as zoonotic genotypes 3 and 4 are grouped in the species Orthohepevirus A. In addition, a large variety of HEV-related viruses has been found in vertebrates including carnivores, rats, bats, and chickens, which were classified in species Orthohepevirus B-D. In 2015, partial genome sequences of a novel hepevirus were detected in feces of red foxes (Vulpes vulpes). However, no further information about virus circulation and the prevalence in foxes was available. We therefore assayed a unique panel of 880 transudates, which was collected from red foxes over 19 years (1993-2012) in Brandenburg, Germany, for HEV-related viral RNA and antibodies. Our results demonstrate a high antibody prevalence of HEV in red foxes, which oscillated annually between 40 and 100%. Molecular screening of the transudates revealed only a single RNA-positive sample, which was assigned to the carnivore species Orthohepevirus C based on the amplified partial sequence. These data indicate that the virus is circulating widely in the fox population and that foxes are carriers of this virus.

West Nile virus epizootic in Germany, 2018.

The summer of 2018 in Germany was the second hottest and driest on record. These generally extremely favorable climatic conditions most likely triggered the further expansion and the efficient propagation of the zoonotic arthropod-borne West Nile virus in many Southern/Southeastern and even Central European countries. WNV infections were detected for the first time in resident wild and aviary birds, such as common blackbirds, northern goshawks and great grey owls in Eastern and Southeastern Germany. The causative WNV strain belonged to the central European subclade II. Phylogeographic analysis indicated a single introduction event of WNV into Germany, most likely in 2016 from Czech Republic, and also a unique non-synonymous mutation in the NS3 gene. Extraordinary high temperatures in 2018 presumably led to decreased averaged extrinsic incubation period values for WNV in mosquitoes, leading to rapid virus amplification and greater transmission risk for vertebrates in Germany. Blood transfusion services and clinicians in Germany should be aware of these possible WNV infection risks in humans especially during late summer.

Co-infection of pigs with Hepatitis E and porcine circovirus 2, Saxony 2016.

Hepatitis E virus (HEV) is a recognized zoonotic disease; autochthonous infections in Europe are caused to a great extent by HEV genotype 3. Pigs and wild boar are the main reservoirs for this genotype and normally they develop no or only subclinical symptoms with mild histopathological lesions. However, co-infections with other pig pathogens can lead to severe cases in pigs, including liver hemorrhage and necrosis. During a monitoring program 2016 in Saxony, Germany, farmed pigs with various clinical outcomes including fatalities were analysed for HEV and concurrent infections. We could detect eight HEV infected pigs from which six were co-infected with porcine circovirus 2 (PCV2). Phylogenetic analysis revealed HEV sub-genotypes 3e and 3f as well as PCV2 genotypes 2b and 2d. A direct correlation of the co-infection to the course of disease could not be determined, but the results provide hints that the immune modulatory effects of PCV2 combined with HEV influence the disease pattern in pigs.

A one-step multiplex real-time RT-PCR for the universal detection of all currently known CCHFV genotypes.

Crimean-Congo hemorrhagic fever (CCHF) is a fatal disease in humans, which is endemic in many countries of Africa, Southern Asia and Southeastern Europe. It is caused by the Crimean-Congo hemorrhagic fever virus (CCHFV), which is an arthropod-borne virus (arbovirus) transmitted by ixodid ticks, mainly of the genus Hyalomma. Animals like hares, hedgehogs, cattle, camels and small ruminants can become infected without developing clinical signs. Seroconversion occurs after a short viremia of up to two weeks, and thus seroprevalence studies in ruminants can be used to reveal risk areas for the human population. Virus detection by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is essential to prove an actual circulation of CCHFV in a country and is also used as diagnostic method for acute human CCHFV infections. In this study, a new universal one-step multiplex real-time RT-qPCR for the sensitive and specific detection of CCHFV is presented. For this purpose, 14 new primers and 2 probes were simultaneously used to detect RNAs representing all six CCHFV genotypes. Additionally, a GC-mirrored sequence within the synthetic RNAs enables the discrimination between true positive samples and unintentional laboratory contaminations. CCHFV negative samples from different animal species and ten different members of the order Bunyavirales were eventually tested to reveal the specificity of the new RT-qPCR.

A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray.

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented.

A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

Serologic and Molecular Survey of Hepatitis E Virus in German Deer Populations.

