Mapping Genetic Markers Associated with Antigenicity and Host Range in H9N2 Influenza A Viruses Infecting Poultry in Pakistan.

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction (P < 0.01) in log2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

Nota de investigación- Mapeo de marcadores genéticos asociados con la antigenicidad y el rango de huéspedes en los virus de la influenza tipo A subtipo H9N2 que infectan a la avicultura en Pakistán. El objetivo del presente estudio fue mapear la diversidad genética en la glicoproteína hemaglutinina (HA) de los virus de la influenza A (IAV) del subtipo H9N2. Se aislaron veinticinco virus de influenza H9N2 de pollos de engorde de marzo a julio del 2019. Se amplificó el gene HA y se realizó un análisis filogenético para determinar la relación evolutiva. Se compararon importantes residuos de aminoácidos antigénicos de la hemaglutinina atribuidos al escape inmunológico y al potencial zoonótico entre los virus de la influenza aviar H9N2. El análisis filogenético reveló que el sublinaje B2 bajo el linaje G1 en Pakistán estaba diversificado, y los aislados de H9N2 recién secuenciados se agruparon en dos clados (A y B). Se observaron mutaciones relacionadas con la variación antigénica y el posible escape inmunológico como los residuos de aminoácidos G72E (1/25, 4%), A180T (3/25, 12%) y A180V (1/25, 4%). Se observó una reducción significativa al doble (P < 0.01) en los títulos de inhibición de la hemaglutinación log2 cuando el virus de la influenza aviar H9N2 albergaba naturalmente el aminoácido V180 en lugar del A180 en la proteína HA. Además, en los últimos 20 años, sustitución completa en los residuos (T127D, D135N y L150N) y sustitución parcial en los residuos (72, 74, 131, 148, 180, 183, 188, 216, 217 y 249, de acuerdo con la numeración de la HA subtipo madura) asociados con cambios en la antigenicidad. La presencia del residuo L216 en todos los aislados de influenza aviar H9N2 y T/V180 en cuatro aislados en el sitio de unión al receptor revela el potencial de estos virus para cruzar la barrera de las especies para infectar a humanos o mamíferos. El estudio actual observó la circulación de variantes antigénicamente diversas del virus de influenza aviar H9N2 que poseen mutaciones potenciales que pueden escapar del sistema inmunológico del huésped.

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.

Seroprevalence of Crimean-Congo haemorrhagic fever among three selected risk human groups in disease-endemic region of Pakistan.

The occurrence of Crimean-Congo haemorrhagic fever (CCHF) in humans is linked with animals living in close vicinity, and information on the incidence of CCHF at the human-animal interface is scarce. Therefore, the current study was designed to identify the high-risk groups of individuals linked with animals in the Chakwal district of Pakistan having a history of CCHF cases in humans. In subject matter, coupled with risk factor analysis, we performed a sero-based CCHF surveillance in three selected risk groups of humans including abattoir workers (n = 137), milkmen (n = 169) and animal handlers (n = 147). Sera samples and questionnaire-based data were collected from each of the participants and screened for anti-CCHFV IgG antibodies using enzyme-linked immunosorbent assay. The highest seroprevalence was observed in animal handlers (n = 14, 9.52%, 95% CI: 4.68-13.99) followed by abattoir workers (n = 9, 6.57%, 95% CI: 2.42-10.72) and milkmen (n = 3, 1.78%, 95% CI: 0.24-4.24). The risk of seropositivity was significantly associated with humans linked with tick-infested animals (OR: 11.0, 95% CI: 1.5-83.0, p = .002), old age >40 years (OR: 6.6, 95% CI: 2.7-16.0, p < .0001), illiteracy (OR: 4.3, 95% CI: 1.5-13.0, p = .004) and humans without knowledge about CCHF (OR: 7.6, 95% CI: 1.8-33.0, p = .0009). The findings of the current study highlighted the seroprevalence of CCHF in high-risk groups of humans living in a disease-endemic area of Pakistan and highlight the need for well-integrated disease surveillance in the future to better comprehend disease control interventions.

Circulation of multiple subtypes (A, G and CRFs 02_AG) of human immunodeficiency virus type 1 (HIV-1) in selected districts of Punjab province, Pakistan.

