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.

Genetic Epidemiology Reveals 3 Chronic Reservoir Areas With Recurrent Population Mobility Challenging Poliovirus Eradication in Pakistan.

BACKGROUND: Pakistan is among 3 countries endemic for wild poliovirus type 1 (WPV1) circulation that are still struggling for eradication of poliomyelitis. Active clinical and environmental surveillance with meticulous laboratory investigations provide insights into poliovirus transmission patterns and genomic diversity to inform decisions for strategic operations required to achieve eradication. METHODS: We analyzed epidemiological and virological data to comprehend the current epidemiological status of WPV1 in Pakistan during 2015-2017. Stool specimens of patients with acute flaccid paralysis (AFP) and sewage samples collected from 60 environmental sites were tested. Viral culturing, intratypic differentiation by real-time polymerase chain reaction, and nucleic acid sequencing of the VP1 region of the poliovirus genome to determine genetic relatedness among WPV1 strains were applied. RESULTS: Poliovirus isolates were grouped into 11 distinct clusters, which had ≥95% nucleotide homology in the VP1 coding region. Most of the poliovirus burden was shared by 3 major reservoirs: Karachi, Peshawar, and Quetta block (64.2% in 2015, 75.4% in 2016, and 76.7% in 2017). CONCLUSIONS: Environmental surveillance reveals importations and pockets of unimmunized children that dictate intensive target mop-up campaigns to contain poliovirus transmission. A decrease in the number of orphan isolates reflects effective combination of AFP and environmental surveillance in Pakistan. The genetic data reflect sustained transmission within reservoir areas, further expanded by periodic importations to areas of high immunity reflected by immediate termination of imported viruses. Improved immunization coverage with high-quality surveillance is vital for global certification of polio eradication.

Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing.

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

Epidemiological and molecular investigation of a measles outbreak in Punjab, Pakistan, 2013-2015.

Despite the availability of an effective vaccine, the measles virus continues to cause significant morbidity and mortality in children worldwide. Molecular characterization of wild-type measles strains is an invaluable component of epidemiological studies or surveillance systems that provides important information pertinent to outbreak linkages and transmission pathways. Serum samples and throat swabs were collected from suspected measles cases from the Punjab province of Pakistan (2013-2015) and further tested for measles immunoglobulin M (IgM) through enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction for molecular characterization. Among the total of 5415 blood samples, 59% tested positive for measles IgM. Males had a higher infection rate (55%) than females (45%), and the highest frequency of positive cases (63%) was found in the age group of 0 to 5 years. Partial sequencing of the nucleoprotein gene showed that 27 strains belonged to the B3 genotype, whereas 2 viruses were identified as D4. On phylogenetic analysis, Pakistani B3 strains were found to be closely related to previously reported indigenous strains and those from neighboring countries of Iran and Qatar. This is the first report on the detection of the measles B3 genotype from Punjab, Pakistan. The current study shows a high burden of measles infections in Punjab province owing to poor routine immunization coverage in major cities. It is imperative that national health authorities adopt strategic steps on an urgent basis for improvement of routine immunization coverage. Molecular epidemiology of the measles viruses circulating in different parts of the country can provide useful data to manage future outbreaks.

A measles outbreak in Sindh, Pakistan caused by a genotype B3 virus.

Measles continues to be a major public health issue causing substantial outbreaks worldwide, mostly affecting young children. Molecular analysis of measles viruses provides important information on outbreak linkages and transmission pathways that can be helpful towards implementation of appropriate control programs. In Pakistan, the control of measles is still tenuous, and progress towards elimination has been irregular and challenging. In the 2013 measles outbreak we received 4,682 sera collected from suspected patients in 23 districts across Sindh. A total of 3,283 samples were confirmed measles positive using IgM ELISA with the highest infection rate in children aged 1-12 months. Males were more affected than females and a visible peak was observed from January to April. Among the 3,283 cases, 59.1% were unvaccinated, 29.6% had received 1 dose and 10.3% had received 2 doses of measles vaccine while 0.85% had an unknown vaccination status. For genotype detection and phylogenetic analysis, 60 throat swab samples were collected from suspected patients below 15 years of age in eight districts of Sindh province. Forty four (73%; 44/60) throat swab samples were successfully genotyped using RT-PCR. Phylogenetic analyses based on partial sequences of the nucleocapsid protein gene revealed that all Pakistani measles virus strains belonged to genotype B3 and were closely related to those isolated from neighboring countries such as Iran, Afghanistan (99.1-100%) and India with 98.6 - 99.6% nucleotide homology. This is the first report on the phylogenetic analysis of measles B3 genotype strains from Pakistan and highlights the need for strengthening the surveillance systems and improving immunization coverage across the country.

