Molecular characterization of rotavirus indicates predominance of G9P[4] genotype among children with acute gastroenteritis: First report after vaccine introduction in Pakistan.

Globally, Group A rotavirus (RVA) is the leading cause of acute gastroenteritis in children under 5 years old, with Pakistan having the highest rates of RVA-related morbidity and mortality. The current study aims to determine the genetic diversity of rotavirus and evaluate the impact of Rotarix-vaccine introduction on disease epidemiology in Pakistan. A total of 4749 children, hospitalized with acute gastroenteritis between 2018 and 2020, were tested at four hospitals in Lahore and Karachi. Of the total, 19.3% (918/4749) cases were tested positive for RVA antigen, with the positivity rate varying annually (2018 = 22.7%, 2019 = 14.4%, 2020 = 20.9%). Among RVA-positive children, 66.3% were under 1 year of age. Genotyping of 662 enzyme-linked immuno sorbent assay-positive samples revealed the predominant genotype as G9P[4] (21.4%), followed by G1P[8] (18.9%), G3P[8] (11.4%), G12P[6] (8.7%), G2P[4] (5.7%), G2P[6] (4.8%), and 10.8% had mixed genotypes. Among vaccinated children, genotypes G9P[4] and G12P[6] were more frequently detected, whereas a decline in G2P[4] was observed. Phylogenetic analysis confirmed the continued circulation of indigenous genotypes detected earlier in the country except G9 and P[6] strains. Our findings highlight the predominance of G9P[4] genotype after the vaccine introduction thus emphasizing continual surveillance to monitor the disease burden, viral diversity, and their impact on control of rotavirus gastroenteritis in children.

Insights into measles virus: Serological surveillance and molecular characterization.

BACKGROUND: Measles has been a significant public health concern in Pakistan, especially in the Khyber Pakhtunkhwa (KPK) province, where sporadic and silent epidemics continue to challenge existing control measures. This study aimed to estimate the prevalence and investigate the molecular epidemiology of the measles virus (MeV) in KPK and explore the vaccination status among the suspected individuals. METHODS: A cross-sectional study was conducted between February and October 2021. A total of 336 suspected measles cases from the study population were analyzed for IgM antibodies using Enzyme-Linked Immunosorbent Assay (ELISA). Throat swabs were randomly collected from a subset of positive cases for molecular analysis. Phylogenetic analysis of MeV isolates was performed using the neighbor-joining method. The vaccination status of individuals was also recorded. RESULTS: Among the suspected participants, 61.0% (205/336) were ELISA positive for IgM antibodies, with a higher prevalence in males (64.17%) compared to females (57.04%). The majority of cases (36.0%) were observed in infants and toddlers, consistent with previous reports. The majority of IgM-positive cases (71.7%) had not received any dose of measles vaccine, highlighting gaps in vaccine coverage and the need for improved immunization programs. Genetic analysis revealed that all MeV isolates belonged to the B3 genotype, with minor genetic variations from previously reported variants in the region. CONCLUSION: This study provides valuable insights into the genetic epidemiology of the MeV in KPK, Pakistan. The high incidence of measles infection among unvaccinated individuals highlights the urgency of raising awareness about vaccine importance and strengthening routine immunization programs.

A hidden dilemma; post COVID-19 first detection of Varicella zoster M4 genotype from Pakistan.

OBJECTIVES: During the COVID-19 pandemic, the risk of childhood infectious diseases was increased. Post-COVID-19 escalation of chickenpox cases, becoming an emerging public health concern. Thus, the study was designed to compare chickenpox prevalence and Varicella zoster virus (VZV) genotypes circulating before, during, and post-COVID-19 in Pakistan. METHODS: A total of 267 lesion specimens collected from tertiary care hospitals, and chickenpox outbreaks from Pakistan were analysed by a two-amplicon approach with phylogenetic analysis. RESULTS: Among suspected cases, overall 178/267 were VZV positive. Majority (84.2 %; 150/178) cases were of post-COVID-19 pandemic time. Small outbreaks occurred soon after COVID-19 in Rawalpindi and Islamabad (Pakistan), 40 positive cases out of 178 cases were outbreak cases. There was first time detection of the M4 genotype, which was significantly associated with disease severity (p = 0.0006) and post-COVID-19 chickenpox outbreaks in 2021 (77.9 %; 46/59; p < 0.00001). However, in pre-COVID-19 only M2 genotype was detected. The M2 prevalence varied from 2019 (100 %; 19/19) to 2022 (3.2 %; 3/91). However, the most prevalent strain of 2022 belonged to the M1 genotype (64.8 %; 59/91). CONCLUSION: A significant rise in chickenpox cases detected soon after COVID-19 in Pakistan, and oscillation of different VZV genotypes with first time detection of M4 genotype is an alarming situation. This demands further detailed genotypic studies on transmission dynamics of a rare M4 with other genotypes to protect the local population and restrict spread in other regions.

