Usage of FTA (R) Classic Cards for Safe Storage, Shipment, and Detection of Arboviruses

Infections caused by arthropod-borne RNA viruses are overrepresented among emerging infectious diseases. Effective methods for collecting, storing, and transporting clinical or biological specimens are needed worldwide for disease surveillance. However, many tropical regions where these diseases are endemic lack analytical facilities and possibility of continuous cold chains, which presents challenges from both a biosafety and material preservation perspective. Whatman (R) FTA (R) Classic Cards may serve as an effective and safe option for transporting hazardous samples at room temperature, particularly for RNA viruses classified as biosafety level (BSL) 2 and 3 pathogens, from sampling sites to laboratories. In this study, we investigated the biosafety and perseverance of representative alpha-and flaviviruses stored on FTA (R) cards. To evaluate the virus inactivation capacity of FTA (R) cards, we used Sindbis virus (SINV), chikungunya virus (CHIKV), and Japanese encephalitis virus (JEV). We inoculated susceptible cells with dilution series of eluates from viral samples stored on the FTA (R) cards and observed for cytopathic effect to evaluate the ability of the cards to inactivate viruses. All tested viruses were inactivated after storage on FTA (R) cards. In addition, we quantified viral RNA of JEV, SINV, and tick-borne encephalitis virus (TBEV) stored on FTA (R) cards at 4 degrees C, 25 degrees C, and 37 degrees C for 30 days using two reverse transcriptase quantitative PCR assays. Viral RNA of SINV stored on FTA (R) cards was not reduced at either 4 degrees C or 25 degrees C over a 30-day period, but degraded rapidly at 37 degrees C. For JEV and TBEV, degradation was observed at all temperatures, with the most rapid degradation occurring at 37 degrees C. Therefore, the use of FTA (R) cards provides a safe and effective workflow for the collection, storage, and analysis of BSL 2- and 3-virus RNA samples, but there is a risk of false negative results if the cards are stored at higher temperatures for long periods of time. Conscious usage of the cards can be useful in disease surveillance and research, especially in tropical areas where transportation and cold chains are problematic

A Mosquito's Guide to Viral Emergence : Unveiling Mosquito-Borne Virus Transmission Dynamics, Vector Competence, and Genetic Evolution

In the complex world of disease transmission, mosquitoes often take centre stage as unwitting actors in the spread of viruses that threaten human and animal health. Imagine this: a tiny mosquito, often overlooked in the grand scheme of nature, carrying within it the potential to cause widespread disease, earning it the title of the world's deadliest animal. This doctoral thesis explores the captivating realm of mosquito-borne viruses, shedding light on their emergence, transmission dynamics, and evolutionary development.Arboviruses such as Japanese encephalitis virus (JEV), Usutu virus (USUV) and chikungunya virus (CHIKV) pose a significant global health problem given their increasing spread, particularly in growing urban areas, and amid today’s climate variations and change. It is important to understand the transmission dynamics of these viruses. In particular, USUV, which is transmitted by Culex mosquitoes, is increasingly being reported in Europe. This warrants an understanding of the vector competence of different European mosquito species to predict and manage potential outbreaks. Similarly, JEV is widespread in Asia and, given the climatic similarities, warrants an investigation into the vector competence of European mosquito species. CHIKV with its different lineages emphasises the need to understand its evolutionary history in order to predict and contain future outbreaks.This work has three objectives. To determine the abundance of JEV vector mosquitoes in Hanoi, Vietnam, to assess the vector competence of Swedish mosquitoes for USUV and JEV, and to analyse the factors that contributed to the 2018 CHIKV outbreak in Thailand. Field collections in Vietnam analysed Culex populations and correlated seasonal abundance with disease incidence. Vector competence experiments revealed USUV transmission potential and emphasised the inability of JEV transmission by Swedish Cx. pipiens. A genomic analysis of CHIKV traced its evolutionary development and provided insight into the dynamics of the 2018 outbreak.The historical context of mosquito-borne viruses emphasises their evolutionary origins and highlights the need for a comprehensive understanding. The results of the thesis contribute to insights into vector abundance dynamics, vector competence, and viral evolution, all critical aspects for disease surveillance and control. Future research approaches include assessing the competence of additional mosquito species, investigating environmental influences on transmission and exploring the immune responses of mosquitoes.Continuous surveillance, molecular epidemiology and innovative methods of vector control are crucial for containing the spread of viruses. Interdisciplinary collaboration and a "One Health" approach are essential for comprehensive disease prevention and control strategies. By improving our understanding of the dynamics of mosquito-borne viruses, we can better protect public health in the face of emerging infectious diseases

A Mosquito's Guide to Viral Emergence : Unveiling Mosquito-Borne Virus Transmission Dynamics, Vector Competence, and Genetic Evolution