Genomic Surveillance Elucidates Persistent Wild Poliovirus Transmission During 2013-2015 in Major Reservoir Areas of Pakistan.

BACKGROUND: Despite tremendous efforts in the fight against polio, Pakistan bears the highest proportion of poliomyelitis cases among the 3 endemic countries including Afghanistan and Nigeria. Apart from insecurity and inaccessibility challenges, the substantial shift of unimmunized children from North Waziristan due to recent military operations was presumed to favor the widespread poliovirus infection in Pakistan. METHODS: To better understand the current epidemiological situation, we analyzed the virologic data of wild poliovirus type 1 (WPV1) strains detected in Pakistan during 2013-2015. RESULTS: Five genetic clusters (A-E) were identified with at least 5% nucleotide divergence in the viral protein 1 (VP1) coding region. Peshawar, Quetta, and Karachi were found to be the major endemic foci where multiple discrete genetic lineages of WPV1 were detected. Phylogenetic analysis suggests that wild poliovirus strains from endemic regions were genetically distant (with 5%-15% VP1 nucleotide divergence) from those detected in North Waziristan cases, excluding the possibility of a recent progenitor of WPV1 instigating single-source transmission across the country. Orphan lineages detected in Rawalpindi, Lahore, Hyderabad, Sukkur, and Jacobabad revealed silent transmission and the need for vigilant surveillance. Sustenance of analogous genetic lineages over a period of 3 years highlights multiple unimmunized foci present to maintain viral genetic diversity. CONCLUSIONS: Our findings are inconsistent with the hypothesis that impoverished populations from North Waziristan serve as a possible determinant of widespread poliomyelitis infection in Pakistan and further emphasize the need to scale-up clinical and environmental surveillance as well as immunization activities.

Seroprevalence of Human Cytomegalovirus (HCMV) infection in pregnant women and outcomes of pregnancies with active infection.

OBJECTIVE: To determine the prevalence of cytomegalovirus in pregnant women and types of overt congenital infection in neonates. METHODS: This cross-sectional study was conducted at the Pakistan Institute of Medical Sciences and Federal Government Services Hospital in Islamabad, Pakistan, from March 2010 to June 2011, and comprised blood samples of pregnant women. Seroprevalence of human cytomegalovirus, immunoglobulin G and immunoglobulin M was determined by enzyme-linked immunosorbent assay while its deoxyribonucleic acid was detected by nested polymerase chain reaction.The congenital human cytomegalovirus infection was also identified in newborn babies from actively infected pregnant women. SPSS 18 was used for data analysis. RESULTS: Of the 409 pregnant women enrolled, 399(97.55%) were seropositive for cytomegalovirus immunoglobulinG and 52(12.71%) for immunoglobulinM, while cytomegalovirus deoxyribonucleic acid was detected in 82(20%). Of the cytomegalovirus immunoglobulinM-positive women, sera of 40(80%) had immunoglobulinG avidity >50%. The remaining 12(23%) sera had avidity assay value <50%. Among the 82(20%) infected pregnant women, 70(85.4%) were successfully followed up. Among them, the virus was isolated from 41(58.5%) newborns babies, of which 15(21%) were symptomatic while 26(47.2%) were asymptomatic. Of the former, 4(26.6%) had hepatosplenomegaly. CONCLUSIONS: Human cytomegalovirus infection in pregnant women was the main reason of congenital defects among neonates.

'Human bocavirus in Pakistani children with gastroenteritis'.

Human Bocaviruses (HBoV) have been detected in human respiratory and gastrointestinal infections worldwide. Four genotypes of HBoV (HBoV1-4) have been described; HBoV-1 is associated with respiratory tract infections while HBoV-2, -3, and -4 genotypes are considered as entero-pathogenic although the exact role largely remains unclear. The global prevalence of HBoV has been reported, but the epidemiological data from Pakistan is largely unavailable to date. This study was conducted to understand the genetic diversity and disease prevalence of HBoV in hospitalized Pakistani children with acute diarrhea. During 2009, a total of 365 stool samples were collected from children hospitalized with gastrointestinal symptoms (as per WHO case definitions) at Rawalpindi General Hospital, Pakistan. Demographic and clinical data were recorded using a standardized questionnaire. The samples were tested for HBoV and rotavirus using real-time RT-PCR and ELISA, respectively. There were 47 (13%) samples positive for HBoV with 98% (n = 46) showing co-infection with rotavirus. HBoV-1 was the most frequently detected and was found in 94% samples followed by HBoV-2 and HBoV-3 genotypes. The mean age of infected children was 7.57 ± 5.4 months while detection was more frequent in males (n = 32, 68%). All cases recovered after 2.43 ± 1.0 mean days of treatment. On phylogenetic analysis, HBoV strains from Pakistan clustered closely with viruses from neighboring Bangladesh and China. These findings represent the first known epidemiological study in Pakistan to investigate the role of HBoV in acute gastroenteritis. The clinical data demonstrates that HBoV is not significantly associated with gastroenteritis alone and predominantly co-infections with rotavirus are found.

