Infectious viral load in unvaccinated and vaccinated patients infected with SARS-CoV-2 WT, Delta and Omicron

Background Viral load (VL) is one determinant of secondary transmission of SARS-CoV-2. Emergence of variants of concerns (VOC) Alpha and Delta was ascribed, at least partly, to higher VL. Furthermore, with parts of the population vaccinated, knowledge on VL in vaccine breakthrough infections is crucial. As RNA VL is only a weak proxy for infectiousness, studies on infectious virus presence by cell culture isolation are of importance. Methods We assessed nasopharyngeal swabs of COVID-19 patients for quantitative infectious viral titres (IVT) by focus-forming assay and compared to overall virus isolation success and RNA genome copies. We assessed infectious viral titres during the first 5 symptomatic days in a total of 384 patients: unvaccinated individuals infected with pre-VOC SARS-CoV-2 (n= 118) or Delta (n= 127) and vaccine breakthrough infections with Delta (n= 121) or Omicron (n=18). Findings Correlation between RNA copy number and IVT was low for all groups. No correlation between IVTs and age or sex was seen. We observed higher RNA genome copies in pre-VOC SARS-CoV-2 compared to Delta, but significantly higher IVTs in Delta infected individuals. In vaccinated vs. unvaccinated Delta infected individuals, RNA genome copies were comparable but vaccinated individuals have significantly lower IVTs, and cleared virus faster. Vaccinated individuals with Omicron infection had comparable IVTs to Delta breakthrough infections. Interpretation Quantitative IVTs can give detailed insights into virus shedding kinetics. Vaccination was associated with lower infectious titres and faster clearance for Delta, showing that vaccination would also lower transmission risk. Omicron vaccine breakthrough infections did not show elevated IVTs compared to Delta, suggesting that other mechanisms than increase VL contribute to the high infectiousness of Omicron. Funding This work was supported by the Swiss National Science Foundation 196644, 196383, NRP (National Research Program) 78 Covid-19 Grant 198412, the Fondation Ancrage Bienfaisance du Groupe Pictet and the Fondation Privée des Hôpitaux Universitaires de Genève.

An in vitro and in vivo approach for the isolation of Omicron variant from human clinical specimens

Due to failure of virus isolation of Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, we infected Syrian hamsters and then passage into Vero CCL-81 cells. The Omicron sequences were studied to assess if hamster could incorporate any mutation to changes its susceptibility. L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene and absence of V17I mutation in E gene was observed in sequences of hamster passage unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequence which suggests usefulness of these isolates in future studies.

Isolation and Molecular Characterization of Turkey Pox Virus from Maharashtra, India: A Review

Avian pox diseases are contagious and slow spreading viral infections in birds. The present study was aim to, isolate and molecular characterization of turkeypox virus from a clinical case. Ten out of the twelve scab lesions sample collected from clinically suspected cases were positive for avian pox viurs (APV) based on virus isolation and polymerase chain reaction. We conducted genetic characterization of the APV strain. The phylogenetic analyses of P4b gene APV genome indicated that, avian poxviruses fragments sequenced in this study clustered along the A clade of avipoxviruses, genetically related to Indian fowl pox virus isolated from chicken, showing 99% homology.

A one health approach to investigating an outbreak of alimentary tick-borne encephalitis in a non-endemic area in France (Ain, Eastern France): a longitudinal serological study in livestock, detection in ticks, and the first TBE virus isolation and molecular characterization

Tick borne encephalitis virus geographic range and human incidence is increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of TBE in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurized raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and to witness in real time the seroconversion of recently exposed individuals and the excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for as a source of dietary contamination in France, obtained its full-length genome sequence, and found that it does not cluster very closely neither with the isolate circulating in Alsace nor with any other isolate within the European lineage. TBEV is now a notifiable human disease in France, which should facilitate surveillance of TBEV incidence and distribution throughout France.

