Nachweis und Charakterisierung potentiell zoonotischer enteraler Viren in Nutztieren und Beeinflussung ihrer Ausscheidung durch Probiotika

Infections with a variety of enteric viruses can cause clinical disease in humans and livestock, leading to gastroenteritis, encephalitis or hepatitis. For some of these viruses, a zoonotic potential has been supposed, because of a close genetic relationship of strains detected in humans and animals. Therefore, the possibility of a direct or indirect transmission of those viruses between livestock and humans is suspected. Particularly, the foodborne transmission of potentially zoonotic viruses is of increasing interest, as the number of foodborne viral diseases in humans increased in the last decades according to the World Health Organization. However, the prevention and control of many foodborne viral infections is currently hampered by the lack of knowledge about the distinct transmission pathways of the respective viruses and the importance of specific reservoir hosts. In addition, strategies to prevent virus transmission from livestock to humans are currently based exclusively on general hygienic measures. In this study, the distribution of selected enteric viruses with suspected zoonotic potential in pigs and poultry should be assessed. The presence of astrovirus (AstV), encephalomyocarditis virus (EMCV), norovirus genogroup II (NoV GII), group A rotavirus (GARV) and hepatitis E virus (HEV) should be analysed in pigs from Germany. The distribution of avian rotaviruses of groups A and D (AvRV-A and -D) should be assessed in poultry from different regions of the world. Sensitive detection systems had to be developed for these purposes. The detected viruses should be compared with known strains from humans and livestock in order to estimate their zoonotic potential. As one option to decrease enteric virus excretion, the effects of feeding pigs with the probiotic bacterium Enterococcus faecium (E. faecium) NCIMB 10415 should be investigated. Sensitive and specific real-time RT-PCR assays were developed, which were able to detect human as well as porcine strains of the respective viruses. The detection limits of the assays ranged from 15 to 78 molecules per PCR reaction. For avian group A and D rotaviruses, real-time RT-PCR assays were developed here for the first time. Although these assays were shown to be more sensitive than conventional detection methods available for avian rotaviruses, their sensitivity was still low for some of the analysed strains. The analysis of faecal samples from chickens and turkeys originating from different countries of Europe and from Bangladesh showed, that AvRV-A and -D are highly prevalent (58.8% and 65.9%, respectively) in the flocks. The investigation of faecal samples of pigs at slaughter age originating from slaughterhouses of three different regions in Germany resulted in the detection of all viruses, which were tested for. Particularly, NoV GII and AstV were found with high detection rates in slaughter pigs (14.2% and 20.8%, respectively). GARV, EMCV and HEV were detected in only a small number of pigs at slaughter age (0.8%, 4.2% and 2.5%, respectively). The phylogenetic analyses of genome parts of the detected pig viruses showed a high sequence identity of 91% and 90% to known human strains of HEV and GARV, respectively. This finding supports the zoonotic potential of these viruses. In contrast, the detected strains of AstV and NoV GII clearly clustered together with typical porcine virus strains, thus questioning their ability of zoonotic transmission to humans. For EMCV, no sequences could be generated for comparison. In an experimental feeding trial with sows and their piglets, no significant differences were recorded in the excretion of HEV, EMCV and NoV GII between the E. faecium NCIMB 10415 fed group and the control group. However, AstV was only detected in the control group and GARV was shed significantly later and with lower amounts in the probiotic feeding group. An activation of specific T cell populations was found in the probiotic feeding group, which may explain some of the effects caused by feeding with E. faecium NCIMB 10415. It can be concluded from the study, that potentially zoonotic viruses are present in faecal samples of pigs of different age in Germany, including pigs at slaughter. The detected GARV and HEV strains were closely related to human viruses, thus indicating a potential for their zoonotic transmission. Although the prevalence of these human-related viruses was rather low, the detection of those viruses in pigs at slaughter should raise awareness on the improvement of hygienic standards in the meat-producing and -processing industry. One way for decreasing excretion of some of the viruses may be the application of probiotics; however, further studies have to confirm the results and should aim to elucidate the distinct mechanisms of action. In addition, other potentially zoonotic viruses like kobuvirus and sapovirus should be included in future investigations. The detection of AvRV-A and -D with high prevalence in poultry worldwide, as shown here, should result in efforts to further characterize these viruses in more detail in order to estimate their zoonotic potential.Infektionen mit verschiedenen enteralen Viren können beim Menschen und Nutztier zu Gastroenteritis, Enzephalitis oder Hepatitis führen. Einige dieser im Mensch und Tier detektierten Viren zeigen eine enge genetische