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Viral metagenomic analysis was used to identify a previously uncharacterized parvovirus species, “HBoV2,” whose closest phylogenetic relative is the human bocavirus (HBoV). HBoV2 has a genomic organization identical to that of HBoV but has only 78%, 67%, and 80% identity, respectively, with the latter's NS1, NP1, and VP1/VP2 proteins. The study used polymerase chain reaction to detect HBoV2 sequences in 5 of 98 stool samples from Pakistani children and in 3 of 699 stool samples from Edinburgh. Nearly-full-length genome sequencing revealed the presence of 3 HBoV2 genotypes and evidence of recombination between genotypes. Further studies are necessary to identify anatomical sites of HBoV2 replication and potential associations with clinical symptoms or disease.

Enteroviruses are common human pathogens occasionally associated with severe disease, notoriously paralytic poliomyelitis caused by poliovirus. Other enterovirus serotypes such as enterovirus A71 and D68 have been linked to severe neurological syndromes. New enterovirus serotypes continue to emerge, some believed to be derived from nonhuman primates. However, little is known about the circulation patterns of many enterovirus serotypes and, in particular, the detailed enterovirus composition of sewage samples. We used a next-generation sequencing approach analyzing reverse transcriptase polymerase chain reaction products synthesized directly from sewage concentrates. We determined whole-capsid genome sequences of multiple enterovirus strains from all 4 A to D species present in environmental samples from the United Kingdom, Senegal, and Pakistan. Our results indicate complex enterovirus circulation patterns in human populations with differences in serotype composition between samples and evidence of sustained and widespread circulation of many enterovirus serotypes. Our analyses revealed known and divergent enterovirus strains, some of public health relevance and genetically linked to clinical isolates. Enteroviruses identified in sewage included vaccine-derived poliovirus and enterovirus D-68 stains, new enterovirus A71 and coxsackievirus A16 genogroups indigenous to Pakistan, and many strains from rarely reported serotypes. We show how this approach can be used for the early detection of emerging pathogens and to improve our understanding of enterovirus circulation in humans.

What is already known about this topic? Pakistan is one of two countries (including Afghanistan) where wild poliovirus type 1 (WPV1) transmission has never been interrupted. What is added by this report? WPV1 cases in Pakistan decreased from 147 in 2019 and 84 in 2020 to a single case in 2021 but increased to 14 cases in 2022 as of July 31. These 14 WPV1 cases are clustered among children in southern Khyber Pakhtunkhwa province, many of whom have never received poliovirus vaccine (zero-dose children). What are the implications for public health practice? Ensuring the highest quality vaccination activities in priority areas of Pakistan will enable the polio program to improve the chances of interrupting ongoing transmission of WPV1.

Group A rotavirus (RVA) associated gastroenteritis is a major cause of infantile morbidity and mortality, globally. Pakistan had the highest rates of gastroenteritis among kids, every year. Our study aimed to assess the RVA disease burden and circulating genotypes in Rawalpindi, before the vaccine’s introduction in Pakistan. Stool samples were collected from children < 5 years of age admitted at Benazir Bhutto Hospital, Rawalpindi, from November 2014 to May 2015. Of the 300 stool samples, 47% of children were found positive for RVA antigen on ELISA, with the highest prevalence (52%) in infants less than 7 months of age. Rotavirus positive cases through real-time PCR were 65.5%. Fever and diarrhea were significantly related to RVA infection when compared to RVA-negative cases ( P  = 0.02). It is the first report on an upsurge of G12P[6] (17.24%) along with the rise of previously declining G3 in the current epidemiological area. The other prevalent types were G1P[8] (12.07%), G1P[4] (6.90%), G1P[6] (5.17%), G3P[6] (5.17%), followed by G2P[6], G3P[4], G9P[4], and G12P[8] each found with a prevalence of (3.45%). This study reports G3P[4], G3P[6] and G12P[4] for the first time in Pakistan. Mixed genotype infections were found in 21% of cases. G12P[6], which was the predominant genotype in this study, was found to be significantly associated with fever ( P  = 0.03). This study provides valuable data on the significantly highest prevalence of RVA-associated gastroenteritis in kids of Rawalpindi, Pakistan, and elucidates the vast diversity of circulating RVA genotypes. The reported disease burden, genotypes, and clinical symptoms would enable public health dealers to cope with the severity of the disease. It also provides an evolutionary trend of changing genotypes in the country.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

