Risk of infection with arboviruses in a healthy population in Pakistan based on seroprevalence.

Infectious diseases caused by arboviruses are a public health concern in Pakistan. However, the studies on data prevalence and threats posed by arboviruses are limited. This study investigated the seroprevalence of arboviruses in a healthy population in Pakistan, including severe fever with thrombocytopenia syndrome virus (SFTSV), Crimean-Congo hemorrhagic fever virus (CCHFV), Tamdy virus (TAMV), and Karshi virus (KSIV) based on a newly established luciferase immunoprecipitation system (LIPS) assays, and Zika virus (ZIKV) by enzyme-linked immunosorbent assays (ELISA). Neutralizing activities against these arboviruses were further examined from the antibody positive samples. The results showed that the seroprevalence of SFTSV, CCHFV, TAMV, KSIV, and ZIKV was 17.37%, 7.58%, 4.41%, 1.10%, and 6.48%, respectively, and neutralizing to SFTSV (1.79%), CCHFV (2.62%), and ZIKV (0.69%) were identified, as well as to the SFTSV-related Guertu virus (GTV, 0.83%). Risk factors associated with the incidence of exposure and levels of antibody response were analyzed. Moreover, co-exposure to different arboviruses was demonstrated, as thirty-seven individuals were having antibodies against multiple viruses and thirteen showed neutralizing activity. Males, individuals aged ≤40 years, and outdoor workers had high risk of exposure to arboviruses. All these results reveal the substantial risks of infection with arboviruses in Pakistan, and indicate the threat from co-exposure to multiple arboviruses. The findings raise the need for further epidemiologic investigation in expanded regions and populations and the necessity to improve health surveillance in Pakistan.

Genotypic analysis of hepatitis E virus (HEV) from sporadic symptomatic cases in Pakistan.

Pakistan is the fifth most populous nation in the world and faces several challenges, including devastating floods, sub-optimal sanitary conditions, clustered accommodations, and unregulated cross-border movements. These drastic population shifts make it vulnerable to the efficient spread of the Hepatitis E virus (HEV). The current study analyzed the genotypic characteristics and variants of the Hepatitis E virus circulating in the population of Pakistan. A total of 75 ELISA-IgM positive samples were collected from three metropolitan cities: Lahore, Peshawar, and Karachi, and subjected to viral RNA extraction. The amplification of the HEV RNA-dependent RNA polymerase (RdRp) region was done using Nested PCR and degenerate primers. Out of the total, 40% of the samples were positive for HEV RNA. Sequencing and phylogenetic analysis identified the new HEV isolates as Subtype 1 g, a subtype within an existing HEV genotype 1. This shift warrants investigation into its impact on clinical manifestation and disease severity. Importantly, this study marks the first HEV subtype analysis in Pakistan, contributing valuable insights into subtype diversity and prevalence in the region.

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.

Dynamic variations in COVID-19 with the SARS-CoV-2 Omicron variant in Kazakhstan and Pakistan.

BACKGROUND: The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) and the Omicron variant presents a formidable challenge for control and prevention worldwide, especially for low- and middle-income countries (LMICs). Hence, taking Kazakhstan and Pakistan as examples, this study aims to explore COVID-19 transmission with the Omicron variant at different contact, quarantine and test rates. METHODS: A disease dynamic model was applied, the population was segmented, and three time stages for Omicron transmission were established: the initial outbreak, a period of stabilization, and a second outbreak. The impact of population contact, quarantine and testing on the disease are analyzed in five scenarios to analysis their impacts on the disease. Four statistical metrics are employed to quantify the model's performance, including the correlation coefficient (CC), normalized absolute error, normalized root mean square error and distance between indices of simulation and observation (DISO). RESULTS: Our model has high performance in simulating COVID-19 transmission in Kazakhstan and Pakistan with high CC values greater than 0.9 and DISO values less than 0.5. Compared with the present measures (baseline), decreasing (increasing) the contact rates or increasing (decreasing) the quarantined rates can reduce (increase) the peak values of daily new cases and forward (delay) the peak value times (decreasing 842 and forward 2 days for Kazakhstan). The impact of the test rates on the disease are weak. When the start time of stage II is 6 days, the daily new cases are more than 8 and 5 times the rate for Kazakhstan and Pakistan, respectively (29,573 vs. 3259; 7398 vs. 1108). The impact of the start times of stage III on the disease are contradictory to those of stage II. CONCLUSIONS: For the two LMICs, Kazakhstan and Pakistan, stronger control and prevention measures can be more effective in combating COVID-19. Therefore, to reduce Omicron transmission, strict management of population movement should be employed. Moreover, the timely application of these strategies also plays a key role in disease control.