Verwandtschaft zueinander und stehen deswegen unter Verdacht ein zoonotisches Potential zu besitzen. Eine direkte oder indirekte Übertragung zwischen Tier und Mensch erscheint deshalb möglich. Die Übertragung von potentiell zoonotischen Viren durch Lebensmittel ist hierbei von besonderem Interesse, da die Zahl der lebensmittelbedingten Viruserkrankungen beim Menschen in den letzten Jahrzenten, Angaben der Weltgesundheitsorganisation zufolge, gestiegen ist. Die Prävention und Kontrolle von durch Lebensmittel übertragenen Virusinfektionen ist jedoch schwierig, weil die genauen Übertragungswege der Viren sowie die Bedeutung spezifischer Reservoirwirte oft nur wenig bekannt sind. Darüber hinaus sind Strategien zur Verhinderung der Virusübertragung vom Nutztier auf den Menschen derzeit ausschließlich auf allgemeine Hygienemaßnahmen beschränkt. In dieser Studie sollte die Verbreitung ausgewählter enteraler Viren, für die ein zoonotisches Potential angenommen wird, in Schweinen und Geflügel ermittelt werden. Schweine aus Deutschland sollten auf Astrovirus (AstV), Enzephalomyokarditis Virus (EMCV), Norovirus Genogruppe II (NoV GII), Gruppe A Rotavirus (GARV) und Hepatitis E Virus (HEV) hin untersucht werden. Des weiteren sollte die Verbreitung aviärer Rotaviren der Gruppen A und D (AvRV-A und -D) in Geflügel aus unterschiedlichen Regionen weltweit untersucht werden. Zu diesem Zweck sollten sensitive Nachweismethoden entwickelt werden. Die detektierten Viren sollten mit bekannten Stämmen aus Mensch und Tier verglichen werden, um deren zoonotisches Potential einschätzen zu können. Als eine Möglichkeit, die Ausscheidung von Viren zu vermindern, sollte der Einfluss der Fütterung mit dem probiotischen Bakterium Enterococcus faecium (E. faecium) NCIMB 10415 im Schwein untersucht werden. Sensitive und spezifische Real-time RT-PCR Assays zum Nachweis humaner sowie entsprechender porziner Virusstämme wurden entwickelt. Die Nachweisgrenzen der Assays lagen zwischen 15 und 78 Molekülen pro PCR-Ansatz. Im Rahmen dieser Studie wurden erstmals Real-time RT-PCR Assays zum Nachweise aviärer Rotaviren der Gruppen A und D entwickelt. Obwohl die Sensitivität dieser Assays im Vergleich zu konventionellen Methoden zur Detektion aviärer Rotaviren nachweislich höher war, wurden einige der Stämme mit nur geringer Sensitivität detektiert. Die Untersuchung von aus unterschiedlichen Ländern Europas und aus Bangladesch stammenden Kotproben von Hühnern und Puten zeigte, dass AvRV-A und -D mit hoher Prävalenz (58,8 % und 65,9 %) in den Beständen vorkommen. Bei der Untersuchung von Kotproben von Schweinen aus drei unterschiedlichen Schlachthöfen in Deutschland konnten alle Viren, auf die die Proben untersucht wurden, nachgewiesen werden. Insbesondere NoV GII und AstV wurden mit hohen Detektionsraten in Schlachtschweinen vorgefunden (14,2 % und 20,8 %). GARV, EMCV und HEV hingegen wurden nur in einer geringen Anzahl von Schlachtschweinen detektiert (0,8 %, 4,2 % und 2,5 %). Anschließende phylogenetische Analysen von partiellen Genomsequenzen zeigten eine hohe Sequenzhomologie der detektierten HEV- und GARV-Stämme von jeweils 91 % und 90 % zu bekannten humanen Stämmen. Diese Ergebnisse stützen die Annahme eines zoonotischen Potentials dieser Viren. Im Gegensatz dazu waren die detektierten NoV GII- und AstV-Stämme jeweils nur entfernt mit humanen Viren verwandt und zeigten hohe Sequenzhomologien mit typisch porzinen Stämmen, so dass eine mögliche zoonotische Übertragung auf den Menschen fraglich erscheint. Für EMCV konnten keine Sequenzen für phylogenetische Analysen generiert werden. In einem experimentellen Fütterungsversuch mit Sauen und deren Ferkeln konnten keine signifikanten Unterschiede der Ausscheidung von HEV, EMCV und NoV GII zwischen E. faecium NCIMB 10415-gefütterten Tieren und der Kontrollgruppe beobachtet werden. Hingegen wurde AstV nur in der Kontrollgruppe detektiert und GARV wurde von Tieren der Probiotika-gefütterten Gruppe signifikant später und in geringerer Menge ausgeschieden. In der Probiotika-gefütterten Gruppe konnte außerdem eine Aktivierung spezifischer T-Zell-Populationen nachgewiesen werden, die einige der durch die Fütterung mit E. faecium NCIMB 10415 verursachten Effekte erklären könnte. Aus den Ergebnissen dieser Studien kann geschlossen werden, dass potentiell zoonotische Viren in deutschen Schweinen unterschiedlichen Alters, darunter auch bei Schweinen im Schlachtalter, nachgewiesen werden können. Die detektierten HEV- und GARV-Stämme waren eng mit humanen Viren verwandt, was für eine mögliche zoonotische Übertragung spricht. Auch wenn die Prävalenz der GARV- und HEV-Stämme eher niedrig war, sollte der Nachweis dieser Viren in Schlachtschweinen die Aufmerksamkeit auf verbesserte hygienische Maßnahmen in der Fleischproduzierenden und -verarbeitenden Industrie lenken. Die Anwendung von Probiotika könnte eine Möglichkeit zur Verminderung der Ausscheidung einiger dieser Viren bieten. Diese Ergebnisse sollten jedoch in weiteren Untersuchungen bestätigt werden; außerdem sollte der genaue Wirkmechanismus des Probiotikums aufgeklärt werden. In zukünftigen Studien sollten auch weitere potentiell zoonotische Viren wie Kobuviren und Sapoviren eingeschlossen werden. Die hier ermittelten hohen Prävalenzen aviärer Rotaviren der Gruppen A und D im Geflügel sollten Ausgangspunkt für weitere Untersuchungen sein, um diese Viren zu charakterisieren und ihr zoonotisches Potential besser einschätzen zu können

