Clinical Validation of an Alternative Specimen Collection Kit for SARS-CoV-2 Testing at Fox Chase Cancer Center.

Background: Supply chain disruptions during the COVID-19 pandemic have affected the availability of components for specimen collection kits to detect SARS-CoV-2. Plastic injection molding offers a rapid and cheap method for mass production of swabs for upper respiratory tract sampling. Local production of virus transport medium increases flexibility to assemble sample collection kits if the medium provides appropriate stability for SARS-CoV-2 detection. Methods: A locally produced virus transport medium and a novel injection molded plastic swab were validated for SARS-CoV-2 detection by reverse-transcription quantitative polymerase chain reaction. Both components were compared to standard counterparts using viral reference material and representative patient samples. Results: Clinical testing showed no significant differences between molded and flocked swabs. Commercial and in-house virus transport media provided stable test results for over 40 days of specimen storage and showed no differences in test results using patient samples. Conclusions: This collection kit provides new supply chain options for SARS-CoV-2 testing.

Specific Bacterial Cell Wall Components Influence the Stability of Coxsackievirus B3.

Enteric viruses infect the mammalian gastrointestinal tract and lead to significant morbidity and mortality worldwide. Data indicate that enteric viruses can utilize intestinal bacteria to promote viral replication and pathogenesis. However, the precise interactions between enteric viruses and bacteria are unknown. Here, we examined the interaction between bacteria and coxsackievirus B3, an enteric virus from the picornavirus family. We found that bacteria enhance the infectivity of coxsackievirus B3 (CVB3) in vitro. Notably, specific bacteria are required, as Gram-negative Salmonella enterica, but not Escherichia coli, enhanced CVB3 infectivity and stability. Investigating the cell wall components of both S. enterica and E. coli revealed that structures in the O-antigen or core of lipopolysaccharide, a major component of the Gram-negative bacterial cell wall, were required for S. enterica to enhance CVB3. To determine if these requirements were necessary for similar enteric viruses, we investigated if S. enterica and E. coli enhanced infectivity of poliovirus, another enteric virus in the picornavirus family. We found that while E. coli did not enhance the infectivity of CVB3, E. coli enhanced poliovirus infectivity. Overall, these data indicate that distinct bacteria enhance CVB3 infectivity and stability, and specific enteric viruses may have differing requirements for their interactions with specific bacterial species. IMPORTANCE Previous data indicate that several enteric viruses utilize bacteria to promote intestinal infection and viral stability. Here, we show that specific bacteria and bacterial cell wall components are required to enhance infectivity and stability of coxsackievirus B3 in vitro. These requirements are likely enteric virus specific, as the bacteria for CVB3 differ from poliovirus, a closely related virus. Therefore, these data indicate that specific bacteria and their cell wall components dictate the interaction with various enteric viruses in distinct mechanisms.

Real-time PCR assay as a simple and efficient tool for viral stability study.

Aim: For oncolytic virus trials, regulatory agencies often require pharmaceutical industry to evaluate risks of released viruses from patients to environment. This study was to establish a real-time PCR method to assess viral shedding and viral stability in human urine. Results/methodology: Herein, we describe an incubation of viral drug product in human urine and use of real-time PCR as a simple, efficient and high throughput assay to assess the level and stability of a nonenveloped and single stranded RNA virus. The viral stability issue is critical to the collection, transport, storage and testing of clinical samples. Discussion/conclusion: In summary, this simple method provides useful viral stability information at various temperatures and detergents. A similar approach may apply to other RNA viruses (including SARS-CoV-2).

Comparative characterization of the reassortant Orthobunyavirus Ngari with putative parental viruses, Bunyamwera and Batai: in vitro characterization and ex vivo stability.

Bunyamwera (BUNV), Batai (BATV) and Ngari (NRIV) are mosquito-borne viruses that are members of the genus Orthobunyavirus in the order Bunyavirales. These three viruses are enveloped with single-stranded, negative-sense RNA genomes consiting of three segments, denoted as Small (S), Medium (M) and Large (L). Ngari is thought to be the natural reassortant progeny of Bunyamwera and Batai viruses. The relationship between these 'parental' viruses and the 'progeny' poses an interesting question, especially given that there is overlap in their respective transmission ecologies, but differences in their infection host ranges and pathogenesis. We compared the in vivo kinetics of these three viruses in a common laboratory system and found no significant difference in growth kinetics. There was, however, a tendency of BATV to have smaller plaques than either BUNV or NRIV. Furthermore, we determined that all three viruses are stable in extracellular conditions and retain infectivity for a week in non-cellular media, which has public health and biosafety implications. The study of this understudied group of viruses addresses a need for basic characterization of viruses that have not yet reached epidemic transmission intensity, but that have the potential due to their infectivity to both human and animal hosts. These results lay the groundwork for future studies of these neglected viruses of potential public and One Health importance.

SARS-CoV-2 infection: the environmental endurance of the virus can be influenced by the increase of temperature.