Coding-Complete Genome Sequence of a SARS-CoV-2 Strain Isolated in Gilgit, Pakistan.

Here, we report the coding-complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate obtained from a nasopharyngeal swab from the first patient with COVID-19 in Gilgit, Pakistan.

Insight of transcriptional regulators reveals the tolerance mechanism of carpet-grass (Axonopus compressus) against drought.

BACKGROUND: Carpet grass [Axonopus compressus (L.)] is an important warm-season perennial grass around the world and is known for its adaptability to varied environmental conditions. However, Carpet grass lacks enough data in public data banks, which confined our comprehension of the mechanisms of environmental adaptations, gene discovery, and development of molecular markers. In current study, the DEGs (differentially expressed genes) in Axonopus compressus under drought stress (DS) were identified and compared with CK (control) by RNA-Seq. RESULTS: A total of 263,835 unigenes were identified in Axonopus compressus, and 201,303 (also added to the numbers of the remaining 2 databases) a sequence of unigenes significantly matched in at least one of the seven databases. A total of 153,697 (58.25%) unigenes classified to 144 KEGG pathways, and 7444 unigenes were expressed differentially between DS and CK, of which 4249 were up-regulated and 3195 were down-regulated unigenes. Of the 50 significantly enriched GO terms, 18, 6, and 14 items were related to BP, CC, and MF respectively. Analysis of KEGG enrichment revealed 2569 DEGs involved in 143 different pathways, under drought stress. 2747 DEGs were up-regulated and 2502 DEGs were down-regulated. Moreover, we identified 352 transcription factors (TFs) in Axonopus compressus, of which 270 were differentially expressed between CK and DS. The qRT-PCR validation experiment also supports the transcriptional response of Axonopus compressus against drought. Accuracy of transcriptome unigenes of Axonopus compressus was assessed with BLAST, which showed 3300 sequences of Axonopus compressus in the NCBI. CONCLUSION: The 7444 unigenes were found to be between DS and CK treatments, which indicate the existence of a strong mechanism of drought tolerance in Axonopus compressus. The current findings provide the first framework for further investigations for the particular roles of these unigenes in Axonopus compressus in response to drought.

Reverse vaccinology assisted designing of multiepitope-based subunit vaccine against SARS-CoV-2.

BACKGROUND: Coronavirus disease 2019 (COVID-19) linked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause severe illness and life-threatening pneumonia in humans. The current COVID-19 pandemic demands an effective vaccine to acquire protection against the infection. Therefore, the present study was aimed to design a multiepitope-based subunit vaccine (MESV) against COVID-19. METHODS: Structural proteins (Surface glycoprotein, Envelope protein, and Membrane glycoprotein) of SARS-CoV-2 are responsible for its prime functions. Sequences of proteins were downloaded from GenBank and several immunoinformatics coupled with computational approaches were employed to forecast B- and T- cell epitopes from the SARS-CoV-2 highly antigenic structural proteins to design an effective MESV. RESULTS: Predicted epitopes suggested high antigenicity, conserveness, substantial interactions with the human leukocyte antigen (HLA) binding alleles, and collective global population coverage of 88.40%. Taken together, 276 amino acids long MESV was designed by connecting 3 cytotoxic T lymphocytes (CTL), 6 helper T lymphocyte (HTL) and 4 B-cell epitopes with suitable adjuvant and linkers. The MESV construct was non-allergenic, stable, and highly antigenic. Molecular docking showed a stable and high binding affinity of MESV with human pathogenic toll-like receptors-3 (TLR3). Furthermore, in silico immune simulation revealed significant immunogenic response of MESV. Finally, MEV codons were optimized for its in silico cloning into the Escherichia coli K-12 system, to ensure its increased expression. CONCLUSION: The MESV developed in this study is capable of generating immune response against COVID-19. Therefore, if designed MESV further investigated experimentally, it would be an effective vaccine candidate against SARS-CoV-2 to control and prevent COVID-19.

Serological and Molecular Investigation of Brucellosis in Breeding Equids in Pakistani Punjab.