Characterization of a hepatitis C virus genotype 1 divergent isolate from an HIV-1 coinfected individual in Germany assigned to a new subtype 1o

Background HCV exhibits a high genetic diversity and is classified into 7 genotypes which are further divided into 86 confirmed subtypes. However, there are multiple isolates with unassigned subtypes. We aimed to amplify and characterize the full-length genome sequence of an HCV genotype 1 (HCV-1) divergent isolate (DE/17–0414) in Germany. Methods The HCV infection was detected in an HIV-1-positive German female within an HCV/HIV-coinfection study using a commercially available antigen-antibody HCV ELISA kit and confirmed by an in-house quantitative real-time RT-PCR assay. Preliminary genotyping was done by sequencing and phylogenetic analysis on partial NS5B region. The full-length genome sequence was determined by consensus RT-PCR assays. Resistance-associated substitutions (RASs) were analyzed using the web-based tool Geno2pheno[HCV]. Results Partial NS5B region of the isolate DE/17–0414 showed more than 95% identity to 73–08460349-1 l and HCV_Fr_003 from France and QC316 from Canada. Full-length genome analysis of the DE/17–0414 strain showed 91.8% identity to QC316 but less than 79.6% to other HCV-1 strains. Phylogenetic analyses demonstrated that DE/17–0414, 73–08460349-1 l, HCV_Fr_003, and QC316 formed a separate subcluster within HCV-1. DE/17–0414 had a distinct 3 amino acids insertion at the N-terminal of hypervariable region 1 (HVR1) within viral envelope glycoprotein 2 (E2) and several potential antiviral RASs among the NS3 and NS5A genes. Conclusions We identified and analyzed an HCV-1 divergent isolate derived from an HIV-1 coinfected individual in Germany, which will be assigned to a new HCV-subtype 1o. Our understanding of the origin and transmission dynamics of this new subtype 1o requires further assessments from patients worldwide.Peer Reviewe