Detection of multiple cocirculating wild poliovirus type 1 lineages through environmental surveillance: impact and progress during 2011-2013 in Pakistan.

BACKGROUND: The environmental surveillance has proven to be a useful tool to identify poliovirus circulation in different countries and was started in Pakistan during July 2009 to support the acute flaccid paralysis (AFP) surveillance system. METHODS: Sewage samples were collected from 27 environmental sampling (ENV) sites and processed for poliovirus isolation through 2-phase separation method. Poliovirus isolates were identified as Sabin-like or wild type through real-time polymerase chain reaction (PCR). Wild-type strains were subjected to VP1 gene sequencing and phylogenetic analysis performed using MEGA 5.0. RESULTS: During 2011-2013, a total of 668 samples were collected from 4 provinces that resulted in 40% of samples positive for wild poliovirus type-1 (WPV-1). None of the samples were positive for WPV-3. The areas with high frequency of WPV-1 detection were Karachi-Gadap (69%), Peshawar (82%), and Rawalpindi (65%), whereas the samples from Quetta and Sukkur remained negative for WPV during 2013. Phylogenetic analysis revealed 3 major clusters with multiple poliovirus lineages circulating across different country areas as well as in bordering areas of Afghanistan. CONCLUSIONS: Environmental surveillance in Pakistan has been proven to be a powerful tool to detect WPV circulation in the absence of poliomyelitis cases in many communities. Our findings emphasize the need to continue and expand such surveillance activities to other high-risk areas in the country.

Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA.

BACKGROUND: The use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures. METHODS: VIDISCA is normally combined with next generation sequencing, however, we set up a simplified VIDISCA which can be used in case next generation sequencing is not possible. Stool samples of 10 patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. To further characterize the viruses, VIDISCA-positive samples were amplified and sequenced with gene specific primers. RESULTS: Simplified VIDISCA detected seven viruses (70%) and the proportion of eukaryotic viral sequences from each sample ranged from 8.3 to 45.8%. Human enterovirus EV-B97, EV-B100, echovirus-9 and echovirus-21, human parechovirus type-3, human astrovirus probably a type-3/5 recombinant, and tetnovirus-1 were identified. Phylogenetic analysis based on the VP1 region demonstrated that the human enteroviruses are more divergent isolates circulating in the community. CONCLUSION: Our data support that a simplified VIDISCA protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. Also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. Redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients.

Genetic analysis and epidemiology of Crimean Congo Hemorrhagic fever viruses in Baluchistan province of Pakistan.

BACKGROUND: Pakistan is considered as an endemic country for Crimean-Congo Hemorrhagic fever with numerous outbreaks and sporadic cases reported during the past two decades. Majority of cases are reported from Baluchistan province with subsequent transmissions to non-endemic regions mainly through infected animals directly or via infested ticks. We hereby describe the molecular investigations of CCHF cases reported during 2008 in Quetta city of Baluchistan province. METHODS: Serum Samples from 44 patients, with clinical signs of hemorrhagic fever attending a tertiary care hospital in Quetta city, were collected and tested for CCHF virus antigen and genomic RNA, using capture IgM EIA kit and standard RT-PCR assay, respectively. The partial S-gene fragments were directly sequenced to get information related to the prevailing CCHFV genotypes and their molecular epidemiology in Pakistan. RESULTS: Out of the total forty four, sixteen (36%) samples were found positive for CCHF IgM. Similarly, viral RNA was detected in six (16%) samples. Phylogenetic analysis revealed that all study viruses belong to genotype Asia-1 with closest similarity (99-100%) to the previously reported strains from Pakistan, Afghanistan and Iran. CONCLUSION: We conclude that CCHF virus remains endemic within Baluchistan and its neighboring regions of Afghanistan warranting a need of incessant surveillance activities.

Molecular identification and characterization of a new type of bovine enterovirus.

Bovine enteroviruses belong to the family Picornaviridae. Little is known about their pathogenic potential; however, they cause asymptomatic infections in cattle and are excreted in feces. In the present study, viruses isolated from environmental samples were sequenced. According to phylogenetic analyses and standard picornavirus nomenclature, these isolates constitute a new type of bovine enterovirus serogroup A.

Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan.