Brucellosis is an important zoonosis worldwide. Equines are susceptible to the infection when in close contact with infected animals. The objective of our study was to update the existing knowledge and detect and differentiate the causative agent of brucellosis in breeding equines in Punjab, Pakistan. A cross-sectional study was designed to evaluate the occurrence and etiology of the infection in the equine population in three districts. A total of 448 equine sera were collected from three prefectures viz. Sahiwal, Khanewal, and Okara of the Punjab Province of Pakistan. Ninety-six (21.4%) samples were found positive by RBPT, 3.56% (16/448) by iELISA, and 4.24% (19/448) by CFT. Real-time PCR demonstrated the presence of Brucella abortus-DNA in sero-positive samples. Age and location were found as risk factors. The study concludes equine brucellosis seroprevalence in the country where Brucella abortus as the main etiology. Fistulous withers and poll evil cases should be treated with care as they could be hazardous and a source of zoonotic transmission. Routine screening at an early age, vaccination in ruminants, and consumption of pasteurized dairy milk in humans is recommended for prevention of the infection. Specific tests need to be standardized and validated.

Serologic Evidence of Severe Fever with Thrombocytopenia Syndrome Virus and Related Viruses in Pakistan.

We describe the seroprevalence of severe fever with thrombocytopenia syndrome virus (SFTSV) and the association of antibody occurrence with location, sex, and age among the human population in Pakistan. Our results indicate substantial activity of SFTSV and SFTSV-related viruses in this country.

Crimean-Congo Hemorrhagic Fever Virus in Humans and Livestock, Pakistan, 2015-2017.

We detected Crimean-Congo hemorrhagic fever virus infections in 4 provinces of Pakistan during 2017-2018. Overall, seroprevalence was 2.7% in humans and 36.2% in domestic livestock. Antibody prevalence in humans was highest in rural areas, where increased contact with animals is likely.

Sentinel surveillance of selected veterinary and public health pathogens in camel population originating from Southern Punjab province, Pakistan.

An extended range of host susceptibility including camel has been evidenced for some of the important veterinary and public health pathogens, such as brucellosis, peste des petits ruminants (PPR) and bluetongue (BT). However, in disease endemic settings across many parts of the globe, most of the disease control interventions accounts for small and large ruminants, whereas unusual hosts and/or natural reservoirs, such as camels, remain neglected for disease control measures including routine vaccination. Such a policy drawback not only plays an important role in disease epizootiology particularly in settings where disease is endemic, but also serves an obstacle in disease control and subsequent eradication in future. With this background, using pre-validated ELISA and molecular assays [multiplex PCR, reverse transcriptase (RT)-PCR and real-time (rt)-PCR], we conducted a large-scale pathogen- and antibody-based surveillance for brucellosis, peste des petits ruminants and bluetongue in camel population (n = 992) originating from a wide geographical region in southern part of the Punjab province, Pakistan. Varying in each of the selected districts, the seroprevalence was found to be maximum for bluetongue [n = 697 (70.26%, 95% CI: 67.29-73.07)], followed by PPR [n = 193 (19.46%, 95% CI: 17.07-22.09)] and brucellosis [n = 66 (6.65%, 95% CI: 5.22-8.43)]. Odds of seroprevalence were more significantly associated with pregnancy status (non-pregnant, OR = 2.23, 95% CI: 1.86-5.63, p<0.01), farming system (mixed-animal, OR = 2.59, 95% CI: 1.56-4.29, p<0.01), breed (Desi, OR = 1.97, 95% CI: 1.28-4.03, p<0.01) and farmer education (illiterate, OR = 3.17, 95% CI: 1.45-6.93, p<0.01) for BTV, body condition (normal, OR = 3.54, 95% CI: 1.92-6.54, p<0.01) and breed (Desi, OR = 2.19, 95% CI: 1.09-4.40, p<0.01) for brucellosis, and feeding system for PPR (grazing, OR = 2.75, 95% CI: 1.79-4.22, p<0.01). Among the total herds included (n = 74), genome corresponding to BT virus (BTV) and brucellosis was detected in 14 (18.92%, 95 CI: 11.09-30.04) and 19 herds (25.68%, 95% CI: 16.54-37.38), respectively. None of the herds was detected with genome of PPR virus (PPRV). Among the positive herds, serotype 1, 8 and 11 were detected for BTV while all the herds were exclusively positive to B. abortus. Taken together, the study highlights the role of potential disease reservoirs in the persistence and transmission of selected diseases in their susceptible hosts and, therefore, urges necessary interventions (e.g., inclusion of camels for vaccine etc.) for the control of diseases from their endemic setting worldwide.

