Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Placental Sampling for Understanding Viral Infections - A Simplified Protocol for the COVID-19 Pandemic / Coleta placentária para entender infecções virais - Um protocolo simplificado para a pandemia de COVID-19

Abstract Objective The coronavirus disease 2019 (COVID-19) is a pandemic viral disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The impact of the disease among the obstetric population remains unclear, and the study of the placenta can provide valuable information. Adequate sampling of the placental tissue can help characterize the pathways of viral infections. Methods A protocol of placental sampling is proposed, aiming at guaranteeing representativity of the placenta and describing the adequate conservation of samples and their integrity for future analysis. The protocol is presented in its complete and simplified versions, allowing its implementation in different complexity settings. Results Sampling with the minimum possible interval from childbirth is the key for adequate sampling and storage. This protocol has already been implemented during the Zika virus outbreak. Conclusion A protocol for adequate sampling and storage of placental tissue is fundamental for adequate evaluation of viral infections on the placenta. During the COVID-19 pandemic, implementation of this protocol may help to elucidate critical aspects of the SARS-CoV-2 infection.

Resumo Objetivo A doença do novo coronavírus (COVID-19) é uma doença viral pandêmica causada pelo coronavírus da síndrome respiratória aguda 2 (SARS-CoV-2). O impacto da doença entre a população obstétrica ainda é incerto, e o estudo da placenta pode fornecer informações valiosas. Assim, a coleta adequada do tecido placentário pode ajudar a caracterizar algumas propriedades das infecções virais. Métodos Um protocolo de coleta placentária é proposto, objetivando a garantia de representatividade da placenta, descrevendo a maneira de conservação adequada das amostras, e visando garantir sua integridade para análises futuras. O protocolo é apresentado em suas versões completa e simplificada, permitindo sua implementação em diferentes configurações de infraestrutura. Resultados A amostragem com o intervalo mínimo possível do parto é essencial para coleta e armazenamento adequados. Esse protocolo já foi implementado durante a epidemia de vírus Zika. Conclusão Um protocolo para coleta e armazenamento adequados de tecido placentário é fundamental para a avaliação adequada de infecções virais na placenta. Durante a pandemia de COVID-19, a implementação deste protocolo pode ajudar a elucidar aspectos críticos da infecção por SARS-CoV-2.

Towards quality and order in human papillomavirus research.

The International Human Papillomavirus (HPV) Reference Center supports quality and order in HPV research and diagnostics. Notably, the center assigns HPV type numbers to novel HPV types, maintains a reference clone repository, and issues international proficiency panels for HPV genotyping. The established HPV types, currently up to HPV225, belong to 5 different genera: alpha (65 types), beta (54 types), gamma (98 types), mu (3 types) and nu (1 type). Since 2014, 23 novel types have been established, 82.6% of which belong to the gamma genus. Reference clones have been provided to 44 different research laboratories and the global proficiency program for HPV genotyping has seen an increasing participation (currently 146 laboratories) and complete proficiency has increased over time (from 26% to 59% of datasets). In summary, an increasing complexity of the HPVs requires international efforts to support a recognized quality and order among HPV types.

Continuing global improvement in human papillomavirus DNA genotyping services: The 2013 and 2014 HPV LabNet international proficiency studies.

BACKGROUND: Accurate and internationally comparable human papillomavirus (HPV) DNA detection and typing services are essential for HPV vaccine research and surveillance. OBJECTIVES: This study assessed the proficiency of different HPV typing services offered routinely in laboratories worldwide. STUDY DESIGN: The HPV Laboratory Network (LabNet) has designed international proficiency panels that can be regularly issued. The HPV genotyping proficiency panels of 2013 and 2014 contained 43 and 41 coded samples, respectively, composed of purified plasmids of sixteen HPV types (HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68a and 68b) and 3 extraction controls. Proficient typing was defined as detection in both single and multiple infections of 50 International Units of HPV 16 and HPV 18 and 500 genome equivalents for the other 14 HPV types, with at least 97% specificity. RESULTS: Ninety-six laboratories submitted 136 datasets in 2013 and 121 laboratories submitted 148 datasets in 2014. Thirty-four different HPV genotyping assays were used, notably Linear Array, HPV Direct Flow-chip, GenoFlow HPV array, Anyplex HPV 28, Inno-LiPa, and PGMY-CHUV assays. A trend towards increased sensitivity and specificity was observed. In 2013, 59 data sets (44%) were 100% proficient compared to 86 data sets (59%) in 2014. This is a definite improvement compared to the first proficiency panel, issued in 2008, when only 19 data sets (26%) were fully proficient. CONCLUSION: The regularly issued global proficiency program has documented an ongoing worldwide improvement in comparability and reliability of HPV genotyping services.

