SARS-CoV-2 viral clearance and viral load kinetics in young children (1-6 years) compared to adults: Results of a longitudinal study in Germany.

Objective: To investigate SARS-COV-2 viral clearance and viral load kinetics in the course of infection in children aged 1-6 years in comparison with adults. Methods: Prospective cohort study of infected daycare children and staff and their close contacts in households from 11/2020 to 06/2021. Adult participants took upper respiratory tract specimen from themselves and/or their children, for PCR tests on SARS-CoV-2. Data on symptoms and exposure were used to determine the date of probable infection for each participant. We determined (a) viral clearance, and (b) viral load dynamics over time. Samples were taken from day 4-6 to day 16-18 after diagnosis of the index case in the respective daycare group (5 samples per participant). Results: We included 40 children (1-6 years) and 67 adults (18-77 years) with SARS-CoV-2 infection. Samples were available at a mean of 4.3 points of time per participant. Among the participants, the 12-day study period fell in different periods within the individual course of infection, ranging from day 5-17 to day 15-26 after assumed infection.Children reached viral clearance at a median of 20 days after assumed infection (95% CI 17-21 days, Kaplan-Meier Analysis), adults at 23 days (95% CI 20-25 days, difference not significant). In both children and adults, viral load decreased over time with trajectories of the mean viral load not being statistically different between groups. Kaplan-Meier calculations show that from day 15 (95% CI 13-15), 50% of all participants had a viral load <1 million copies/ml, i.e. were no longer infectious or negative. Conclusion: Children aged 1-6 and adults infected with SARS-CoV-2 (wild type and Alpha variant) did not differ significantly in terms of viral load kinetics and time needed to clear the virus. Therefore, containment measures are important also in the daycare settings as long as the pandemic continues.

Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use.

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.

Replication of cowpox virus in macrophages is dependent on the host range factor p28/N1R.

Zoonotic orthopoxvirus infections continue to represent a threat to human health. The disease caused by distinct orthopoxviruses differs in terms of symptoms and severity, which may be explained by the unique repertoire of virus factors that modulate the host's immune response and cellular machinery. We report here on the construction of recombinant cowpox viruses (CPXV) which either lack the host range factor p28 completely or express truncated variants of p28. We show that p28 is essential for CPXV replication in macrophages of human or mouse origin and that the C-terminal RING finger domain of p28 is necessary to allow CPXV replication in macrophages.

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2.

BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.

SARS-CoV-2 Transmissibility Within Day Care Centers-Study Protocol of a Prospective Analysis of Outbreaks in Germany.

Introduction: Until today, the role of children in the transmission dynamics of SARS-CoV-2 and the development of the COVID-19 pandemic seems to be dynamic and is not finally resolved. The primary aim of this study is to investigate the transmission dynamics of SARS-CoV-2 in child day care centers and connected households as well as transmission-related indicators and clinical symptoms among children and adults. Methods and Analysis: COALA ("Corona outbreak-related examinations in day care centers") is a day care center- and household-based study with a case-ascertained study design. Based on day care centers with at least one reported case of SARS-CoV-2, we include one- to six-year-old children and staff of the affected group in the day care center as well as their respective households. We visit each child's and adult's household. During the home visit we take from each household member a combined mouth and nose swab as well as a saliva sample for analysis of SARS-CoV-2-RNA by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and a capillary blood sample for a retrospective assessment of an earlier SARS-CoV-2 infection. Furthermore, information on health status, socio-demographics and COVID-19 protective measures are collected via a short telephone interview in the subsequent days. In the following 12 days, household members (or parents for their children) self-collect the same respiratory samples as described above every 3 days and a stool sample for children once. COVID-19 symptoms are documented daily in a symptom diary. Approximately 35 days after testing the index case, every participant who tested positive for SARS-CoV-2 during the study is re-visited at home for another capillary blood sample and a standardized interview. The analysis includes secondary attack rates, by age of primary case, both in the day care center and in households, as well as viral shedding dynamics, including the beginning of shedding relative to symptom onset and viral clearance. Discussion: The results contribute to a better understanding of the epidemiological and virological transmission-related indicators of SARS-CoV-2 among young children, as compared to adults and the interplay between day care and households.

Equination (inoculation of horsepox): An early alternative to vaccination (inoculation of cowpox) and the potential role of horsepox virus in the origin of the smallpox vaccine.

For almost 150 years after Edward Jenner had published the "Inquiry" in 1798, it was generally assumed that the cowpox virus was the vaccine against smallpox. It was not until 1939 when it was shown that vaccinia, the smallpox vaccine virus, was serologically related but different from the cowpox virus. In the absence of a known natural host, vaccinia has been considered to be a laboratory virus that may have originated from mutational or recombinational events involving cowpox virus, variola viruses or some unknown ancestral Orthopoxvirus. A favorite candidate for a vaccinia ancestor has been the horsepox virus. Edward Jenner himself suspected that cowpox derived from horsepox and he also believed that "matter" obtained from either disease could be used as preventative of smallpox. During the 19th century, inoculation with cowpox (vaccination) was used in Europe alongside with inoculation with horsepox (equination) to prevent smallpox. Vaccine-manufacturing practices during the 19th century may have resulted in the use of virus mixtures, leading to different genetic modifications that resulted in present-day vaccinia strains. Horsepox, a disease previously reported only in Europe, has been disappearing on that continent since the beginning of the 20th century and now seems to have become extinct, although the virus perhaps remains circulating in an unknown reservoir. Genomic sequencing of a horsepox virus isolated in Mongolia in 1976 indicated that, while closely related to vaccinia, this horsepox virus contained additional, potentially ancestral sequences absent in vaccinia. Recent genetic analyses of extant vaccinia viruses have revealed that some strains contain ancestral horsepox virus genes or are phylogenetically related to horsepox virus. We have recently reported that a commercially produced smallpox vaccine, manufactured in the United States in 1902, is genetically highly similar to horsepox virus, providing a missing link in this 200-year-old mystery.