Owing to consistent genetic mutation and recombination, various escape mutants and/or drug-resistant mutants of human immunodeficiency virus (HIV-1) are now emerging worldwide. Therefore, an understanding of the genetic characteristics of prevailing strains, particularly with regard to drug-resistance-associated substitutions, is essential for devising and implementing treatments and disease control interventions in endemic settings such as Pakistan. We processed a total of 130 plasma samples originating from HIV-treatment centers in selected districts of Punjab province, Pakistan. The samples were first screened using an HIV-1 Ag/Ab Combo test followed by amplification of the pol gene (1084 bp) from samples that were positive either for the antigen or for both the antigen and antibodies simultaneously. Screening revealed that a total of 45 samples were positive (34.62%; 95% CI: 26.99-43.13) for either antigen or both antigen and antibodies (n = 18, 40%; 95% CI: 27.02-54.55) or for antibodies alone (n = 27, 60%; 95% CI: 45.45-72.98). A largest number of positive samples was from the district of Lahore (n = 19/43, 44.18%; 95% CI: 30.44-58.9) followed by Faisalabad (n= 12/36, 33.33%; 95% CI: 20.21-49.66), Gujranwala (n = 05/23, 21.7%; 95% CI: 9.66-41.9) and Sargodha (n = 09/28, 32.1%; 95% CI: 17.93-50.66). The probability of occurrence of HIV infection was significantly associated with individuals having a history of injecting drug use (68.08%; OR = 11.15; 95% CI: 53.84-79.61, p = 0.0001). Phylogenetic analysis based on the pol gene showed that the sequences from this study clustered into three distinct clades representing recombinant form 02_AG (n = 14, 77.0%; 95% CI: 54.79-91.00), and subtypes A (n = 2, 11.1%; 95% CI: 3.1-32.8) and G (n = 2, 11.1%; 95% CI: 3.1-32.8). Although we screened 18 samples for drug-resistance-associated mutations, except for an accessory mutation (M46K) in the protease (PR) region in one subject, we found a lack of drug-resistance-associated substitutions in the PR region. On the other hand, we found two subjects (2/18) carrying a resistance-associated mutation (V106I) conferring a low level of resistance against non-nucleoside reverse transcriptase inhibitors. The present study shows that multiple subtypes of HIV-1 are present in the affected population. Continuous disease surveillance coupled with evaluation of drug resistance at higher resolution should be done in future studies.

Seroprevalence of Bluetongue virus in small and large ruminants in Punjab province, Pakistan.

Bluetongue (BT) is a vector-borne disease of immense economic importance for small and large ruminants. Despite frequent disease reports from neighboring countries, a little is known about current disease status and prevalent serotypes in Pakistan. We screened a total of 1312 healthy animals for group-specific antibodies and serotype-specific genome for BT virus through competitive ELISA and real-time PCR, respectively. An overall prevalence of group-specific VP7 antibodies [28.81% (n = 378/1312, 95% CI = 26.4-31.4)] was observed. The prevalence was higher in goats [40.75% (n = 194/476, 95% CI = 36.4-45.3)] followed by buffalo [29.34% (n = 81/276, 95% CI = 24.3-34.9)], sheep [18.40% (n = 60/326, 95% CI = 14.5-22.9)] and cattle [17.94% (n = 42/234, 95% CI = 13.56-23.4)]. The odds of seropositivity were more in buffalo of Nili breed (OR = 2.06, 95% CI = 1.19-3.58) as well as those found with a presence of vector (OR = 2.04, 95% CI = 1.16-3.59). Buffalo and cattle with history of abortion [(OR = 3.95, 95% CI = 1.33-11.69) and (OR = 5.89, 95% CI = 1.80-19.27) respectively] were much likely to be infected with the disease. Serotype 8 was detected in all animal species while, serotypes 4 and 6 were detected in sheep, 2, 6 and 11 in goat, and 2 and 16 in buffalo. The study concludes a much frequent exposure of different serotypes of Bluetongue virus (BTV) in small and large ruminants and indicates its expansion to enzootic range worldwide.

Prevalence and Phylogenetics of H9n2 in Backyard and Commercial Poultry in Pakistan.