Are vaginal swabs comparable to cervical smears for human papillomavirus DNA testing?

OBJECTIVE: Human papillomavirus (HPV) testing is widely incorporated into cervical cancer screening strategies. Current screening requires pelvic examination for cervical sampling, which may compromise participation. The acceptance could be raised by introducing testing on vaginal swabs. We explored the interchangeability of vaginal swabs and cervical smears for HPV testing, by means of a prospective study conducted in female sex workers (FSWs). Besides, we report on the occurrence of 32 different HPV genotypes in FSW with low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL). METHODS: Paired physician-collected vaginal swabs and cervical smears from 303 FSW were tested for HPV using the Abbott RealTime High-Risk HPV assay. Cervical cytology was examined on cervical smears. In case of HSIL/LSIL cytological classification (n=52), both samples were genotyped using INNO-LiPa HPV Genotyping Extra II. RESULTS: The overall prevalence of high-risk (HR)-HPV was 51%. In FSW with HSIL/LSIL cervical cytology, the sensitivity and specificity of vaginal samples for the detection of HR-HPV was 100% and 70% and for probable HR-HPV 100% and 91%. The mean number of genotypes identified in vaginal samples (mean=3.5; 95% confidence interval [CI]=2.8-4.2) was significantly higher than in cervical smear samples (mean=2.6; 95% CI=2.1-3.0) (p=0.001). The most frequently encountered HR-HPV genotypes were HPV16, 31, 51, and 52. CONCLUSION: As our study shows that vaginal swabs are equivalent to cervical smears for the detection of (probable) HR-HPV, vaginal swabs can be used for HPV testing in cervical cancer screening strategies. Given the acceptance of vaginal sampling, this finding offers an opportunity to boost screening coverage.

An evaluation of clinical performance of FTA cards for HPV 16/18 detection using cobas 4800 HPV Test compared to dry swab and liquid medium.

BACKGROUNDS: Effective dry storage and transport media as an alternative to conventional liquid-based medium would facilitate the accessibility of women in the low-resource settings to human papillomavirus (HPV)- based cervical cancer screening. OBJECTIVE: To evaluate analytical and clinical performance of indicating FTA™ Elute Cartridge (FTA card) for the detection of HPV16/18 and cervical precancerous lesions and cancer compared to dry swab and liquid medium. STUDY DESIGN: Ninety patients with abnormal cytology and/or HPV infection were included for analysis. Three specimens of cervical exfoliated cells from each woman were randomly collected by FTA card, dry swab or liquid-based medium prior to colposcopy examination. The subsequent HPV DNA tests were performed on cobas 4800 HPV platform. RESULTS AND CONCLUSIONS: High-risk HPV (hrHPV) positivity rate was 63.3%, 62.2% and 65.6% for samples collected by FTA card, dry swab and liquid medium, respectively. The overall agreements and kappa values for the detection of hrHPV, HPV 16 and HPV 18 between FTA card and liquid-based medium were 88.9% (κ=0.76), 97.8% (κ=0.94) and 100% (κ=1.0),respectively; between FTA card and dry swab were 92.1% (κ=0.83), 94.5% (κ=0.87) and 100% (κ=1.0), respectively. The performances of hrHPV tested by FTA card, dry swab, and liquid-based medium for detecting CIN2+ were comparable in terms of the sensitivity and specificity. The specificity of detection of CIN2+ by HPV16/18 increased by approximately 40% compared to hrHPV for any medium albeit at cost of a moderate loss of sensitivity. Dry medium might offer an alternative to conventional liquid-based medium in the HPV-based cervical cancer screening program especially in low-resource settings but still needs further evaluation.

Diagnosis of Pediatric Acute Adenovirus Infections: Is a Positive PCR Sufficient?