Correction to: Genetic Evidence of Middle East Respiratory Syndrome Coronavirus (MERS-Cov) and Widespread Seroprevalence among Camels in Kenya.

The acknowledgement section in the original article was published incorrectly.

Detection of West Nile virus lineage 1 sequences in blood donors, Punjab Province, Pakistan.

OBJECTIVES: This study was performed to determine the presence of West Nile virus (WNV) in mosquito specimens and human blood donors in Pakistan. METHODS: A total of 4150 mosquito specimens were collected using CO2-baited traps from five selected districts of Punjab Province, Pakistan. The mosquitoes were taxonomically identified using standard morphological keys, resulting in 166 pools. In addition, 1070 serum samples were collected from human blood donors. RNA was extracted from mosquito and human samples and screened for WNV using a reverse transcriptase PCR (RT-PCR). RESULTS: None of the mosquito pools tested positive for WNV, whereas three samples from asymptomatic humans tested positive. To determine the WNV strains, partial sequences were compared against a global representation of 23 WNV sequences. The study strains were determined to come from WNV lineage 1. CONCLUSIONS: This study is novel in reporting the circulation of lineage 1 WNV in Pakistan. Given its ability to transmit from human to human via blood transfusion, this highlights the urgent need for nationwide surveillance to assess the distribution and impact of WNV in Pakistan. Determining the source of human infection will require more extensive mosquito sampling.

Genetic Evidence of Middle East Respiratory Syndrome Coronavirus (MERS-Cov) and Widespread Seroprevalence among Camels in Kenya.

We describe the first genome isolation of Middle East respiratory syndrome coronavirus (MERS-CoV) in Kenya. This fatal zoonotic pathogen was first described in the Kingdom of Saudi Arabia in 2012. Epidemiological and molecular evidence revealed zoonotic transmission from camels to humans and between humans. Currently, MERS-CoV is classified by the WHO as having high pandemic potential requiring greater surveillance. Previous studies of MERS-CoV in Kenya mainly focused on site-specific and archived camel and human serum samples for antibodies. We conducted active nationwide cross-sectional surveillance of camels and humans in Kenya, targeting both nasal swabs and plasma samples from 1,163 camels and 486 humans collected from January 2016 to June 2018. A total of 792 camel plasma samples were positive by ELISA. Seroprevalence increased with age, and the highest prevalence was observed in adult camels (82.37%, 95% confidence interval (CI) 79.50-84.91). More female camels were significantly seropositive (74.28%, 95% CI 71.14-77.19) than male camels (P < 0.001) (53.74%, 95% CI 48.48-58.90). Only 11 camel nasal swabs were positive for MERS-CoV by reverse transcription-quantitative PCR. Phylogenetic analysis of whole genome sequences showed that Kenyan MERS-CoV clustered within sub-clade C2, which is associated with the African clade, but did not contain signature deletions of orf4b in African viruses. None of the human plasma screened contained neutralizing antibodies against MERS-CoV. This study confirms the geographically widespread occurrence of MERS-CoV in Kenyan camels. Further one-health surveillance approaches in camels, wildlife, and human populations are needed.

Countrywide Survey for MERS-Coronavirus Antibodies in Dromedaries and Humans in Pakistan.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050) samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged > 10 years (81.37%) followed by those aged 3.1-10 years (78.65%) and ≤ 3 years (58.19%). Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016-2017. Among the sampled population, 840 humans were camel herders. Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.

Terminal drought and seed priming improves drought tolerance in wheat.