In the complex world of disease transmission, mosquitoes often take centre stage as unwitting actors in the spread of viruses that threaten human and animal health. Imagine this: a tiny mosquito, often overlooked in the grand scheme of nature, carrying within it the potential to cause widespread disease, earning it the title of the world's deadliest animal. This doctoral thesis explores the captivating realm of mosquito-borne viruses, shedding light on their emergence, transmission dynamics, and evolutionary development.Arboviruses such as Japanese encephalitis virus (JEV), Usutu virus (USUV) and chikungunya virus (CHIKV) pose a significant global health problem given their increasing spread, particularly in growing urban areas, and amid today’s climate variations and change. It is important to understand the transmission dynamics of these viruses. In particular, USUV, which is transmitted by Culex mosquitoes, is increasingly being reported in Europe. This warrants an understanding of the vector competence of different European mosquito species to predict and manage potential outbreaks. Similarly, JEV is widespread in Asia and, given the climatic similarities, warrants an investigation into the vector competence of European mosquito species. CHIKV with its different lineages emphasises the need to understand its evolutionary history in order to predict and contain future outbreaks.This work has three objectives. To determine the abundance of JEV vector mosquitoes in Hanoi, Vietnam, to assess the vector competence of Swedish mosquitoes for USUV and JEV, and to analyse the factors that contributed to the 2018 CHIKV outbreak in Thailand. Field collections in Vietnam analysed Culex populations and correlated seasonal abundance with disease incidence. Vector competence experiments revealed USUV transmission potential and emphasised the inability of JEV transmission by Swedish Cx. pipiens. A genomic analysis of CHIKV traced its evolutionary development and provided insight into the dynamics of the 2018 outbreak.The historical context of mosquito-borne viruses emphasises their evolutionary origins and highlights the need for a comprehensive understanding. The results of the thesis contribute to insights into vector abundance dynamics, vector competence, and viral evolution, all critical aspects for disease surveillance and control. Future research approaches include assessing the competence of additional mosquito species, investigating environmental influences on transmission and exploring the immune responses of mosquitoes.Continuous surveillance, molecular epidemiology and innovative methods of vector control are crucial for containing the spread of viruses. Interdisciplinary collaboration and a "One Health" approach are essential for comprehensive disease prevention and control strategies. By improving our understanding of the dynamics of mosquito-borne viruses, we can better protect public health in the face of emerging infectious diseases

Vector competence of Swedish Culex pipiens mosquitoes for Usutu virus

Usutu virus (USUV) is an emerging mosquito-borne flavivirus with increasing prevalence in Europe. Understanding the role of mosquito species in USUV transmission is crucial for predicting and controlling potential outbreaks. This study aimed to assess the vector competence of Swedish Culex pipiens for USUV. The mosquitoes were orally infected with an Italian strain of USUV (Bologna 2009) and infection rates (IR), dissemination rates (DR), and transmission rates (TR) were evaluated over 7 to 28 days post-infection. The study revealed that Swedish Cx. pipiens are susceptible to USUV infection, with a gradual decrease in IR over time. However, the percentage of mosquitoes with the ability to transmit the virus remained consistent across all time points, indicating a relatively short extrinsic incubation period. Overall, this research highlights the potential of Swedish Cx. pipiens as vectors for USUV and emphasizes the importance of surveillance and monitoring to prevent future outbreaks of mosquito-borne diseases

SARS-CoV-2 in hospital indoor environments is predominantly non-infectious

Background The ongoing SARS-CoV-2 pandemic has spread rapidly worldwide and disease prevention is more important than ever. In the absence of a vaccine, knowledge of the transmission routes and risk areas of infection remain the most important existing tools to prevent further spread. Methods Here we investigated the presence of the SARS-CoV-2 virus in the hospital environment at the Uppsala University Hospital Infectious Disease ward by RT-qPCR and determined the infectivity of the detected virus in vitro on Vero E6 cells. Results SARS-CoV-2 RNA was detected in several areas, although attempts to infect Vero E6 cells with positive samples were unsuccessful. However, RNase A treatment of positive samples prior to RNA extraction did not degrade viral RNA, indicating the presence of SARS-CoV-2 nucleocapsids or complete virus particles protecting the RNA as opposed to free viral RNA. Conclusion Our results show that even in places where a moderate concentration (Ct values between 30 and 38) of SARS-CoV-2 RNA was found; no infectious virus could be detected. This suggests that the SARS-CoV-2 virus in the hospital environment subsides in two states; as infectious and as non-infectious. Future work should investigate the reasons for the non-infectivity of SARS-CoV-2 virions