Vaccinomics Approach for Multi-Epitope Vaccine Design against Group A Rotavirus Using VP4 and VP7 Proteins.

Rotavirus A is the most common cause of Acute Gastroenteritis globally among children <5 years of age. Due to a segmented genome, there is a high frequency of genetic reassortment and interspecies transmission which has resulted in the emergence of novel genotypes. There are concerns that monovalent (Rotarix: GlaxoSmithKline Biologicals, Rixensart, Belgium) and pentavalent (RotaTeq: MERCK & Co., Inc., Kenilworth, NJ, USA) vaccines may be less effective against non-vaccine strains, which clearly shows the demand for the design of a vaccine that is equally effective against all circulating genotypes. In the present study, a multivalent vaccine was designed from VP4 and VP7 proteins of RVA. Epitopes were screened for antigenicity, allergenicity, homology with humans and anti-inflammatory properties. The vaccine contains four B-cell, three CTL and three HTL epitopes joined via linkers and an N-terminal RGD motif adjuvant. The 3D structure was predicted and refined preceding its docking with integrin. Immune simulation displayed promising results both in Asia and worldwide. In the MD simulation, the RMSD value varied from 0.2 to 1.6 nm while the minimum integrin amino acid fluctuation (0.05-0.1 nm) was observed with its respective ligand. Codon optimization was performed with an adenovirus vector in a mammalian expression system. The population coverage analysis showed 99.0% and 98.47% in South Asia and worldwide, respectively. These computational findings show potential against all RVA genotypes; however, in-vitro/in-vivo screening is essential to devise a meticulous conclusion.

First report of coinfection and whole-genome sequencing of norovirus and sapovirus in an acute gastroenteritis patient from Pakistan.

Acute gastroenteritis is one of the most common diseases in infants and children in developing countries including Pakistan. In Pakistan, rotavirus (RVA) is known to contribute significantly to pediatric diarrheal illness, but the contribution of other viruses is still unclear. In the current study we have identified a case of mixed infection of norovirus (NoV) and sapovirus (SaV) in a 2-year-old child with acute gastroenteritis. The sample was initially processed for the detection of group A RVA through ELISA followed by NoV using RT-PCR assay. The sample tested positive for NoV RNA and was later subjected to whole-genome sequencing using meta-genome approach on Miseq (Illumina) platform. Sequencing results revealed GII.15 genotype of NoV that clustered with viruses from China and USA from 2017 to 2021. We also retrieved the complete genome of SaV (GI.1 genotype) from the same sample and phylogenetic analysis showed clustering with strains reported from Japan, South Korea, US, and Taiwan during 2012-2016. This is the first report from Pakistan that confirms coinfection of NoV and SaV and elucidates their whole genomes. We recommend initiation of NoV and SaV surveillance program to ascertain disease burden and explore genetic diversity, especially as RVA vaccines have been included in national immunization program.

Breakthrough cases of Omicron and Delta variants of SARS-CoV-2 during the fifth wave in Pakistan.