BACKGROUND: Human adenovirus (HAdV), especially species C (HAdV-C), can be detected incidentally by polymerase chain reaction in nasopharyngeal (NP) samples, making it difficult to interpret clinical significance of a positive result. We classified patients into groups based on HAdV culture positivity from respiratory specimens and the presence of an identified co-pathogen. We hypothesized that HAdV-C would be over-represented and viral burden would be lower in patients most likely to have incidental detection (ie, with a negative viral culture and documented co-pathogen). METHODS: Immunocompetent children with HAdV + nasopharyngeal specimens were classified into 4 groups: group I (HAdV culture (+) and no co-infection), group II (culture (+) and co-infection), group III (culture (-) and no co-infection) and group IV (culture (-) and co-infection). Viral burden (cycle threshold) and species were compared among groups. RESULTS: Of 483 nasopharyngeal specimens, HAdV was isolated in culture in 252 (52%); co-infection was found in 265 (55%) patients. Group I (most consistent with acute disease) had significantly lower cycle thresholds (median 23.9; interquarile range 22.2-28.1) compared with group IV (most consistent with incidental detection; median 37.3; interquarile range 35.3-38.9; P < 0.0001). HAdV-C accounted for 41% samples of group I and 83% of group IV (P < 0.0001). We identified a subset of 22 patients with bacterial or fungal co-pathogens, 18 of whom had no growth on viral culture (group IV) with a median cycle threshold of 37.4 (interquarile range 33.9-39.2). CONCLUSIONS: Species identification and viral burden may assist in interpretation of a positive HAdV result. Low viral burden with HAdV-C may be consistent with incidental detection.

International standardization and classification of human papillomavirus types.

Established Human Papillomavirus (HPV) types, up to HPV202, belong to 49 species in five genera. International standardization in classification and quality standards for HPV type designation and detection is ensured by the International HPV Reference Center. The center i) receives clones of potentially novel HPV types, re-clones and re-sequences them. If confirmed, an HPV type number is assigned and posted on www.hpvcenter.se. ii) distributes reference clone samples, for academic research, under Material Transfer Agreements agreed with the originator. iii) provides preliminary checking of whether new sequences represent novel types iv) issues international proficiency panels for HPV genotyping. The rate of HPV type discovery is increasing, probably because of metagenomic sequencing. γ-genus today contains 79HPV types and 27 species, surpassing ∝ and ß genera with 65 and 51HPV types, respectively. Regular issuing of proficiency panels based on HPV reference clones has resulted in global improvement of HPV genotyping services.

Technical standards for hepatitis B virus X protein (HBx) research.

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing hepatocellular carcinoma (HCC). The life cycle of HBV is complex and has been difficult to study because HBV does not infect cultured cells. The HBV regulatory X protein (HBx) controls the level of HBV replication and possesses an HCC cofactor role. Attempts to understand the mechanism(s) that underlie HBx effects on HBV replication and HBV-associated carcinogenesis have led to many reported HBx activities that are likely influenced by the assays used. This review summarizes experimental systems commonly used to study HBx functions, describes limitations of these experimental systems that should be considered, and suggests approaches for ensuring the biological relevance of HBx studies.

Global improvement in genotyping of human papillomavirus DNA: the 2011 HPV LabNet International Proficiency Study.

Accurate and internationally comparable human papillomavirus (HPV) DNA genotyping is essential for HPV vaccine research and for HPV surveillance. The HPV Laboratory Network (LabNet) has designed international proficiency studies that can be issued regularly and in a reproducible manner. The 2011 HPV genotyping proficiency panel contained 43 coded samples composed of purified plasmids of 16 HPV types (HPV6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68a, and -68b) and 3 extraction controls. Tests that detected 50 IU of HPV16 and HPV18 and 500 genome equivalents for the other 14 HPV types in both single and multiple infections were considered proficient. Ninety-six laboratories worldwide submitted 134 data sets. Twenty-five different HPV genotyping assay methods were used, including the Linear Array, line blot/INNO-LiPA, PapilloCheck, and PCR Luminex assays. The major oncogenic HPV types, HPV16 and HPV18, were proficiently detected in 97.0% (113/116) and 87.0% (103/118) of the data sets, respectively. In 2011, 51 data sets (39%) were 100% proficient for the detection of at least one HPV type, and 37 data sets (28%) were proficient for all 16 HPV types; this was an improvement over the panel results from the 2008 and 2010 studies, when <25 data sets (23% and 19% for 2008 and 2010, respectively) were fully proficient. The improvement was also evident for the 54 laboratories that had also participated in the previous proficiency studies. In conclusion, a continuing global proficiency program has documented worldwide improvement in the comparability and reliability of HPV genotyping assay performances.

