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.

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.

Robust and sensitive subtype-generic HIV-1 pol genotyping for use with dried serum spots in epidemiological studies.

HIV-1 genotyping of larger pol-fragments from dried serum/plasma spots (DSS/DPS) is often hindered by RNA-degradation during transportation at ambient temperature. We evaluated an in-house HIV-1 DSS/DPS-genotyping assay designed in two shorter overlapping fragments covering all resistance mutations in protease and reverse transcriptase. Validation criteria such as specificity, detection limit, accuracy, reproducibility and storage conditions were assessed using reference plasma samples prepared as DPS and clinical DSS from the German molecular HIV-1 surveillance processed under real-life transportation conditions. The specificity was 100% for both samples types, and the experimental DPS detection limit of 1000 copies/ml yielded a 98.7% (3,329/3373) success rate for DSS (including all subtypes) above this detection limit. Accuracy for DPS compared to the gold standard was 99.1% and the reproducibility was 100% for DPS replicates and 99.9% for DSS pairs. Storage of DPS at room temperature was possible for 90 or 30 days and at -20 °C for at least 180 or 90 days at viral loads of 10,000 or 1000 copies/ml, respectively. The HIV-1 pol-genotyping assay presented here is a sensitive, robust and subtype generic tool for a large-scale population-based HIV-1 drug resistance surveillance for the use of DSS/DPS.

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.

Detection of rotavirus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of bovine species A rotavirus strains.

Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and animals worldwide. In this study, 425 faecal samples were collected between 1999 and 2013 from diarrhoeic livestock and companion animals at different locations in Germany and tested for RVs. A previously published real-time RT-PCR assay was optimized for detection of a larger variety of RV species A (RVA) strains, and real-time RT-PCR assays for detection of RV species B (RVB) and C (RVC) were newly developed. The detection limits of the assays were 1.54×10(2), 3.95×10(2) and 3.60×10(3) genome copies for RVA, RVB and RVC, respectively. RVA was identified in 85.2% of bovine samples, 51.2% of porcine samples, 50.0% of feline samples, 43.2% of equine samples and 39.7% of canine samples. RVB was found in 3.0% of bovine samples, 2.7% of equine samples and 1.6% of porcine samples. RVC was detected in 31.0% of porcine samples, 21.7% of feline samples, 9.0% of canine samples and 6.0% of bovine samples. For genotyping, 101 RVA-positive bovine samples were further analysed by semi-nested RT-PCR. Genotype combination G6P[5] was most frequently detected (67.3% of samples), followed by G6P[11] (13.9%), G10P[5] (4.0%), G8P[11] (3.0%), G6P[1] (1.0%), and G10P[11] (1.0%). Mixed RVA infections were detected in 5.9% of samples; no or incomplete typing was possible in 4.0% of the samples. This first overview on RV species and RVA genotypes in diarrhoeic livestock and companion animals from Germany indicates a broad circulation of a large variety of RVs.

Metagenomic identification of novel enteric viruses in urban wild rats and genome characterization of a group A rotavirus.

Rats are known as reservoirs and vectors for several zoonotic pathogens. However, information on the viruses shed by urban wild rats that could pose a zoonotic risk to human health is scare. Here, intestinal contents from 20 wild Norway rats (Rattus norvegicus) collected in the city of Berlin, Germany, were subjected to metagenomic analysis of viral nucleic acids. The determined faecal viromes of rats consisted of a variety of known and unknown viruses, and were highly variable among the individuals. Members of the families Parvoviridae and Picobirnaviridae represented the most abundant species. Novel picornaviruses, bocaviruses, sapoviruses and stool-associated circular ssDNA viruses were identified, which showed only low sequence identity to known representatives of the corresponding taxa. In addition, noroviruses and rotaviruses were detected as potential zoonotic gastroenteritis viruses. However, partial-genome sequence analyses indicated that the norovirus was closely related to the recently identified rat norovirus and the rotavirus B was closely related to the rat rotavirus strain IDIR; both viruses clustered separately from respective human virus strains in phylogenetic trees. In contrast, the rotavirus A sequences showed high identity to human and animal strains. Analysis of the nearly complete genome of this virus revealed the known genotypes G3, P[3] and N2 for three of the genome segments, whereas the remaining eight genome segments represented the novel genotypes I20-R11-C11-M10-A22-T14-E18-H13. Our results indicated a high heterogeneity of enteric viruses present in urban wild rats; their ability to be transmitted to humans remains to be assessed in the future.