Surveillance of H9N2 is currently focused on areas central to the commercial poultry industry. This study determined the prevalence of H9N2 virus in commercial and backyard poultry flocks in Punjab Province, Pakistan. Oral and tracheal swabs were collected from commercial and backyard poultry from January 2015 through June 2016. Antisera against H5, H7, H9, and Newcastle disease viruses were used for virus identification. Molecular confirmation was made by reverse transcription PCR. Avian influenza virus subtypes H5 and H7 were not detected. The H9N2 virus was isolated in 5.7% of 905 tested flocks (5-10 birds/flock). Prevalence in commercial and backyard poultry was 6.7% of 687 flocks and 2.7% of 218 flocks, respectively. Hemagglutinin and neuraminidase-gene-based phylogenetic analysis of commercial and backyard poultry isolates showed 100% homology. Within sublineage B2 of Pakistan, identity among most recent isolates (2015) was 100%, compared to 75%-99% identity with previously isolated viruses (2010-12), indicating continued virus evolution. Most of the previously reported and currently studied viruses were isolated near the Pakistan-India border. Phylogenetic analysis showed that Pakistani and Indian isolates were closely related, indicating that avian influenza virus transmission may occur across this border.

Prevalencia y filogenia del virus de la influenza H9N2 en avicultura de traspatio y comercial en Pakistán. La vigilancia del virus de influenza H9N2 se centra actualmente en áreas centrales de la industria avícola comercial. Este estudio determinó la prevalencia del virus de influenza H9N2 en parvadas comerciales y de traspatio en la provincia de Punjab, Pakistán. Se recolectaron hisopos orales y traqueales de aves comerciales y de traspatio desde enero del 2015 hasta junio del 2016. Se usaron antisueros contra los virus de la enfermedad H5, H7, H9 y contra Newcastle para la identificación del virus. La confirmación molecular se realizó mediante transcripción reversa y PCR. No se detectaron los subtipos H5 y H7 del virus de la influenza aviar. El virus H9N2 se aisló en 5.7% de 905 parvadas analizadas (5-10 aves/parvada). La prevalencia en aves comerciales y domésticas fue del 6.7% de 687 bandadas y de 2.7% de 218 bandadas, respectivamente. El análisis filogenético basado en el gene de la hemaglutinina y de la neuraminidasa de los aislamientos de aves comerciales y de traspatio mostró 100% de identidad genética. Dentro del sublinaje B2 de Pakistán, la identidad entre los aislados más recientes (2015) fue del 100%, en comparación con el 75% al 99% de identidad con los virus aislados previamente (años 2010 al 2012), lo que indica la continua evolución del virus. La mayoría de los virus previamente reportados y estudiados actualmente se aislaron cerca de la frontera entre Pakistán e India. El análisis filogenético mostró que los aislamientos de Pakistán e India estaban estrechamente relacionados, lo que indica que la transmisión del virus de la influenza aviar puede ocurrir a través de esta frontera.

Concurrent oxytocin in women needing second dinoprostone.

OBJECTIVE: To reduce average induction delivery internal in patients with poor Bishop score without compromising fetomaternal outcome (in terms of birth weight, NICU admission, maternal complications and mode of delivery). STUDY DESIGN: A descriptive study. PLACE AND DURATION OF STUDY: Department of Obstetrics and Gynaecology, Pakistan Atomic Energy Commission (PAEC) General Hospital, Islamabad, from February to December 2009. METHODOLOGY: All patients needing 2nd dinoprostone pessary for induction of labour were included in the study. Patients with gestation below 37 weeks, those with intra-uterine growth restriction, bad obstetric history, previous uterine scar and patients in whom Bishop score improved for amniotomy after 1st dinoprostone pessary, were excluded. Data was collected on a special proforma where all variables were defined. RESULTS: Out of 90 patients, 44 (48.8%) had spontaneous vertex deliveries and 12 (13.3%) had instrumental deliveries so a total vaginal deliveries occurred in 56 (62.2%) patients. Thirty four patients (37.8%) had emergency caesarean sections. Main indication for cesarean was failure to progress in 1st stage of labour followed by fetal distress. There were 3 failed inductions. Only 2 patients had hyperstimulation. NICU admission were 8 and all babies were discharged healthy from nursery with no case of early neonatal death. CONCLUSION: Concurrent oxytocin with 2nd dinoprostone in patients with poor Bishop scores (initial scores 2 and 3) resulted in more vaginal birth and comparatively shorter induction delivery time with almost negligible fetomaternal complications.