COVID-19 pandemic has severely affected Pakistan with 1,557,134 cases as of August 4, 2022. However, the data regarding breakthrough infections in Pakistan is scant. Hence, the objective was to analyze SARS-CoV-2 breakthrough infections with respect to vaccines and variants during the fifth wave in Pakistan. Therefore, the Department of Virology (NIH, Pakistan) genotyped 2,467 randomly selected individuals between November 2021 and February 2022 using the SNPsig® SARS-CoV-2 (EscapePLEX) kit (PrimerDesign, UK). P681R and K417N mutations were used to distinguish delta and omicron. Data on the patient's age, gender, date of collection, variant, and vaccination status were analyzed using Statistical Package for Social Sciences (SPSS) software. Among 2,467 genotyped samples, Omicron was detected in 58.6% (n = 1445), Delta in 40.4% (n = 998) and undetermined/wildtype variant in 24 samples. The vaccination status of omicron-positive patients showed (49.7%; n = 718/1445) and Delta-positive patients (39.67%; n = 396/998) to be fully vaccinated. Of note, a high percentage 85% of breakthrough cases (n = 947) were identified among fully vaccinated individuals (n = 1114). Among them, 85.9% (n = 617/718) belonged to omicron and 83.3% (n = 330/396) to delta. Moreover, 76.7% (n = 855) of vaccinated individuals (n = 1114) received Sinopharm (n = 432) and Sinovac (n = 423) vaccines. The majority of breakthrough subjects who contracted Omicron were vaccinated with Sinopharm (93.0%, n = 256) and delta with Cansino (100%, n = 44). Individuals vaccinated with Sinovac showed the most frequent breakthrough cases for both Omicron and Delta variant between the 4th and 6th months (n = 278) after primary vaccination as compared to the 7th to 9th months (n = 24) category. While in case of Sinopharm, maximum breakthrough cases occurred between 7th to 9th months (n = 234) as compared to the 4th to 6th months (n = 120) after primary vaccination. Omicron and Delta breakthrough cases in men (n = 364 and 193) are more frequently seen than women (n = 253 and 138) respectively and breakthrough majority cases (n = 392) occurred in individuals aged 18-33 years. Breakthrough cases limiting monitoring in Pakistan impose a substantial constraint on policymakers' ability to take timely effective decisions. Since the current study consists of only a 2,467-genotyped sample, comprehensive data should be analyzed.

Molecular detection and genetic characterization of human metapneumovirus strains circulating in Islamabad, Pakistan.

Lower respiratory illness is one of the leading causes of death among children in low- and high-income countries. Human metapneumovirus (hMPV) is a key contributor to respiratory illnesses commonly reported among children and causes serious clinical complications ranging from mild respiratory infections to severe lower respiratory tract anomalies mainly in the form of bronchiolitis and pneumonia. However, due to the lack of a national surveillance system, the clinical significance of hMPV remains obscure in the Pakistani population. This study was conducted to screen throat swabs samples collected from 127 children reported with respiratory symptoms at a tertiary care hospital in Islamabad. Out of 127, 21 (16.5%) samples were positive for hMPV with its genotype distribution as A2a (10%), A2b (20%), B1 (10%), and B2 (60%). Phylogenetic analysis showed that the hMPV viruses were closely related to those reported from neighboring countries including India and China. This work will contribute to a better understanding of this virus, its diagnosis, and the handling of patients in clinical setups. Further studies at a large-scale are warranted for a better understanding of the disease burden and epidemiology of hMPV in Pakistan.

Isolation and molecular characterization of Adenovirus in suspected acute flaccid paralysis patients: A preliminary report from Pakistan.

Human adenoviruses (HAdVs) usually cause asymptomatic or mild infection, but infrequently, they are responsible for various severe syndromes including neurological disorders. Various research studies have investigated the association of HAdVs with acute flaccid paralysis (AFP). The purpose of this study was to investigate the genetic diversity of HAdVs and their association with AFP. Stool samples from patients ≤ 12 years of age with suspected AFP were collected from all over Pakistan within the framework of poliovirus surveillance. Poliovirus- and enterovirus-negative samples were screened for HAdVs. For virus isolation, the human epithelial cell line HEp-2c was used, culture-positive samples were screened by nested PCR assay, and partial hexon gene sequences were used for genotype identification. Out of 172 samples, 94 were positive by virus isolation, 89 were positive by PCR, and 32 isolates were genotyped successfully. Phylogenetic analysis showed that the HAdVs belonged to species A (HAdV-A12 and A31), B (HAdV-B3 and B7), C (HAdV-C1 and C6), D (HAdV-D19 and D93), and F (HAdV-F41), showing 99-100% nucleotide sequence identity and 98.3-100% amino acid sequence identity). Most of these genotypes have been reported previously in AFP cases, but this is the first report of the detection of HAdV-D93 in stool samples from AFP cases. The detection of a significant fraction of the HAdVs genotypes indicates that these genetically distinct genotypes are circulating in Pakistan and suggests their possible role in the pathogenesis of AFP.

Conserved Antigenic Structure of Contemporary Wild Poliovirus Type 1 Strains Endemic in Pakistan.