Seasonal recurrence of cowpox virus outbreaks in captive cheetahs (Acinonyx jubatus).

Cowpox virus infections in captive cheetahs (Acinonyx jubatus) with high morbidity and mortality have already been reported in the UK and Russia in the 1970s. However, most of the reported cases have been singular events. Here, we report a total of five cowpox virus outbreaks in cheetahs in the same safari park in Denmark between 2010 and 2014. Nine cheetahs showed varying severity of clinical disease; two of them died (22%). All episodes occurred between August and October of the respective year. No other carnivores kept at the same institution nor the keepers taking care of the animals were clinically affected. The clinical picture of cowpox was confirmed by extensive laboratory investigations including histopathological and molecular analyses as well as cell culture isolation of a cowpox virus. High anti-orthopoxvirus antibody titers were detected in all 9 diseased cheetahs compared to seven contact cheetahs without clinical signs and 13 cheetahs not in direct contact. Additionally, whole genome sequencing from one sample of each cluster with subsequent phylogenetic analysis showed that the viruses from different outbreaks have individual sequences but clearly form a clade distinct from other cowpox viruses. However, the intra-clade distances are still larger than those usually observed within clades of one event. These findings indicate multiple and separate introductions of cowpox virus, probably from wild rodent populations, where the virus keeps circulating naturally and is only sporadically introduced into the cheetahs. Sero-positivity of voles (Arvicola amphibious) caught in zoo grounds strengthens this hypothesis. As a consequence, recommendations are given for medical and physical management of diseased cheetahs, for hygienic measures as well as for pre-shipment isolation before cheetah export from zoo grounds.

Berlin Squirrelpox Virus, a New Poxvirus in Red Squirrels, Berlin, Germany.

Near Berlin, Germany, several juvenile red squirrels (Sciurus vulgaris) were found with moist, crusty skin lesions. Histology, electron microscopy, and cell culture isolation revealed an orthopoxvirus-like infection. Subsequent PCR and genome analysis identified a new poxvirus (Berlin squirrelpox virus) that could not be assigned to any known poxvirus genera.

A Cross-Sectional Serosurvey of Anti-Orthopoxvirus Antibodies in Central and Western Africa.

Since the eradication of smallpox and the subsequent discontinuation of the worldwide smallpox vaccination program, other Orthopoxviruses beside Variola virus have been increasingly representing a risk to human health. To investigate the extent of natural contact with Orthopoxviruses and possible demographic risk factors for such an exposure, we performed a cross-sectional serosurvey of anti-Orthopoxvirus IgG antibodies in West and Central Africa. To this end, people living in forest regions in Côte d'Ivoire (CIV, n = 737) and the Democratic Republic of the Congo (COD, n = 267) were assigned into groups according to their likely smallpox vaccination status. The overall prevalence of anti-Orthopoxvirus antibodies was 51% in CIV and 60% in COD. High rates of seropositivity among the vaccinated part of the population (80% in CIV; 96% COD) indicated a long-lasting post vaccination immune response. In non-vaccinated participants, seroprevalences of 19% (CIV) and 26% (COD) indicated regular contact with Orthopoxviruses. Multivariate logistic regression revealed that the antibody level in the vaccinated part of the population was higher in COD than in CIV, increased with age and was slightly higher in females than males. In the unvaccinated part of the population none of these factors influenced antibody level significantly. In conclusion, our results confirm expectedly high anti-Orthopoxvirus seroprevalences in previously smallpox-vaccinated people living in CIV and the COD but more unexpectedly imply regular contact with Orthopoxviruses both in Western and Central Africa, even in the absence of recognized outbreaks.

External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014 - 2016 Ebola outbreak in West Africa.

During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied.

[On-site detection of bioterrorism-relevant agents : Rapid detection methods for viruses, bacteria and toxins - capabilities and limitations]. / Vor-Ort-Nachweis bioterroristisch relevanter Agenzien : Schnelle Nachweismethoden für Viren, Bakterien und Toxine ­ Potenziale und Grenzen.

In Europe, besides the threat of terrorist attacks involving conventional methods such as explosive devices and automatic weapons, there is also a potential threat of terrorist groups using non-conventional material like biological agents in the scope of future attacks. Consequently, rapid and reliable detection systems for biological agents are being developed and tested continuously to inform crisis management. For environmental detection, a broad spectrum of different laboratory-based techniques has been developed for relevant biological agents. However for environmental samples, fast and reliable on-site detection methods are desired by first responders for rapid assessment.Based on different functional principles, generic, immunological and nucleic-acid-based on-site detection methods can be distinguished. Those should be facile, fast, sensitive, and specific. However, commercially available kits usually have limited sensitivity and often have not been validated independently. Furthermore in this context, the multitude of relevant biological agents that potentially have to be considered present in complex environmental matrices poses a serious challenge for reliable detection. Therefore, detailed knowledge of the specific scope of applications and the limitations of different analytical systems is necessary to evaluate the results obtained purposefully.The aim of this article is to provide an overview of the analytical principles, benefits and limitations of prevailing on-site environmental detection systems for bioterrorism-relevant viruses, bacteria and toxins. Despite promising developments the informative value of currently available on-site tests is still limited. Thus, expert laboratories have to conduct confirmatory testing.

Pitfalls in PCR troubleshooting: Expect the unexpected?

PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.