Enhanced isolation of influenza viruses in qualified cells improves the probability of well-matched vaccines

Influenza vaccines are utilised to combat seasonal and pandemic influenza. The key to influenza vaccination currently is the availability of candidate vaccine viruses (CVVs). Ideally, CVVs reflect the antigenic characteristics of the circulating virus, which may vary depending upon the isolation method. For traditional inactivated egg-based vaccines, CVVs are isolated in embryonated chicken eggs, while for cell-culture production, CVV’s are isolated in either embryonated eggs or qualified cell lines. We compared isolation rates, growth characteristics, genetic stability and antigenicity of cell and egg CVV’s derived from the same influenza-positive human clinical respiratory samples collected from 2008–2020. Influenza virus isolation rates in MDCK33016PF cells were twice that of eggs and mutations in the HA protein were common in egg CVVs but rare in cell CVVs. These results indicate that fully cell-based influenza vaccines will improve the choice, match and potentially the effectiveness, of seasonal influenza vaccines compared to egg-based vaccines.

Avian Influenza Virus Status and Maternal Antibodies in Nestling White Ibis (Eudocimus albus)

The White Ibis (Eudocimus albus), a nomadic wading bird, has increased its exploitation of urban habitats in South Florida, United States, and has recently established several urban breeding colonies. Certain characteristics of ibis ecology could position them in the natural cycle of the avian influenza virus (AIV). In fact, experimentally infected ibises were shown to be competent hosts for multiple AIV subtypes, and seroconversion to AIV has been documented in adult ibises in natural populations. However, the mechanisms of transmission and the timing of infection are unclear as we have yet to isolate AIV from a free-living ibis. To investigate the age-specific AIV dynamics of ibis, we captured nestlings (n = 115) weekly for 1–4 weeks from urban and natural settings in 2020 and 2021. We collected choanal/cloacal swabs for rRT-PCR and virus isolation, and plasma to screen for maternal AIV antibodies. AIV was not detected in any individual by virus isolation; however, maternal antibodies to AIV were detected in 95% of nestlings, with varying rates of catabolism. These results confirm that nestlings are afforded maternal antibodies from adults at rates reflective of higher adult seroprevalence than previously documented and that nestlings in breeding colonies may have some degree of protection and are unlikely to become infected with AIV.

Marburg Virus Persistence on Fruit as a Plausible Route of Bat to Primate Filovirus Transmission

Marburg virus (MARV), the causative agent of Marburg virus disease, emerges sporadically in sub-Saharan Africa and is often fatal in humas. The natural reservoir for this zoonotic virus is the frugivorous Egyptian rousette bat (Rousettus aegyptiacus) that when infected, sheds virus in the highest amounts in oral secretions and urine. Being fruit bats, these animals forage nightly for ripened fruit throughout the year, including those types often preferred by humans. During feeding, they continually discard partially eaten fruit on the ground that could then be consumed by other Marburg virus susceptible animals or humans. In this study, using qRT-PCR and virus isolation, we tested fruit discarded by Egyptian rousette bats experimentally infected with a natural bat isolate of Marburg virus. We then separately tested viral persistence on fruit varieties commonly cultivated in sub-Saharan Africa using a recombinant Marburg virus expressing the fluorescent ZsGreen1. Marburg virus RNA was repeatedly detected on fruit in the food bowls of the infected bats and viable MARV was recovered from inoculated fruit for up to 6 h.

Prolonged SARS-CoV-2 Positivity in Immunocompetent Patients: Virus Isolation, Genomic Integrity, and Transmission Risk

In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset.

Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

Background Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step. Methods In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets. Results Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9–96.1%) and 67.4% sensitive (95% CI: 51.5–80.9%) for E gene and RdRp gene, respectively. Conclusion Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

Culture-Independent Detection of Poliovirus in Stool Samples by Direct RNA Extraction

The GPLN is a global surveillance system composed of 146 laboratories in 92 countries, in each of the six World Health Organization regions. Laboratory surveillance for PV relies on virus isolation by cell culture to identify PV in stool samples from AFP cases.