Prevalence and persistence of transmitted drug resistance mutations in the German HIV-1 Seroconverter Study Cohort

The prevalence of transmitted drug resistance (TDR) in antiretroviral therapy (ART)-naïve individuals remains stable in most developed countries despite a decrease in the prevalence of acquired drug resistance. This suggests that persistence and further transmission of HIV-1 that encodes transmitted drug resistance mutations (TDRMs) is occurring in ART-naïve individuals. In this study, we analysed the prevalence and persistence of TDRMs in the protease and reverse transcriptase-sequences of ART-naïve patients within the German HIV-1 Seroconverter Study Cohort who were infected between 1996 and 2017. The prevalence of TDRMs and baseline susceptibility to antiretroviral drugs were assessed using the Stanford HIVdb list and algorithm. Mean survival times of TDRMs were calculated by Kaplan-Meier analysis. The overall prevalence of TDR was 17.2% (95% CI 15.7–18.6, N = 466/2715). Transmitted NNRTI resistance was observed most frequently with 7.8% (95% CI 6.8–8.8), followed by NRTI resistance (5.0%, 95% CI 4.2–5.9) and PI resistance (2.8%, 95% CI 2.2–3.4). Total TDR (OR = 0.89, p = 0.034) and transmitted NRTI resistance (OR = 0.65, p = 0.000) decreased between 1996 and 2017 but has remained stable during the last decade. Viral susceptibility to NNRTIs (6.5%-6.9% for individual drugs) was mainly reduced, while <3% of the recommended NRTIs and PIs were affected. The longest mean survival times were calculated for the NNRTI mutations K103N (5.3 years, 95% CI 4.2–5.6) and E138A/G/K (8.0 years, 95% CI 5.8–10.2 / 7.9 years, 95% CI 5.4–10.3 / 6.7 years, 95% CI 6.7–6.7) and for the NRTI mutation M41L (6.4 years, 95% CI 6.0–6.7).The long persistence of single TDRMs indicates that onward transmission from ART-naïve individuals is the main cause for TDR in Germany. Transmitted NNRTI resistance was the most frequent TDR, showing simultaneously the highest impact on baseline ART susceptibility and on TDRMs with prolonged persistence. These results give cause for concern regarding the use of NNRTI in first-line regimens.Peer Reviewe

Feeding of the probiotic bacterium <it>Enterococcus faecium</it> NCIMB 10415 differentially affects shedding of enteric viruses in pigs

Abstract Effects of probiotic bacteria on viral infections have been described previously. Here, two groups of sows and their piglets were fed with or without feed supplementation of the probiotic bacterium Enterococcus faecium NCIMB 10415. Shedding of enteric viruses naturally occurring in these pigs was analyzed by quantitative real-time RT-PCR. No differences between the groups were recorded for hepatitis E virus, encephalomyocarditis virus and norovirus. In contrast, astrovirus was exclusively detected in the non-supplemented control group. Rotavirus was shedded later and with lower amounts in the probiotic piglet group (p p p p p p < 0.1) B cell populations. The results indicate that probiotic bacteria could have effects on virus shedding in naturally infected pigs, which depend on the virus type. These effects seem to be caused by immunological changes; however, the distinct mechanism of action remains to be elucidated.</p

Decreased emergence of HIV-1 drug resistance mutations in a cohort of Ugandan women initiating option B+ for PMTCT