On Dec 22, 2016, NIH, Pakistan's reference public health laboratory serving as WHO's Collaborating Center for Research and Training in Viral Diagnostics, confirmed the first three cases of chikungunya virus infection by use of the Trioplex real-time PCR assay, which simultaneously tests for dengue and Zika viruses as well.3 This assay is approved for in-vitro diagnostic use under the US Food and Drug Administration (FDA) emergency-use authorisation only and its use is limited to qualified...

Despite concerted global vaccination efforts, wild poliovirus remains endemic in two countries in 2024, Pakistan and Afghanistan. This study uses phylogeographic analysis of poliovirus genetic and epidemiological data from clinical and wastewater surveillance to identify the causes of poliovirus persistence and routes of spread over the last decade (2012 to 2023). Poliovirus genetic diversity declined after 2020, with one of two major genetic clusters dying out, and recent detections are now closely related genetically. High-risk and hard-to-access regions have sustained polio transmission over the past decade, even when interrupted elsewhere. Karachi, one of the most densely populated cities globally, has acted as a hub for the amplification and spread of poliovirus to other regions, many of which we show to be dead-end for onwards transmission despite frequent virus detection. Phylogenetic analysis has long been central to the polio surveillance network, and advancing the approaches used can provide critical epidemiological insights to accelerate eradication efforts.

During December 2016-May 2017, an outbreak of chikungunya virus infection occurred across Pakistan. The East/Central/South African genotype was predominant. This study provides baseline data on the virus strain and emphasizes the need for active surveillance and implementation of preventive interventions to contain future outbreaks.

Being an endemic country for Crimean Congo hemorrhagic fever (CCHF), this study aimed to explore the genetic diversity of CCHF virus (CCHFV) detected in Pakistan during 2019. Serum samples from patients with clinical signs of hemorrhagic fever attending tertiary care hospitals in Pakistan were tested for CCHFV RNA using real-time PCR at Department of Virology, National Institute of Health. The partial S-gene fragments were directly sequenced to determine the prevailing CCHFV genotypes and their molecular epidemiology in Pakistan. During January-December 2019, 280 samples from suspected CCHF patients were tested and 28 (10%) were found positive on real-time PCR. Positive cases were detected from 14 districts and across all four provinces of Pakistan with majority reported during August-September. The mean age of CCHFV positive patients was 37.25 years (range 5-65 years) with a high frequency in males (92.8%; n = 26) and a case fatality rate of 40.7% was observed. Phylogenetic analysis showed that S- segment of 2019 PAK CCHFV strains (n = 13) belonged to Asia-1 genotype and clustered with regional strains from Iran, Oman, and Afghanistan. We conclude that Asia-1 genotype of CCHF virus remains endemic in Pakistan. Our findings emphasize to establish a laboratory based surveillance program to monitor the disease burden and identify outbreak hotspots for effective control.

Aim: The biosynthesis of chromium oxide nanoparticles (Cr2O3 NPs), using Hyphaene thebaica as a bioreductant, for assessment of their potential nanomedicinal applications. Materials & methods: Biosynthesized Cr2O3 NPs were characterized by x-ray diffraction, Fourier-transform infrared spectroscopy, energy dispersive x-ray spectroscopy, scanning and transmission electron microscopy, selected area electron diffraction, UV–Vis spectroscopy and ζ-potential measurement. In vitro assays were used to assess the biological properties of Cr2O3 NPs. Results: Nanoparticles with size approximately 25–38 nm were obtained with a characteristic Cr–O vibration at 417 cm-1. A broad spectrum antimicrobial potential and antioxidant nature is reported. Slight inhibition of polio virus and biocompatibility at low doses was observed. Conclusion: We conclude a multifunctional nature of biogenic Cr2O3 NPs.