Plants retain the preceding abiotic stress memory that may aid in attainment of tolerance to subsequent stresses. This study was conducted to evaluate the influence of terminal drought memory (drought priming) and seed priming in improving drought tolerance in wheat (Triticum aestivum L.). During first growing season, wheat was planted in field under optimal (well-watered) and drought stress imposed at reproductive stage (BBCH growth stage 49) until maturity (BBCH growth stage 83). Seeds collected from both sources were subjected to hydropriming or osmopriming (with 1.5% CaCl2 solution); while, dry seed was taken as control. Treated and control seeds, from both sources, were sown in soil filled pots. After the completion of seedling emergence, pots were maintained at 50% water holding capacity (drought) or 100% water holding capacity (well-watered). Drought stress suppressed the plant growth (2-44%), perturbed water relations (1-18%) and reduced yield (192%); however, osmolytes accumulation (3-14%) and malondialdehyde contents (26-29%) were increased under drought. The crop raised from the seeds collected from terminal drought stressed plants had better growth (5-63%), improved osmolyte accumulation (13-45%), and lower lipid peroxidation (3%) than the progeny of well-watered crop. Seed priming significantly improved the crop performance under drought stress as compared to control. However, osmopriming was more effective than hydropriming in this regard as it improved leaf area (9-43%), tissue water status (2-47%), osmolytes accumulation (6-48%) and grain yield (14-79%). In conclusion, terminal drought induced modifications in seed composition and seed priming improved transgenerational drought tolerance through improvement in tissue water status and osmolytes accumulation, and decrease in lipid peroxidation.

Author Correction: Effect of Plant Density, Boron Nutrition and Growth Regulation on Seed Mass, Emergence and Offspring Growth Plasticity in Cotton.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Effect of Plant Density, Boron Nutrition and Growth Regulation on Seed Mass, Emergence and Offspring Growth Plasticity in Cotton.

Seed nutrients reserves have direct relationship with seed functional traits and influence offspring performance. Effects of plant density, foliage boron (B) nutrition and mepiquat chloride (MC) growth regulation on seed nutrients reserves, seed mass and production, and emergence and offspring growth traits of cotton were studied in two years field experiment. Seed nutrients reserves and seed mass were decreased at higher maternal plant density relative to lower plant density with concomitant decrease in emergence and offspring seedling growth. However, maternal foliage B nutrition and MC growth regulation enhanced seed nutrients reserves, seed mass, emergence and offspring seedling growth performance. There was a significant positive relationship between seed mass and seed nutrients reserves indicating that changes in nutrient availability/uptake in response to maternal ecological factors determine variation in seed functional traits. Nonetheless, seed mass was positively correlated with emergence percentage and negatively with emergence timing. Furthermore, variation in offspring seedling growth traits with seed mass indicated the significance of initial seed nutrients reserves for early seedling vigour and establishment. In conclusion, lower maternal plant density, B nutrition and MC growth regulation ensued in higher emergence and offspring seedling growth of cotton because of higher seed nutrient reserves and seed mass.

Correction to: Molecular detection of viruses in Kenyan bats and discovery of novel astroviruses, caliciviruses and rotaviruses.

The affiliation listed for Cecilia Waruhiu is incorrect. The byline and affiliation line should appear as shown above.

Metagenomic Virome Analysis of Culex Mosquitoes from Kenya and China.

Many blood-feeding arthropods are known vectors of viruses that are a source of unprecedented global health concern. Mosquitoes are an integral part of these arthropod vectors. Advancements in next-generation sequencing and bioinformatics has expanded our knowledge on the richness of viruses harbored by arthropods. In the present study, we applied a metagenomic approach to determine the intercontinental virome diversity of Culex quinquefasciatus and Culex tritaeniorhynchus in Kwale, Kenya and provinces of Hubei and Yunnan in China. Our results showed that viromes from the three locations were strikingly diverse and comprised 30 virus families specific to vertebrates, invertebrates, plants, and protozoa as well as unclassified group of viruses. Though sampled at different times, both Kwale and Hubei mosquito viromes were dominated by vertebrate viruses, in contrast to the Yunnan mosquito virome, which was dominated by insect-specific viruses. However, each virome was unique in terms of virus proportions partly influenced by type of ingested meals (blood, nectar, plant sap, environment substrates). The dominant vertebrate virus family in the Kwale virome was Papillomaviridae (57%) while in Hubei it was Herpesviridae (30%) and the Yunnan virome was dominated by an unclassified viruses group (27%). Given that insect-specific viruses occur naturally in their hosts, they should be the basis for defining the viromes. Hence, the dominant insect-specific viruses in Kwale, Hubei, and Yunnan were Baculoviridae, Nimaviridae and Iflaviridae, respectively. Our study is preliminary but contributes to growing and much needed knowledge, as mosquito viromes could be manipulated to prevent and control pathogenic arboviruses.