BACKGROUND: Elimination of poliovirus in Pakistan and Afghanistan is challenged by notions against the role of oral poliovirus vaccine (OPV) in eradicating contemporary wild poliovirus (WPV) strains. METHODS: A total of 1055 WPV type 1 (WPV1) strains isolated between 2013 and 2018 were categorized into 68 antigenic groups and tested for neutralization by OPV-derived antibodies. Molecular docking was conducted to determine neutralization efficiency of antibodies against WPV. The clinical significance of WPV1 variants was assessed to ascertain their role in patient outcomes. RESULTS: We found that 88% of WPV1 strains isolated from paralytic children belonged to a single antigenic lineage identical to the WPV1 strain detected in 1993. WPV1 antigenic variants were effectively neutralized by OPV-derived antibodies, with geometric mean titers comparable to the neutralization titers found for 3 strains in OPV (OPV1-3, 7.96-9.149 [95% confidence interval, 6.864-10.171]; WPV1 strains, 7.542-8.786 [6.493-9.869]). Docking examination underscored a strong antigen-antibody interaction despite variations within the viral protein 1 epitopes. There was no significant association (P = .78) with clinical prognosis among patients infected with antigenically diverse WPV1 strains and patient outcomes, including death. CONCLUSIONS: Our findings substantiate the robustness of OPV for neutralizing the contemporary WPV1 strains endemic in Pakistan and Afghanistan. Vaccination coverage must be augmented to achieve early eradication.

Genomic surveillance reveals the detection of SARS-CoV-2 delta, beta, and gamma VOCs during the third wave in Pakistan.

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide and gained significant importance due to their high transmissibility and global spread, thus meriting close monitoring. In Pakistan, limited information is available on circulation of these variants as the alpha variant has been reported the main circulating lineage. The current study was designed to detect and explore the genomic diversity of SARS-CoV-2 lineages circulating during the third wave of the pandemic in the indigenous population. From May 01 to June 09, 2021, a total of 16 689 samples were tested using TaqPath™ COVID-19 kit for the presence of SARS-CoV-2. Overall, 2562 samples (15.4%) were COVID-19 positive. Out of these positive samples, 2124 (12.7%) did not show the spike gene amplification (spike gene target failure ([SGTF]), whereas 438 (2.6%) showed spike gene amplification (non-SGTF). A subset (n = 58/438) of non-SGTF samples were randomly selected for whole-genome sequencing. Among VOCs, 45% (n = 26/58) were delta, 46% (n = 27/58) were beta, and one was gamma variant. The delta variant cases were reported mainly from Islamabad (n = 15; 58%) followed by Rawalpindi and Azad Kashmir (n = 1; 4% each). Beta variant cases originated mainly from Karachi (n = 8; 30%) and Islamabad (n = 11; 41%) and the gamma variant case was reported in a traveler from Italy. The delta, beta, and gamma variants possessed lineage-specific spike mutations. Notably, two rare mutations (E484Q and L5F) were found in the delta variant. Furthermore, in the beta variant, two significant rare non-synonymous spike mutations (A879S and K444R) were also reported. High prevalence of beta and delta variants in local population may increase the number of cases in the near future and provides an early warning to national health authorities to take timely decisions and devise suitable interventions to contain a possible fourth wave.

Prevalence and Associated Risk Factor of COVID-19 and Impacts of Meteorological and Social Variables on Its Propagation in Punjab, Pakistan.

The current study identifies the spatial distribution of COVID-19 cases and its association with meteorological and social variables in Punjab (densely populated province of Pakistan). To identify the COVID-19 propagation, the weekly growth, recovery, and deaths rate have also been calculated. The geographic information system (GIS) has used to determine COVID-19 impacts on gender (male/female), age groups, and causalities over an affected population (km-2) for the period of 11th March to 12th August, 2020 in each district of province. Our results show that 43 peak days (where daily positive cases were above 900) have been observed in Punjab during 27th May to 8th July, 2020. The high population density districts, i.e., Lahore and Islamabad, have been affected (five persons per square kilometers) due to COVID-19, whereas the maximum death tolls (> 50 persons per millions) have also been observed in these urban districts. The meteorological variables (temperature, humidity, heat index, and ultraviolet index) show negative significant relationship to basic reproduction number (R0), whereas daily COVID-19 cases are positively correlated to aerosols concentration at 95% confidence level. The government intervention (stringency index) shows a positive impact to reduce the COVID-19 cases over the province. Keeping in view the COVID-19 behavior and climatology of the region, it has been identified that the COVID-19 cases may likely to increase during the dry period (high concentration of aerosols) i.e., October-December, 2020 and post-spring season (April to June), 2021 in urban areas of Pakistan. This study provides an overview on districts vulnerability that would help the policy makers, health agencies to plan their activities to reduce the COVID-19 impacts.