Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence

Tick-borne encephalitis virus (TBEV) is a flavivirus transmitted by ticks. Serological screenings in animals are performed to estimate the prevalence and distribution of TBEV. Most screenings consist of a primary screening by ELISA, followed by confirmation of positive samples by plaque reduction neutralization tests (PRNTs). In this study, 406 wild boar sera were tested with 2 regularly used commercial ELISAs for flavivirus screening in animals (Immunozym FSME (TBEV) IgG All Species (Progen) and ID Screen West Nile Competition (Innovative Diagnostics)) and PRNTs for TBEV and USUTU virus. The results showed that the Immunozym and IDScreen ELISAs had low relative sensitivities of 23% and 20%, respectively, compared to the PRNT results. The relative specificities were 88% and 84% due to cross reactions with USUTU virus-specific antibodies. The minimal TBEV prevalence in our sample set was 8.6% when determined by PRNT. When the screening approach of ELISA testing followed by PRNT confirmation was applied, a TBEV seroprevalence of only 2.0% and 1.7% was found. The suboptimal performance of the ELISAs was confirmed by testing sera collected from experimentally TBEV-infected sheep. While the PRNT detected TBEV specific antibodies in 94% of samples collected between 7 and 18 days post-infection, the ELISAs classified only 50% and 31% of the samples as positive. Both routinely used ELISAs for TBEV antibody screening in animal sera were shown to have a low sensitivity, potentially leading to an underestimation of the true prevalence, and furthermore cross-react with other flavivirus antibodies

Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence

Tick-borne encephalitis virus (TBEV) is a flavivirus transmitted by ticks. Serological screenings in animals are performed to estimate the prevalence and distribution of TBEV. Most screenings consist of a primary screening by ELISA, followed by confirmation of positive samples by plaque reduction neutralization tests (PRNTs). In this study, 406 wild boar sera were tested with 2 regularly used commercial ELISAs for flavivirus screening in animals (Immunozym FSME (TBEV) IgG All Species (Progen) and ID Screen West Nile Competition (Innovative Diagnostics)) and PRNTs for TBEV and USUTU virus. The results showed that the Immunozym and IDScreen ELISAs had low relative sensitivities of 23% and 20%, respectively, compared to the PRNT results. The relative specificities were 88% and 84% due to cross reactions with USUTU virus-specific antibodies. The minimal TBEV prevalence in our sample set was 8.6% when determined by PRNT. When the screening approach of ELISA testing followed by PRNT confirmation was applied, a TBEV seroprevalence of only 2.0% and 1.7% was found. The suboptimal performance of the ELISAs was confirmed by testing sera collected from experimentally TBEV-infected sheep. While the PRNT detected TBEV specific antibodies in 94% of samples collected between 7 and 18 days post-infection, the ELISAs classified only 50% and 31% of the samples as positive. Both routinely used ELISAs for TBEV antibody screening in animal sera were shown to have a low sensitivity, potentially leading to an underestimation of the true prevalence, and furthermore cross-react with other flavivirus antibodies

Marek’s Disease Virus Virulence Genes Encode Circular RNAs

Circular RNAs (circRNAs) are a recently rediscovered class of functional noncoding RNAs that are involved in gene regulation and cancer development. Next-generation sequencing approaches identified circRNA fragments and sequences underlying circularization events in virus-induced cancers. In the present study, we performed viral circRNA expression analysis and full-length sequencing in infections with Marek’s disease virus (MDV), which serves as a model for herpesvirus-induced tumorigenesis. We established inverse PCRs to identify and characterize circRNA expression from the repeat regions of the MDV genome during viral replication, latency, and reactivation. We identified a large variety of viral circRNAs through precise mapping of full-length circular transcripts and detected matching sequences with several viral genes. Hot spots of circRNA expression included the transcriptional unit of the major viral oncogene encoding the Meq protein and the latency-associated transcripts (LATs). Moreover, we performed genome-wide bioinformatic analyses to extract back-splice junctions from lymphoma-derived samples. Using this strategy, we found that circRNAs were abundantly expressed in vivo from the same key virulence genes. Strikingly, the observed back-splice junctions do not follow a unique canonical pattern, compatible with the U2-dependent splicing machinery. Numerous noncanonical junctions were observed in viral circRNA sequences characterized from in vitro and in vivo infections. Given the importance of the genes involved in the transcription of these circRNAs, our study contributes to our understanding and complexity of this deadly pathogen. IMPORTANCE Circular RNAs (circRNAs) were rediscovered in recent years both in physiological and pathological contexts, such as in cancer. Viral circRNAs are encoded by at least two human herpesviruses, the Epstein Barr virus and the Kaposi’s Sarcoma-associated herpesvirus, both associated with the development of lymphoma. Marek’s disease virus (MDV) is a well-established animal model to study virus-induced lymphoma but circRNA expression has not been reported for MDV yet. Our study provided the first evidence of viral circRNAs that were expressed at key steps of the MDV lifecycle using genome-wide analyses of circRNAs. These circRNAs were primarily found in transcriptional units that corresponded to the major MDV virulence factors. In addition, we established a bioinformatics pipeline that offers a new tool to identify circular RNAs in other herpesviruses. This study on the circRNAs provided important insights into major MDV virulence genes and herpesviruses-mediated gene dysregulation

Marek’s Disease Virus Virulence Genes Encode Circular RNAs

International audienceCircular RNAs (circRNAs) are a recently rediscovered class of functional noncoding RNAs that are involved in gene regulation and cancer development. Next-generation sequencing approaches identified circRNA fragments and sequences underlying circularization events in virus-induced cancers. In the present study, we performed viral circRNA expression analysis and full-length sequencing in infections with Marek's disease virus (MDV), which serves as a model for herpesvirus-induced tumorigenesis. We established inverse PCRs to identify and characterize circRNA expression from the repeat regions of the MDV genome during viral replication, latency, and reactivation. We identified a large variety of viral circRNAs through precise mapping of full-length circular transcripts and detected matching sequences with several viral genes. Hot spots of circRNA expression included the transcriptional unit of the major viral oncogene encoding the Meq protein and the latency-associated transcripts (LATs). Moreover, we performed genome-wide bioinformatic analyses to extract back-splice junctions from lymphoma-derived samples. Using this strategy, we found that circRNAs were abundantly expressed in vivo from the same key virulence genes. Strikingly, the observed back-splice junctions do not follow a unique canonical pattern, compatible with the U2-dependent splicing machinery. Numerous noncanonical junctions were observed in viral circRNA sequences characterized from in vitro and in vivo infections. Given the importance of the genes involved in the transcription of these circRNAs, our study contributes to our understanding and complexity of this deadly pathogen. IMPORTANCE Circular RNAs (circRNAs) were rediscovered in recent years both in physiological and pathological contexts, such as in cancer. Viral circRNAs are encoded by at least two human herpesviruses, the Epstein Barr virus and the Kaposi's Sarcoma-associated herpesvirus, both associated with the development of lymphoma. Marek's disease virus (MDV) is a well-established animal model to study virus-induced lymphoma but circRNA expression has not been reported for MDV yet. Our study provided the first evidence of viral circRNAs that were expressed at key steps of the MDV lifecycle using genome-wide analyses of circRNAs. These circRNAs were primarily found in transcriptional units that corresponded to the major MDV virulence factors. In addition, we established a bioinformatics pipeline that offers a new tool to identify circular RNAs in other herpesviruses. This study on the circRNAs provided important insights into major MDV virulence genes and herpesviruses-mediated gene dysregulation

SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab.

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.status: Published onlin

SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab.

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results