BACKGROUND/AIM: Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. METHODS: In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. RESULTS: One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. CONCLUSION: Our results emphasize that there was a persistent transmission of wild type 1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk population groups which include migrants, internally displaced people, and refugees. The implication of this study is to maintain high quality mass immunization with oral polio vaccine (OPV) in order to interrupt chains of virus transmission in both countries to endorse substantial progress in Eastern-Mediterranean region.

A highly prevalent and genetically diversified Picornaviridae genus in South Asian children.

Viral metagenomics focused on particle-protected nucleic acids was used on the stools of South Asian children with nonpolio acute flaccid paralysis (AFP). We identified sequences distantly related to Seneca Valley virus and cardioviruses that were then used as genetic footholds to characterize multiple viral species within a previously unreported genus of the Picornaviridae family. The picornaviruses were detected in the stools of >40% of AFP and healthy Pakistani children. A genetically diverse and highly prevalent enteric viral infection, characteristics similar to the Enterovirus genus, was therefore identified substantially expanding the genetic diversity of the RNA viral flora commonly found in children.

Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.

Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

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.

Cardioviruses are genetically diverse and cause common enteric infections in South Asian children.

Cardioviruses cause enteric infections in mice and rats which when disseminated have been associated with myocarditis, type 1 diabetes, encephalitis, and multiple sclerosis-like symptoms. Cardioviruses have also been detected at lower frequencies in other mammals. The Cardiovirus genus within the Picornaviridae family is currently made up of two viral species, Theilovirus and Encephalomyocarditis virus. Until recently, only a single strain of cardioviruses (Vilyuisk virus within the Theilovirus species) associated with a geographically restricted and prevalent encephalitis-like condition had been reported to occur in humans. A second theilovirus-related cardiovirus (Saffold virus [SAFV]) was reported in 2007 and subsequently found in respiratory secretions from children with respiratory problems and in stools of both healthy and diarrheic children. Using viral metagenomics, we identified RNA fragments related to SAFV in the stools of Pakistani and Afghani children with nonpolio acute flaccid paralysis (AFP). We sequenced three near-full-length genomes, showing the presence of divergent strains of SAFV and preliminary evidence of a distant recombination event between the ancestors of the Theiler-like viruses of rats and those of human SAFV. Further VP1 sequencing showed the presence of five new SAFV genotypes, doubling the reported genetic diversity of human and animal theiloviruses combined. Both AFP patients and healthy children in Pakistan were found to be excreting SAFV at high frequencies of 9 and 12%, respectively. Further studies are needed to examine the roles of these highly common and diverse SAFV genotypes in nonpolio AFP and other human diseases.

Genomic characterization of novel human parechovirus type.

Using a simple metagenomic approach, we identified a divergent human parechovirus (HPeV) in the stool of a child in Pakistan. Genomic characterization showed this virus was distinct enough from reported HPeV types to qualify as candidate prototype for the seventh HPeV type.

Identification of measles virus genotype B3 associated with outbreaks in Islamabad, Pakistan, 2013-2015.

BACKGROUND: Measles virus infection remains a significant cause of childhood mortality and morbidity despite continued global efforts and the availability of a safe and effective vaccine. Molecular analysis of indigenous measles viruses could provide critical information on outbreak linkages and transmission pathways that can aid the implementation of appropriate control programs in Pakistan. METHODS: Blood samples and throat swabs were collected from subjects suspected with measles in Islamabad, Pakistan from 2013 to 2015. Serum samples were tested for the presence of measles immunoglobulin M (IgM) antibodies using enzyme-linked immunosorbent assay (ELISA) while throat swabs were used for the isolation (Vero/SLAM cell line) and subsequent characterization and phylogenetic analysis of measles strains. RESULTS: Of 373 blood samples, 66% tested positive for measles IgM. Male subjects were more often infected (58%) than female (42%) with the highest frequency of positive cases (63%) in the 0-5-years age group. Among the positive cases, only 13% had received one or two doses of the measles vaccine, while 87% were unvaccinated. Of 80 throat swabs, 29 (36%) showed a measles virus-specific cytopathic effect (CPE) and were characterized as genotype B3 through partial sequencing of the nucleoprotein (N) gene. Phylogenetic analysis revealed the Pakistani B3 strains to be closely related to strains from neighboring countries (Iran and Afghanistan) as well as with B3 viruses from the USA, Germany, and the UK. CONCLUSIONS: The study results showed that despite the availability of an effective vaccine, the burden of measles infections is very high in Pakistan due to poor routine immunization coverage even in major cities, including the capital city of Islamabad. It is imperative that national health authorities take urgent strategic steps to improve routine immunization and implement adequate molecular identification methods to tackle future measles outbreaks.