Temporal study of wastewater surveillance from September 2020 to March 2021: an estimation of COVID-19 patients in Lahore, Pakistan.

The first aim of study was to quantify the viral load in the wastewater samples by RT-qPCR testing in Lahore population to estimate the number of patients affected and predict the next resurgence of COVID-19 wave in the city. The second aim of the study was to determine the hotspot areas of Lahore which remained positive more often for virus with high viral load. In this study, n = 420 sewage samples were collected on an average of two weeks intervals from 30 different sewage water disposal stations (14 sampling events) from Sept 2020 to March 2021. RNA was extracted and quantified by RT-qPCR without concentrating the virus in samples. Number of positive disposal sites (7-93%), viral load from sewage samples (100.296 to 103.034), and estimated patients (660-17,030) ranged from low to high according to the surge and restrain of 2nd and 3rd COVID-19 waves in the country. The viral load and estimated patients were reported high in January 2021 and March 2021 which were similar to the peak of 2nd and 3rd waves in Pakistan. Site 18 (Niaz Baig village DS) showed the highest viral load among all sites. Findings of the present study helped to estimate the number of patients and track the resurgence in COVID-19 waves in Lahore particularly, and in Punjab generally. Furthermore, it emphasizes the role of wastewater-based epidemiology to help policymakers strengthen the quarantine measures along with immunization to overcome enteric viral diseases. Local and national stake holders should work in collaboration to improve the environmental hygiene to control the disease.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

Renessans Helps in Early Clearance of SARS-CoV-2: In-Vivo Activity of the Iodine Complex in Rhesus macaque.

Iodine complexes have known antimicrobial properties along with reported in-vitro antiviral activity for several viruses. Renessans is one such product with iodine complexes and ascorbic acid. The present study was designed to determine its efficacy for SARS-CoV-2 in Rhesus macaque. Rhesus macaque were assigned to: A) prophylactic group (n = 3), (B) treatment group (n = 3), (C) infection control group (n = 4), and (D) negative control group (n = 4). Groups A, B, and C were challenged with 2 × 106 TCID of SARS-CoV-2. The prophylactic group (A) was administered Renessans from 5 days before infection till 8 days postinfection (DPI). The treatment group (B) was administered Renessans from 3 till 8 DPI. Group C was administered water-insoluble fractions only. Nasal swabs from all monkeys of groups A, B, and C remained positive for SARS-CoV-2 till 2 and 7 DPI, while the swabs became negative for groups A and B at 14 DPI. Likewise, fecal matter of monkeys in group A returned negative results during the experiment, while that of group B had significantly decreased viral load (101.5 genome copies/mL) compared to group C (103 genome copies/mL). Hence, it is concluded that Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2.

A Cross-Sectional Survey of Knowledge, Attitude, and Practices of University Students in Pakistan Regarding COVID-19.

The COVID-19 pandemic is striking the world with serious public health and socioeconomic complications. The pandemic has influenced all forms of daily life, including educational institutions. Therefore, this cross-sectional survey was conducted to understand knowledge, attitudes, and practices related to COVID-19 among the students of the University of Veterinary and Animal Sciences, Lahore. The data was collected using an online self-directed questionnaire. The survey form includes six items about sociodemographic characteristics, 14 knowledge-based questions, seven questions on attitude, and eight questions on practices. The sample number was calculated using the Raosoft sample size calculator. A total number of 3,854 students, including 1,823 men and 2,031 women, were engaged in this survey, having student representation from all the provinces in the country. The data were analyzed using a chi-square test. A total of 97% of the students knew that the etiological agent of COVID-19 is a virus and that it is a disease of the respiratory system (94%). Many students kept visiting their relatives during the lockdown (45%), and their relatives kept visiting them at home (59%). The responses from the students varied a lot on specific questions about the transmission of the virus. Women tended to have less information regarding precautionary travel measures (p < 0.01), but supplemental knowledge of prevention of disease transmission from positive patients (p < 0.01). Conclusively, the majority of the university students surveyed had imperative knowledge, a good attitude, and active practice in response to the COVID-19 outbreak. Moreover, the KAP scores have varied by demography, gender, and the number of family members. Therefore, continuous awareness of preventative behaviors should be disseminated regularly in emergencies.

An in vitro antiviral activity of iodine complexes against SARS-CoV-2.

Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID50. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 101.43 TCID50 and 101.58 TCID50 for the syrup and capsule, respectively, as compare to virus infected control cells 106.08 TCID50 and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

A comparative phylogenomic analysis of SARS-CoV-2 strains reported from non-human mammalian species and environmental samples.

Coronaviruses (CoVs) infect a wide range of domestic and wild mammals. These viruses have a potential and tendency to cross-species barriers and infect humans. Novel human coronavirus 2019-nCoV (hCoV-19) emerged from Wuhan, China, and has caused a global pandemic. Genomic features of SARS-CoV-2 may attribute inter-species transmission and adaptation to a novel host, and therefore is imperative to explicate the evolutionary dynamics of the viral genome and its propensity for differential host selection. We conducted an in silico analysis of all the coding gene sequences of SARS-CoV-2 strains (n = 39) originating from a range of non-human mammalian species, including pangolin, bat, dog, cat, tiger, mink, mouse, and the environmental samples such as wastewater, air and surface samples from the door handle and seafood market. Compared to the reference SARS-CoV-2 strain (MN908947; Wuhan-Hu-1), phylogenetic and comparative residue analysis revealed the circulation of three variants, including hCoV-19 virus from humans and two hCoV-19-related precursors from bats and pangolins. A lack of obvious differences as well as a maximum genetic homology among dog-, cat-, tiger-, mink-, mouse-, bat- and pangolin-derived SARS-CoV-2 sequences suggested a likely evolution of these strains from a common ancestor. Several residue substitutions were observed in the receptor-binding domain (RBD) of the spike protein, concluding a promiscuous nature of the virus for host species where genomic alternations may be required for the adaptation to novel host/s. However, such speculation needs in vitro investigations to unleash the influence of substitutions towards species-jump and disease pathogenesis.