Coronaviruses (CoVs) cause a broad spectrum of diseases in domestic and wild animals, poultry, and rodents, ranging from mild to severe enteric, respiratory, and systemic disease, and also cause the common cold or pneumonia in humans. Seven coronavirus species are known to cause human infection, 4 of which, HCoV 229E, HCoV NL63, HCoV HKU1 and HCoV OC43, typically cause cold symptoms in immunocompetent individuals. The others namely SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus) were zoonotic in origin and cause severe respiratory illness and fatalities. On 31 December 2019, the existence of patients with pneumonia of an unknown aetiology was reported to WHO by the national authorities in China. This virus was officially identified by the coronavirus study group as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the present outbreak of a coronavirus-associated acute respiratory disease was labelled coronavirus disease 19 . COVID-19's first cases were seen in Turkey on March 10, 2020 and was number 47,029 cases and 1006 deaths after 1 month. Infections with SARS-CoV-2 are now widespread, and as of 10 April 2020, 1,727,602 cases have been confirmed in more than 210 countries, with 105,728 deaths.

Following the announcement of SARS-CoV-2 worldwide pandemic spread by WHO on March 11, 2020, wastewater based epidemiology received great attention in several countries: The Netherlands [Medama et al., 2020; K-Lodder et al., 2020], USA [Wu et al., 2020; Memudryi et al., 2020], Australia [Ahmed et al., 2020], France [Wurtzer et al., 2020], China [Wang et al., 2020], Spain [Randazzo et al., 2020; Walter et al., 2020], Italy (La Rosa et al., 2020; Rimoldi et al., 2020) and Israel [Or et al., 2020], performed analysis in wastewaters by using different virus concentration techniques. Turkey took its place among these countries on 7th of May, 2020 by reporting SARS-CoV-2 RT-qPCR levels at the inlet of seven (7) major municipal wastewater treatment plants (WWTPs) of Istanbul [Alpaslan Kocamemi et al., 2020], which is a metropole with 15.5 million inhabitants and a very high population density (2987 persons/km2) and having about 65 % of Covid-19 cases in Turkey. Sludges that are produced in WWTPs should be expected to contain SARS-CoV-2 virus as well. There has not yet been any study for the fate of SAR-CoV-2 in sludges generated from WWTPs. Knowledge about the existing of SARS-CoV-2 in sludge may be useful for handling the sludge during its dewatering, stabilizing and disposal processes. This information will also be valuable in case of sludges that are used as soil conditioners in agriculture or sent to landfill disposal. In wastewater treatment plants, generally two different types of sludges are generated; primary sludge (PS) and waste activated sludge (WAS). PS forms during the settling of wastewater by gravity in the primary settling tanks. Little decomposition occurs during primary sludge formation. Since most of the inorganic part of the wastewater is removed in the earlier grit removal process, the PS consists of mainly organic material that settles. The PS is about 1-2 % solids by weight. In the biological treatment part of the WWTPs, the biomass that forms in the anaerobic, anoxic and oxic zones of the process is settled in final clarifiers by gravity and returned to the beginning of the biological process so that it is not washed off. The waste activated sludge (WAS) is the excess part of the biomass that grows in this secondary treatment process. It has to be removed from the process not to increase the mixed liquor suspended solids concentration (bacteria concentration) in the secondary process more than a fixed value. The WAS is about 0.6 - 0.9 % solids by weight. This work aims to find whether SARS-CoV-19 is present in the PS and WAS before it is dewatered and sent to anaerobic or aerobic digester processes or to thermal drying operations. For this purpose, on the 7th of May 2020, two (2) PS samples were collected from Ambarlı and Tuzla WWTPs, seven (7) WAS samples were collected from Terkos, Ambarlı, Atakoy I & II, Pasakoy II, Buyukcekmece and Tuzla I WWTPs. Polyethylene glycol 8000 (PEG 8000) adsorption [Wu et al., 2020] SARS-Cov-2 concentration method was used for SARS-CoV-2 concentration after optimization. [Alpaslan Kocamemi et al., 2020]. Real time RT-PCR diagnostic panel validated by US was used to quantify SARS-CoV-2 RNA in primary and waste activated sludge samples taken from WWTPs in Istanbul. All samples were tested positive. Titers of SARS-CoV-2 have been detected ranging copies between 1.17E4 to 4.02x104 per liter.

Background Vaccines that incorporate multiple SARS‐CoV‐2 antigens can further broaden the breadth of virus‐specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS‐CoV‐2 VLP vaccine that incorporates the four structural proteins of SARS‐CoV‐2. Methods VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable‐resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. Results Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS‐CoV‐2. Alum adsorbed, K3‐CpG ODN‐adjuvanted VLPs elicited high titer anti‐S, anti‐RBD, anti‐N IgG, triggered multifunctional Th1‐biased T‐cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. Conclusion These data suggest that VLPs expressing all four structural protein antigens of SARS‐CoV‐2 are immunogenic and can protect animals from developing COVID‐19 infection following vaccination.

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Since the announcement of the pandemic of Covid-19 by WHO on March 11, 2020, the countries have started to monitor surveillance of SARS-CoV-2 through medical tests. However, people with no and very light symptoms are usually not medically tested or never hospitalized and they are missed. In the study of Wu et al. [1], it was realized that the urine and faeces of all infected people contain SARS-CoV-2. After that, sewage, and sludge-based SARS-CoV-2 surveillance studies have gained significant importance around the world (Fig.1). SARS-CoV-2 was detected in wastewaters in The Netherlands [2,3,4], USA [1,5,6,7, 8, 9, 10], Australia [11], France [12, 13, 14], China [15], Spain [16,17,18,19,20], Italy [21, 22,23], Israel [24], Turkey[25], Germany[26] , Japan [27,28], India [29,30], Pakistan [31], Brazil [32,33], Chile [34], Denmark, France, Belgium[35], Equator [36] and Sweden [37] using different virus concentration techniques. Published data show that high concentrations of the SARS-CoV-2 RNA reaches to wastewater treatment plants (WWTPs). On 7th of May 2020, Turkey took its place among a few country which have been started wastewater based surveillance studies at the early stages of pandemic by reporting SARS-CoV-2 RT-qPCR levels of major WWTPs of Istanbul [25]. Turkey [38] first detected SARS-CoV-2 in both primary and waste activated sludges of Istanbul WWTPs. Later, USA [39] and Spain [40] were also studied on sludge samples. There are also studies evaluating the SARS-CoV-2 in WWTPs effluents [10,13,14, 28, 29,30, 34, 36]. This study aimed to scan distribution of Covid-19 through Turkey by SARS-CoV-2 measurements in influent, effluent and sludge samples of WWTPs. The influent, effluent and sludge samples were collected from main WWTPs located in 81 cities of Turkey through May 2020- July 2020. Among those 81 cities, Istanbul metropole with 15.5 million inhabitants was chosen as the pilot city since 65% of all cases in Turkey were present here. Hence, all treatment plants in Istanbul were scanned through the study. The viral activity tests were also conducted for the influent, effluent and sludge samples resulting high qPCR.