OBJECTIVES: To evaluate whether the increase of temperature can influence the environmental endurance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Virus was inoculated on a plastic surface and harvested at predefined time-points in parallel at 20°C-25°C (room temperature; RT) and at 28°C (June temperature; JT). Samples were tested by TCID50 titres on Vero cells. RESULTS: Our results confirm that fomite transmission of the emerging SARS-CoV-2 is possible: the virus reserved its ability to infect cells for up to 84 hours at both RT and JT on a plastic surface, with TCID50 viral titres of 0.67 and 0.25 log10, respectively. At RT, an important reduction in the viral titre, from 4 log10 to 3 log10 TCID50, was observed during the first 24-36 hours. At JT, the same decay was observed more rapidly (between 8 and 12 hours), The rate of viral inactivation by D-value was 24.74 hours at RT and 12.21 hours at JT. CONCLUSIONS: This remarkable difference between the two temperatures suggests that virus vitality can be influenced by the environmental temperature and that the hot season could reduce the probability of COVID-19 transmission.

Viral uptake and stability in Crassostrea gigas oysters during depuration, storage and steaming.

More stable than bacteria in environmental samples, enteric viruses are generally related to outbreaks of gastroenteritis caused by the consumption of contaminated oysters. This study evaluated: i) the dynamic processes of enteric viral models bioaccumulation by Crassostrea gigas oysters artificially contaminated; ii) the stability of these viruses in oysters in controlled temperature conditions and iii) the effect of UV light in inactivating these viruses in depurated oysters. Plaque assay (PA) was used to assess the infectivity of both viral models. Cell culture coupled with RT-qPCR (ICC-RT-qPCR) was used to measure infectious adenovirus type 2 (HAdV-2) genomes and qPCR to measure genome copies of murine norovirus (MNV-1). The virus uptake through bioaccumulation behave differently: HAdV-2 reached its peak of uptake faster than MNV-1. Both viruses showed high stability in oysters when maintained under 4 °C, but were completely inactivated in steamed oysters. The HAdV-2 was completely inactivated after 12 h of depuration with UV light and after 24 h without UV light. After 72 h of depuration, MNV-1 was still detected in both tanks, probably due to the stronger interaction of this virus with the oyster's tissues. This study demonstrated the importance of a secure depuration time in ensuring a clean and safe product, and that the steaming process is the safest way to prepare oysters for consumption.

Influence of Tattoo Ink on Hepatitis C Virus Infectiousness.

Hepatitis C virus (HCV) is a blood-borne virus and is most frequently transmitted through large or repeated direct percutaneous exposures to infected blood. The 2 most common exposures associated with transmission of HCV are blood transfusion and intravenous drug abuse. The association between HCV transmission and other suspected risk factors such as tattooing is more controversial. Although HCV can survive for days to weeks in suspension or on inanimate surfaces, its stability in tattooing supplies remains elusive. Here, we analyzed the influence of tattoo ink on HCV infectiousness.

Interaction of human adenoviruses and coliphages with kaolinite and bentonite.

Human adenoviruses (hAdVs) are pathogenic viruses responsible for public health problems worldwide. They have also been used as viral indicators in environmental systems. Coliphages (e.g., MS2, ΦX174) have also been studied as indicators of viral pollution in fecally contaminated water. Our objective was to evaluate the distribution of three viral fecal indicators (hAdVs, MS2, and ΦΧ174), between two different phyllosilicate clays (kaolinite and bentonite) and the aqueous phase. A series of static and dynamic experiments were conducted under two different temperatures (4, 25°C) for a time period of seven days. HAdV adsorption was examined in DNase I reaction buffer (pH=7.6, and ionic strength (IS)=1.4mM), whereas coliphage adsorption in phosphate buffered saline solution (pH=7, IS=2mM). Moreover, the effect of IS on hAdV adsorption under static conditions was evaluated. The adsorption of hAdV was assessed by real-time PCR and its infectivity was tested by cultivation methods. The coliphages MS2 and ΦΧ174 were assayed by the double-layer overlay method. The experimental results have shown that coliphage adsorption onto both kaolinite and bentonite was higher for the dynamic than the static experiments; whereas hAdV adsorption was lower under dynamic conditions. The adsorption of hAdV increased with decreasing temperature, contrary to the results obtained for the coliphages. This study examines the combined effect of temperature, agitation, clay type, and IS on hAdV adsorption onto clays. The results provide useful new information on the effective removal of viral fecal indicators (MS2, ΦX174 and hAdV) from dilute aqueous solutions by adsorption onto kaolinite and bentonite. Factors enabling enteric viruses to penetrate soils, groundwater and travel long distances within aquifers are important public health issues. Because the observed adsorption behavior of surrogate coliphages MS2 and ΦΧ174 is substantially different to that of hAdV, neither MS2 nor ΦΧ174 is recommended as a suitable model for adenovirus.

Survival of influenza virus on human fingers.

Indirect transmission of the influenza virus via finger contamination with respiratory mucus droplets has been hypothesized to contribute to transmission in the community. Under laboratory conditions, influenza-infected respiratory droplets were reconstituted as close as possible to natural conditions. We investigated experimentally the survival of influenza A (H3N2) and A (H1N1)pdm09 viruses on human fingers. Infectious virus was easily recoverable on all fingers 1 min after fingertip contamination but then decreased very rapidly. After 30 min, infectious virus was detectable in only a small minority of subjects. Infectious viruses were detected for a longer period of time when droplets of larger size containing a higher number of particles were tested or when the viral concentration increased. A rapid decrease in infectiousness was observed when droplet integrity was disrupted. Our findings could help to set up the promotion of hand hygiene to prevent influenza hand contamination.