Hepatitis E virus (HEV) is a human pathogen that is primarily transmitted by the fecal-oral route and causes a usually self-limiting acute viral hepatitis. The virus is endemic in developing countries of Africa, Asia, and Latin America and is responsible for sporadic cases in industrialized countries. In western Europe, an increasing number of autochthonous cases have been associated with zoonotic transmissions of HEV from domestic and wild animals. In Germany, animal reservoirs for HEV have been mainly assigned to domestic pigs and wild boars. To investigate the potential role of deer as a reservoir of HEV, we surveyed HEV-specific antibodies and RNA in deer samples from geographic regions in Germany. We sampled red deer (Cervus elaphus) and roe deer (Capreolus capreolus) during active surveillance in three forest districts in northern Hesse and southern Lower Saxony during 2011-12 and 2012-13, respectively. Additionally, archived samples of red, roe, and fallow deer (Dama dama), collected in 2000-01 in German national parks, were included in the study. Antibody prevalence ranged from 2-3.3% in red deer to 5.4-6.8% in roe deer. Viral RNA was detected in red deer and fallow deer at prevalences of 2.0-6.6% and 4.3%, respectively. The investigation confirmed the presence of HEV infections in three deer species in Germany. Red, roe, and fallow deer should be further monitored to assess their role as hosts and potential reservoirs of HEV in Germany.

Detection of Hepatitis E Virus in Archived Rabbit Serum Samples, Germany 1989.

We detected Hepatitis E virus in serum samples of wild rabbits that were hunted in 1989 around the city of Greifswald, Germany. The recovery of one partial sequence and subsequent phylogenetic analysis indicates a close relationship to rabbit HEV sequences from France and suggests a long-established circulation of rabbit HEV in Europe.

Chronically infected wild boar can transmit genotype 3 hepatitis E virus to domestic pigs.

Hepatitis E virus (HEV) causes acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialized nations. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with the consumption of raw and undercooked products from domestic pig and wild boar. As shown recently, naturally acquired HEV gt3 replicates efficiently in experimentally infected wild boar and is transmissible from a wild boar to domestic pigs. Generally, following an acute infection swine suffer from a transient febrile illness and viremia in connection with fecal virus shedding. However, little is known about sub-acute or chronic HEV infections in swine, and how and where HEV survives the immune response. In this paper, we describe the incidental finding of a chronic HEVgt3 infection in two naturally infected European wild boar which were raised and housed at FLI over years. The wild boar displayed fecal HEV RNA excretion and viremia over nearly the whole observation period of more than five months. The animal had mounted a substantial antibody response, yet without initial clearance of the virus by the immune system. Further analysis indicated a subclinical course of HEV with no evidence of chronic hepatitis. Additionally, we could demonstrate that this chronic wild boar infection was still transmissible to domestic pigs, which were housed together with this animal. Sentinel pigs developed fecal virus shedding accompanied by seroconversion. Wild boar should therefore be considered as an important reservoir for transmission of HEV gt3 in Europe.

Hepatitis E virus genotype 3 diversity: phylogenetic analysis and presence of subtype 3b in wild boar in Europe.

An increasing number of indigenous cases of hepatitis E caused by genotype 3 viruses (HEV-3) have been diagnosed all around the word, particularly in industrialized countries. Hepatitis E is a zoonotic disease and accumulating evidence indicates that domestic pigs and wild boars are the main reservoirs of HEV-3. A detailed analysis of HEV-3 subtypes could help to determine the interplay of human activity, the role of animals as reservoirs and cross species transmission. Although complete genome sequences are most appropriate for HEV subtype determination, in most cases only partial genomic sequences are available. We therefore carried out a subtype classification analysis, which uses regions from all three open reading frames of the genome. Using this approach, more than 1000 published HEV-3 isolates were subtyped. Newly recovered HEV partial sequences from hunted German wild boars were also included in this study. These sequences were assigned to genotype 3 and clustered within subtype 3a, 3i and, unexpectedly, one of them within the subtype 3b, a first non-human report of this subtype in Europe.

Natural and experimental hepatitis E virus genotype 3-infection in European wild boar is transmissible to domestic pigs.

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialised countries. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with domestic pig and wild boar. However, little is known about the course of HEV infection in European wild boar and their role in HEV transmission to domestic pigs. To investigate the transmissibility and pathogenesis of wild boar-derived HEVgt3, we inoculated four wild boar and four miniature pigs intravenously. Using quantitative real-time RT-PCR viral RNA was detected in serum, faeces and in liver, spleen and lymph nodes. The antibody response evolved after fourteen days post inoculation. Histopathological findings included mild to moderate lymphoplasmacytic hepatitis which was more prominent in wild boar than in miniature pigs. By immunohistochemical methods, viral antigens were detected mainly in Kupffer cells and liver sinusoidal endothelial cells, partially associated with hepatic lesions, but also in spleen and lymph nodes. While clinical symptoms were subtle and gross pathology was inconspicuous, increased liver enzyme levels in serum indicated hepatocellular injury. As the faecal-oral route is supposed to be the most likely transmission route, we included four contact animals to prove horizontal transmission. Interestingly, HEVgt3-infection was also detected in wild boar and miniature pigs kept in contact to intravenously inoculated wild boar. Given the high virus loads and long duration of viral shedding, wild boar has to be considered as an important HEV reservoir and transmission host in Europe.