Mitigation of the replication of SARS-CoV-2 by nitric oxide in vitro

The ongoing SARS-CoV-2 pandemic is a global public health emergency posing a high burden on nations' health care systems and economies. Despite the great effort put in the development of vaccines and specific treatments, no prophylaxis or effective therapeutics are currently available. Nitric oxide (NO) is a broad-spectrum antimicrobial and a potent vasodilator that has proved to be effective in reducing SARS-CoV replication and hypoxia in patients with severe acute respiratory syndrome. Given the potential of NO as treatment for SARS-CoV-2 infection, we have evaluated the in vitro antiviral effect of NO on SARS-CoV-2 replication. The NO-donor S-nitroso-N-acetylpenicillamine (SNAP) had a dose dependent inhibitory effect on SARS-CoV-2 replication, while the non S-nitrosated NAP was not active, as expected. Although the viral replication was not completely abolished (at 200 μM and 400 μM), SNAP delayed or completely prevented the development of viral cytopathic effect in treated cells, and the observed protective effect correlated with the level of inhibition of the viral replication. The capacity of the NO released from SNAP to covalently bind and inhibit SARS-CoV-2 3CL recombinant protease in vitro was also tested. The observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosation of the enzyme active site cysteine

Long-distance airborne dispersal of SARS-CoV-2 in COVID-19 wards

Evidence suggests that SARS-CoV-2, as well as other coronaviruses, can be dispersed and potentially transmitted by aerosols directly or via ventilation systems. We therefore investigated ventilation openings in one COVID-19 ward and central ducts that expel indoor air from three COVID-19 wards at Uppsala University Hospital, Sweden, during April and May 2020. Swab samples were taken from individual ceiling ventilation openings and surfaces in central ducts. Samples were subsequently subjected to rRT-PCR targeting the N and E genes of SARS-CoV-2. Central ventilation HEPA filters, located several stories above the wards, were removed and portions analyzed in the same manner. In two subsequent samplings, SARS-CoV-2 N and E genes were detected in seven and four out of 19 room vents, respectively. Central ventilation HEPA exhaust filters from the ward were found positive for both genes in three samples. Corresponding filters from two other, adjacent COVID-19 wards were also found positive. Infective ability of the samples was assessed by inoculation of susceptible cell cultures but could not be determined in these experiments. Detection of SARS-CoV-2 in central ventilation systems, distant from patient areas, indicate that virus can be transported long distances and that droplet transmission alone cannot reasonably explain this, especially considering the relatively low air change rates in these wards. Airborne transmission of SARS-CoV-2 must be taken into consideration for preventive measures

Evaluation of a COVID-19 IgM and IgG rapid test; an efficient tool for 4 assessment of past exposure to SARS-CoV-2

COVID-19 is the most rapidly growing pandemic in modern time, and the need for 21 serological testing is most urgent. Although the diagnostics of acute patients by RT-PCR is 22 both efficient and specific, we are also crucially in need of serological tools for investigating 23 antibody responses and assessing individual and potential herd immunity. We evaluated a 24 commercially available test developed for rapid (within 15 minutes) detection of SARS-CoV-25 2-specific IgM and IgG by 29 PCR-confirmed COVID-19 cases and 124 negative controls. 26 The results revealed a sensitivity of 69.0 % and 93.1 % for IgM and IgG, respectively, based 27 solely on PCR-positivity due to the absence of a serological gold standard. The assay 28 specificities were shown to be 100 % for IgM and 99.2 % for IgG. This indicates that the test 29 is suitable for assessing previous virus exposure, although negative results may be unreliable 30 during the first weeks after infection. More detailed studies on antibody responses during and 31 post infection are urgently needed

Corrigendum to “Presymptomatic viral shedding and infective ability of SARS-CoV-2; a case report” [Heliyon 7, (2), (February 2021), Article e06328]

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The evolutionary and molecular history of a chikungunya virus outbreak lineage

In 2018–2019, Thailand experienced a nationwide spread of chikungunya virus (CHIKV), with approximately 15,000 confirmed cases of disease reported. Here, we investigated the evolutionary and molecular history of the East/Central/South African (ECSA) genotype to determine the origins of the 2018–2019 CHIKV outbreak in Thailand. This was done using newly sequenced clinical samples from travellers returning to Sweden from Thailand in late 2018 and early 2019 and previously published genome sequences. Our phylogeographic analysis showed that before the outbreak in Thailand, the Indian Ocean lineage (IOL) found within the ESCA, had evolved and circulated in East Africa, South Asia, and Southeast Asia for about 15 years. In the first half of 2017, an introduction occurred into Thailand from another South Asian country, most likely Bangladesh, which subsequently developed into a large outbreak in Thailand with export to neighbouring countries. Based on comparative phylogenetic analyses of the complete CHIKV genome and protein modelling, we identified several mutations in the E1/E2 spike complex, such as E1 K211E and E2 V264A, which are highly relevant as they may lead to changes in vector competence, transmission efficiency and pathogenicity of the virus. A number of mutations (E2 G205S, Nsp3 D372E, Nsp2 V793A), that emerged shortly before the outbreak of the virus in Thailand in 2018 may have altered antibody binding and recognition due to their position. This study not only improves our understanding of the factors contributing to the epidemic in Southeast Asia, but also has implications for the development of effective response strategies and the potential development of new vaccines