Detection and characterization of potentially zoonotic viruses in faeces of pigs at slaughter in Germany.

Pigs can harbour a variety of viruses in their gastrointestinal tract. Some of them are closely related to human viruses and are therefore suspected to have a zoonotic potential. Only little is known about the presence of those viruses in pigs at slaughter. However, by contamination of meat with zoonotic viruses during the slaughtering process, food-borne transmission to humans may be possible. Here we analyzed 120 faecal samples of pigs at slaughter from 3 different geographical regions of Germany for the presence of astrovirus (AstV), encephalomyocarditis virus (EMCV), hepatitis E virus (HEV), norovirus genogroup II (NoV GII) and group A rotavirus (GARV). Using real-time RT-PCR, the most frequently detected virus was AstV, which was present in 20.8% of the samples, followed by NoV GII with a detection rate of 14.2%. EMCV, HEV and GARV were found only occasionally with detection rates of 4.2%, 2.5% and 0.8%, respectively. Analyses of partial genome sequences of the viruses indicated that the detected AstV and NoV GII mainly represented typical pig virus strains, which have not been detected in humans so far. However, the GARV and HEV strains were more closely related to human strains. The results indicate that enteric viruses, some of them with zoonotic potential, are present in pig faeces at slaughter. Application of good hygiene practice is necessary to minimize the risk of introducing these viruses into the food and to prevent virus transmission to highly exposed persons such as slaughterers and veterinarians.

Feeding of the probiotic bacterium Enterococcus faecium NCIMB 10415 differentially affects shedding of enteric viruses in pigs.

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 < 0.05); rotavirus-shedding piglets gained less weight than non-infected animals (p < 0.05). Serum titres of anti-rotavirus IgA and IgG antibodies were higher in piglets from the control group, whereas no difference was detected between sow groups. Phenotype analysis of immune cell antigens revealed significant differences of the CD4 and CD8ß (p < 0.05) as well as CD8α and CD25 (p < 0.1) T cell populations of the probiotic supplemented group compared to the non-supplemented control group. In addition, differences were evident for CD21/MHCII-positive (p < 0.05) and IgM-positive (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.

Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh.

Avian rotaviruses (AvRVs) represent a diverse group of intestinal viruses, which are suspected as the cause of several diseases in poultry with symptoms of diarrhoea, growth retardation or runting and stunting syndrome (RSS). To assess the distribution of AvRVs in chickens and turkeys, we have developed specific PCR protocols. These protocols were applied in two field studies investigating faecal samples or intestinal contents of diseased birds derived from several European countries and Bangladesh. In the first study, samples of 166 chickens and 33 turkeys collected between 2005 and 2008 were tested by PAGE and conventional RT-PCR and AvRVs were detected in 46.2%. In detail, 16.1% and 39.2% were positive for AvRVs of groups A or D, respectively. 11.1% of the samples contained both of them and only four samples (2.0%) contained rotaviruses showing a PAGE pattern typical for groups F and G. In the second study, samples from 375 chickens and 18 turkeys collected between 2009 and 2010 were analyzed using a more sensitive group A-specific and a new group D-specific real-time RT-PCR. In this survey, 85.0% were AvRV-positive, 58.8% for group A AvRVs, 65.9% for group D AvRVs and 38.9% for both of them. Although geographical differences exist, the results generally indicate a very high prevalence of group A and D rotaviruses in chicken and turkey flocks with cases of diarrhoea, growth retardation or RSS. The newly developed diagnostic tools will help to investigate the epidemiology and clinical significance of AvRV infections in poultry.