Two new real-time quantitative reverse transcription polymerase chain reaction assays with unique target sites for the specific and sensitive detection of lineages 1 and 2 West Nile virus strains.

Two novel 1-step real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for the simultaneous detection of West Nile virus (WNV) lineage 1 and 2 strains were developed. Primers and the probe of assay 1 target the 5'-untranslated region (UTR), whereas the amplicon of assay 2 is located in the nonstructural region NS2A, which enables an unambiguous and independent WNV diagnosis based on 2 different amplicons. Both assays allow the detection of as few as 2-4 genome copies of WNV strains NY99, Uganda B956, Kunjin, and Sarafend (all cultured on Vero cells). A new synthetic RNA mutant of the 5'-UTR amplicon, which contains 6 twist inverted base-pair changes at the probe attachment site, was used as external calibrator control.

The usefulness of Umelosa hepatitis C vírus qualitative kit as supplemental test for confirmation of hepatitis C virus infection.

Forty voluntary blood donors from two different blood banks in Havana, Cuba, who were repeatedly reactive on the routine screening of antibodies to hepatitis C virus, by Umelisa HCV test, were analyzed for the presence of HCV RNA using a nested PCR assay of the HCV 5' untranslated region, Umelosa HCV qualitative. Sera from 45 patients of a specialized gastroenterology consultation, positive to Umelisa HCV, were also assayed with the Umelosa HCV qualitative, to establish their condition related to the presence of HCV RNA previously to the indication of a treatment or after three, six or twelve months of antiviral therapy. Serum HCV-RNA was detected in 21/40 (52.5%) donors who had repeatedly positive ELISA results, confirming the HCV infection for them. In specialized consultation HCV-RNA was detected by PCR analysis in 30/45 (66%) analyzed sera.

The usefulness of Umelosa hepatitis C vírus qualitative kit as supplemental test for confirmation of hepatitis C virus infection

Quarenta doadores de sangue voluntários de dois bancos de sangue em Havana, Cuba, repetidamente reativos ao exame de rotina para anticorpos contra o vírus da hepatite C, pelo teste Umelisa HCV, foram analisados para a presença de RNA HCV através de um ensaio de PCR da regiäo 5' näo-traduzida do HCV denominado Umelosa HCV qualitativo. Amostras de soro obtidas de 45 pacientes atendidos em consulta gastroenterológica especializada, positivos para Umelisa HCV, também foram avaliados através do Umelosa HCV qualitativo, para estabelecer sua condiçäo em relaçäo à presença de RNA HCV previamente à indicaçäo de um tratamento ou após três, seis ou doze meses de terapia anti-viral. O RNA HCV sérico foi detectado em 21/40 (52,5 por cento) doadores que haviam apresentado resultados ELISA positivos repetidos, confirmando a infecçäo por HCV. Nos pacientes atendidos em consulta especializada, o RNA HCV foi detectado por análise de PCR em 30/45 (66 por cento) amostras de soro analisadas.

Validation of a nested PCR assay UMELOSA HCV CUALITATIVO for the detection of Hepatitis C virus.

An analysis of the performance characteristics of the UMELOSA HCV CUALITATIVO assay for the detection of Hepatitis C virus (HCV) RNA in human serum or plasma is presented. This qualitative in vitro diagnostic test is performed in three steps: (a) extraction of viral RNA, (b) reverse transcription of target RNA to generate complementary DNA followed by a polymerase chain reaction assay coupled with a second round of amplification, and finally (c) fluorescent detection of the amplified DNA by hybridization in a solid phase of an ultramicroplate coated with a complementary to amplified DNA probe. Considering the assay as a limit test for the control of impurities, the following analytical performance parameters were evaluated: specificity, detection limit and robustness. A comparative evaluation of the clinical performance and detection limit of our kit and the commercial AMPLICOR HCV test, version 2.0, was also included in the validation protocol. The assay had a good specificity and did not cross-react with the non-HCV analyzed positive samples. The 95% detection limit was of 101.7IU/ml with 95% confidence interval from 81.0 to 162.8IU/ml. The UMELOSA HCV CUALITATIVO meets the minimal sensitivity requirements for a single unit blood testing of 5000 and 1250IU/ml, defined by the Paul-Ehrlich-Institute in Germany and the Food and Drug Administration in USA, respectively. Compared with the commercial AMPLICOR, the test gave identical results for all analyzed positive and negative samples. In robustness studies there was no cross-contamination between negative samples and these same samples spiked with 10000IU/ml of HCV-RNA.