Background: Since 2012, WHO guidelines for the prevention of mother-to-child transmission (PMTCT) of HIV-1 in resource-limited settings recommend the initiation of lifelong antiretroviral combination therapy (cART) for all pregnant HIV-1 positive women independent of CD4 count and WHO clinical stage (Option B+). However, long-term outcomes regarding development of drug resistance are lacking until now. Therefore, we analysed the emergence of drug resistance mutations (DRMs) in women initiating Option B+ in Fort Portal, Uganda, at 12 and 18 months postpartum (ppm). Methods and findings: 124 HIV-1 positive pregnant women were enrolled within antenatal care services in Fort Portal, Uganda. Blood samples were collected at the first visit prior starting Option B+ and postpartum at week six, month six, 12 and 18. Viral load was determined by real-time RT-PCR. An RT-PCR covering resistance associated positions in the protease and reverse transcriptase HIV-1 genomic region was performed. PCR-positive samples at 12/18 ppm and respective baseline samples were analysed by next generation sequencing regarding HIV-1 drug resistant variants including low-frequency variants. Furthermore, vertical transmission of HIV-1 was analysed. 49/124 (39.5%) women were included into the DRM analysis. Virological failure, defined as >1000 copies HIV-1 RNA/ml, was observed in three and seven women at 12 and 18 ppm, respectively. Sequences were obtained for three and six of these. In total, DRMs were detected in 3/49 (6.1%) women. Two women displayed dual-class resistance against all recommended first-line regimen drugs. Of 49 mother-infant-pairs no infant was HIV-1 positive at 12 or 18 ppm. Conclusion: Our findings suggest that the WHO-recommended Option B+ for PMTCT is effective in a cohort of Ugandan HIV-1 positive pregnant women with regard to the low selection rate of DRMs and vertical transmission. Therefore, these results are encouraging for other countries considering the implementation of lifelong cART for all pregnant HIV-1 positive women

Decreased emergence of HIV-1 drug resistance mutations in a cohort of Ugandan women initiating option B+ for PMTCT

<div><p>Background</p><p>Since 2012, WHO guidelines for the prevention of mother-to-child transmission (PMTCT) of HIV-1 in resource-limited settings recommend the initiation of lifelong antiretroviral combination therapy (cART) for all pregnant HIV-1 positive women independent of CD4 count and WHO clinical stage (Option B+). However, long-term outcomes regarding development of drug resistance are lacking until now. Therefore, we analysed the emergence of drug resistance mutations (DRMs) in women initiating Option B+ in Fort Portal, Uganda, at 12 and 18 months postpartum (ppm).</p><p>Methods and findings</p><p>124 HIV-1 positive pregnant women were enrolled within antenatal care services in Fort Portal, Uganda. Blood samples were collected at the first visit prior starting Option B+ and postpartum at week six, month six, 12 and 18. Viral load was determined by real-time RT-PCR. An RT-PCR covering resistance associated positions in the protease and reverse transcriptase HIV-1 genomic region was performed. PCR-positive samples at 12/18 ppm and respective baseline samples were analysed by next generation sequencing regarding HIV-1 drug resistant variants including low-frequency variants. Furthermore, vertical transmission of HIV-1 was analysed. 49/124 (39.5%) women were included into the DRM analysis. Virological failure, defined as >1000 copies HIV-1 RNA/ml, was observed in three and seven women at 12 and 18 ppm, respectively. Sequences were obtained for three and six of these. In total, DRMs were detected in 3/49 (6.1%) women. Two women displayed dual-class resistance against all recommended first-line regimen drugs. Of 49 mother-infant-pairs no infant was HIV-1 positive at 12 or 18 ppm.</p><p>Conclusion</p><p>Our findings suggest that the WHO-recommended Option B+ for PMTCT is effective in a cohort of Ugandan HIV-1 positive pregnant women with regard to the low selection rate of DRMs and vertical transmission. Therefore, these results are encouraging for other countries considering the implementation of lifelong cART for all pregnant HIV-1 positive women.</p></div

Sample availability.

<p>Sample availability.</p

Outcome of detection of DRMs in three women.

<p>Outcome of detection of DRMs in three women.</p

Outcome sample analysis.

<p>Outcome sample analysis.</p