Genomic DNA of MCF-7 breast cancer cells not an ideal choice as positive control for PCR amplification based detection of Mouse Mammary Tumor Virus-Like Sequences.

The identification of the etiology of breast cancer is a crucial research issue for the development of an effective preventive and treatment strategies. Researchers are exploring the possible involvement of Mouse Mammary Tumor Virus (MMTV) in causing human breast cancer. Hence, it becomes very important to use a consistent positive control agent in PCR amplification based detection of MMTV-Like Sequence (MMTV-LS) in human breast cancer for accurate and reproducible results. This study was done to investigate the feasibility of using genomic DNA of MCF-7 breast cancer cells to detect MMTV-LS using PCR amplification based detection. MMTV env and SAG gene located at the 3' long terminal repeat (LTR) sequences were targeted for the PCR based detection. No amplification was observed in case of the genomic DNA of MCF-7 breast cancer cells. However, the 2.7 kb DNA fragment comprising MMTV env and SAG LTR sequences yielded the products of desired size. From these results it can be concluded that Genomic DNA of MCF-7 cell is not a suitable choice as positive control for PCR or RT-PCR based detection of MMTV-LS. It is also suggested that plasmids containing the cloned genes or sequences of MMTV be used as positive control for detection of MMTV-LS.

HeLa cell heterogeneity and coxsackievirus B3 cytopathic effect: implications for inter-laboratory reproducibility of results.

Concerns over cell line identities and contamination have led investigators to acquire fresh stocks of HeLa CCL-2 cells, but results with the HeLa CCL-2 cells do not always reproduce results with HeLa cells that have long history in the laboratory. When used for TCID(50) assays of Coxsackievirus B3/28 (CVB3/28), HeLa CCL-2 cells returned titers for CVB3/28 that were more than ten-fold lower than titers obtained using laboratory HeLa cells. The viral cytopathic effect was less distinct in the HeLa CCL-2 cultures, suggestive of a mixed population of cells with varied susceptibility to viral cytopathic effect. Analysis of short tandem repeat markers confirmed the identities of the cell lines as HeLa. Subpopulations in the HeLa CCL-2 culture, separated easily based on the speed with which they were released by trypsin-EDTA, differed in their susceptibilities to CVB3/28 cytopathic effect, and in their expression of the Coxsackievirus and adenovirus receptor (CAR). The distinctions between Lab HeLa and HeLa CCL-2 cells were less obvious when infected with CVB3/RD, a strain selected for growth in RD cells. Results that differ among laboratories may be due to the use of HeLa cell strains with different histories, and experiments using HeLa CCL-2 available from the American Type Culture Collection are probably incapable of reproducing many of the published studies of Coxsackievirus that have used HeLa cells with laboratory-dependent histories.

Nucleic acid tests for the detection of alpha human papillomaviruses.

Testing for high-risk types of alpha human papillomaviruses (HPV) is an invaluable part of clinical guidelines for cervical carcinoma screening, management and treatment. In this comprehensive inventory of commercial tests for detection of alpha-HPV, we identified at least 125 distinct HPV tests and at least 84 variants of the original tests. However, only a small subset of HPV tests has documented clinical performance for any of the standard HPV testing indications. For more than 75% of HPV tests currently on the market, no single publication in peer-reviewed literature can be identified. HPV tests that have not been validated and lack proof of reliability, reproducibility and accuracy should not be used in clinical management. Once incorporated in the lab, it is essential that the whole procedure of HPV testing is subject to continuous and rigorous quality assurance to avoid sub-optimal, potentially harmful practices. Manufacturers of HPV tests are urged to put more effort into evaluating their current and future products analytically, using international standards, and for clinical applications, using clinically validated endpoints. To assist with analytical validation, the World Health Organization is developing international standards for HPV types other than HPV16 and HPV18 and is planning development of external quality control panels specifically designed to be used for performance evaluation of current and future HPV tests. There is a need for more competitively priced HPV tests, especially for resource-poor countries, and uniform test validation criteria based on international standards should enable issuing more competitive and fair tender notices for purchasing. Automation systems allowing large-scale testing, as well as further increases in clinical performance, are the main needs in the further improvement of HPV tests. This article forms part of a special supplement entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine Volume 30, Supplement 5, 2012.

Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections.

The aim of this work was to improve the validity of laboratory-developed real-time PCR protocols implemented in the laboratory for molecular diagnosis of opportunistic DNA virus infections using the Simplexa™ extraction and amplification control (SEAC) which allows the monitoring of the whole extraction and amplification process. Herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), and adenovirus (AdV) genomes were investigated in 152 different clinical specimens. The use of the SEAC did not influence the results of the different virus-specific PCRs. The SEAC results showed high reproducibility with a mean Cp value of 31.08±1.44, and were not influenced by the virus-specific PCR performed or the type of clinical specimen tested. The SEAC in the DNA extracts showed high stability during storage at both +4°C and -20°C. These data allowed establishing a new procedure for the validation of viral PCR results. In conclusion, the SEAC provides a reliable option for improving the diagnosis of opportunistic viral infections by laboratory-developed real-time PCR assays in quality assurance programs.

Genotyping external quality assurance in the World Health Organization HIV drug resistance laboratory network during 2007-2010.

The World Health Organization (WHO) has developed a global laboratory network to support human immunodeficiency virus drug resistance genotyping for public health surveillance in resource-limited countries. Blinded proficiency panels are an essential part of a genotyping quality-assurance program and are used to monitor the reliability of genotyping data in the WHO laboratory network. Laboratories in Europe, North America, Asia, Africa, and the Caribbean have tested panels annually since 2007; 103 of 131 submissions (79%) had >99% nucleotide sequence identity and resistance mutation concordance, compared with consensus. Most errors were associated with mixtures in the test specimen, leading to subjectivity in base-calling or amplification bias. Overall, genotyping assays used by the WHO laboratory network are reliable.

Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays.

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits.

The 2010 global proficiency study of human papillomavirus genotyping in vaccinology.

Accurate and internationally comparable human papillomavirus (HPV) DNA genotyping is essential both for evaluation of HPV vaccines and for effective monitoring and implementation of vaccination programs. The World Health Organization (WHO) HPV Laboratory Network (LabNet) regularly issues international proficiency studies. The 2010 HPV genotyping proficiency panel for HPV vaccinology contained 43 coded samples composed of purified plasmids of 16 HPV types (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68a and 68b) and 3 coded extraction controls. Proficient typing was defined as detection in both single and multiple infections of 50 international units (IU) of HPV type 16 (HPV-16) and HPV-18 DNA and 500 genome equivalents (GE) for the other 14 HPV types. Ninety-eight laboratories worldwide submitted a total of 132 data sets. Twenty-four different HPV genotyping assay methods were used, with Linear Array being the most commonly used. Other major assays used were a line blot assay (Inno-LiPa), CLART, type-specific real-time PCR, PCR Luminex, and different microarray assays. Altogether, 72 data sets were proficient for detection of more than 1 type, and only 26 data sets proficiently detected all 16 HPV types. The major oncogenic HPV types, 16 and 18, were proficiently detected in 95.0% (114/120) and 87.0% (94/108) of data sets, respectively. Forty-six data sets reported multiple false-positive results and were considered nonproficient. A trend toward increased sensitivity of assays was seen for the 41 laboratories that participated in both 2008 and 2010. In conclusion, continued global proficiency studies will be required for establishing comparable and reliable HPV genotyping services for vaccinology worldwide.

International collaborative proficiency study of Human Papillomavirus type 16 serology.

We performed an international proficiency study of Human Papillomavirus (HPV) type 16 serology. A common methodology for serology based on virus-like particle (VLP) ELISA was used by 10 laboratories in 6 continents. The laboratories used the same VLP reference reagent, which was selected as the most stable, sensitive and specific VLP preparation out of VLPs donated from 5 different sources. A blinded proficiency panel consisting of 52 serum samples from women with PCR-verified HPV 16-infection, 11 control serum samples from virginal women and the WHO HPV 16 International Standard (IS) serum were distributed. The mean plus 3 standard deviations of the negative control serum samples was the most generally useful "cut-off" criterion for distinguishing positive and negative samples. Using sensitivity of at least 50% and a specificity of 100% as proficiency criteria, 6/10 laboratories were proficient. In conclusion, an international Standard Operating Procedure for HPV serology, an international reporting system in International Units (IU) and a common "cut-off" criterion have been evaluated in an international HPV serology proficiency study.