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.

Genomic Diversity and Evolution of SARS-CoV-2 Lineages in Pakistan.

The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

Evaluation of haematological, serum biochemical and oxidative stress parameters in cattle naturally infected with lumpy skin disease virus.

Lumpy skin disease (LSD) is a vector-borne viral transboundary disease of cattle caused by the LSD virus (LSDV). Despite investigations on clinical and outbreak features of LSDV, information on disease pathogenesis and alternative changes in blood parameters are scarce. Keeping this in view, the current study was designed to determine haematological, serum biochemical, and oxidative stress parameters in naturally infected cattle with LSDV during the recent surge of outbreaks in Punjab, Pakistan. A total of 35 blood samples was collected from polymerase chain reaction-confirmed LSDV-infected cattle for assessment of all parameters. The haematological examination of blood samples showed a significant reduction (p < 0.05) in different variables of erythrogram and leucogram. On the other hand, differences between levels of various serum biochemical parameters with the significant increase in levels of alkaline phosphatase, serum alanine aminotransferase, aspartate aminotransferase, and blood urea nitrogen were observed in LSDV naturally infected cattle. Moreover, malondialdehyde levels for lipid peroxidation and nitrate concentration were markedly elevated whereas glutathione S-transferase fluorescent and serum superoxide dismutase enzymes showed a decrease in levels. The current study suggests that alternations in haematological and serum biochemical parameters following LSDV infection stimulate oxidative stress and such findings may be useful for early and rapid diagnosis and improvement in the treatment strategy of the disease.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

Rate of Multiple Viral and Bacterial CoInfection(s) in Influenza A/H9N2-Infected Broiler Flocks.

Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (Mycoplasma gallisepticum; Mycoplasma synovae; Ornithobacterium rhinotracheale [ORT]) using PCR assays. Upon initial screening for IAV, 35/60 (58.3%) flocks tested positive. The coinfection of IAV/H9N2 with other pathogens was detected in 25 (71.4%) flocks and only IAV/H9N2 was detected in 10 (28.6%) flocks out of total positive IAV flocks (n = 35). IAV subtypes H5 and H7, ILT, and ORT were not detected throughout the study period. The detection rate of double, triple, and quadruple combinations of coinfections with IAV/H9N2 were 37% (13 flocks), 26% (9 flocks), 9% (3 flocks), respectively. Higher average mortality (28.5%) was found in broiler chicken flocks coinfected with viral and/or bacterial pathogens than in flocks where only H9 low pathogenic IAV/H9N2 was detected (20.8%). In conclusion, higher circulation of IAV/H9N2 with other viral and bacterial pathogens may contribute to higher production and economic losses at the farm level.

Nota de investigación- Tasa de coinfecciones virales y bacterianas múltiples en parvadas de pollos de engorde infectadas con virus influenza A/H9N2. Se han reportado varios casos del virus de influenza A de baja patogenicidad H9N2 (IAV/H9N2) en pollos comerciales desde su aparición en 1998 en Pakistán. Sin embargo, recientemente se ha observado un aumento de la mortalidad y complicaciones respiratorias graves en condiciones de campo, lo que sugiere infecciones concomitantes de influenza con patógenos respiratorios virales y/o bacterianos. Por lo tanto, el presente estudio tuvo como objetivo investigar la presencia del virus de influenza aviar H9N2 coinfectando con múltiples patógenos virales y bacterianos en parvadas de pollos de engorde. Se evaluaron 60 parvadas de pollos de engorde con signos respiratorios desde marzo hasta julio del año 2019 en Punjab, Pakistán. Las parvadas sospechosas fueron analizadas para detectar la presencia del virus de influenza aviar utilizando un dispositivo de flujo lateral. Se recolectaron muestras traqueales, cloacales y de médula ósea y se analizaron para detectar siete agentes virales (anemia infecciosa aviar, enfermedad de Newcastle, bronquitis infecciosa, laringeotraqueítis infecciosa [ILT] y subtipos H9, H7 y H5 de influenza aviar) y tres agentes bacterianos (Mycoplasma gallisepticum ; Mycoplasma sinovae; Ornithobacterium rhinotracheale [ORT]) utilizando ensayos de PCR. Tras la detección inicial del virus de la influenza aviar, 35/60 (58.3 %) parvadas resultaron positivas. La coinfección del virus de la influenza H9N2 con otros patógenos se detectó en 25 (71.4 %) parvadas y el virus de influenza aviar H9N2 fue detectado solo en 10 (28.6 %) parvadas del total de parvadas positivas (n = 35). Los subtipos H5 y H7 del virus de influenza, ILT y ORT no se detectaron durante el período de estudio. La tasa de detección de combinaciones dobles, triples y cuádruples de coinfecciones con el virus de influenza H9N2 fue del 37 % (13 parvadas), del 26% (9 parvadas), del 9 % (3 parvadas), respectivamente. Se encontró una mortalidad promedio más alta (28.5 %) en lotes de pollos de engorde coinfectados con patógenos virales y/o bacterianos que en lotes donde solo se detectó al virus de influenza H9 de baja patogenicidad (20.8%). En conclusión, una mayor circulación del virus de influenza aviar H9N2 con otros patógenos virales y bacterianos puede contribuir a mayores pérdidas en la producción y económicas a nivel de granja.

Genetic Characterization of Highly Pathogenic Avian Influenza A(H5N8) Virus in Pakistani Live Bird Markets Reveals Rapid Diversification of Clade 2.3.4.4b Viruses.

The highly pathogenic (HPAI) avian influenza A(H5N1) viruses have undergone reassortment with multiple non-N1-subtype neuraminidase genes since 2008, leading to the emergence of H5Nx viruses. H5Nx viruses established themselves quickly in birds and disseminated from China to Africa, the Middle East, Europe and North America. Multiple genetic clades have successively evolved through frequent mutations and reassortment, posing a continuous threat to domestic poultry and causing substantial economic losses. Live bird markets are recognized as major sources of avian-to-human infection and for the emergence of zoonotic influenza. In Pakistan, the A(H5N1) virus was first reported in domestic birds in 2007; however, avian influenza surveillance is limited and there is a lack of knowledge on the evolution and transmission of the A(H5) virus in the country. We collected oropharyngeal swabs from domestic poultry and environmental samples from six different live bird markets during 2018-2019. We detected and sequenced HPAI A(H5N8) viruses from two chickens, one quail and one environmental sample in two markets. Temporal phylogenetics indicated that all novel HPAI A(H5N8) viruses belonged to clade 2.3.4.4b, with all eight genes of Pakistan A(H5N8) viruses most closely related to 2017 Saudi Arabia A(H5N8) viruses, which were likely introduced via cross-border transmission from neighboring regions approximately three months prior to virus detection into domestic poultry. Our data further revealed that clade 2.3.4.4b viruses underwent rapid lineage expansion in 2017 and acquired significant amino acid mutations, including mutations associated with increased haemagglutinin affinity to human α-2,6 receptors, prior to the first human A(H5N8) infection in Russian poultry workers in 2020. These results highlight the need for systematic avian influenza surveillance in live bird markets in Pakistan to monitor for potential A(H5Nx) variants that may arise from poultry populations.

Prevalence and phylogenetic analysis of Crimean-Congo hemorrhagic fever virus in ticks collected from Punjab province of Pakistan.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease of human that caused by CCHF virus. To study the epidemiological distribution of CCHFV, 2183 tick samples were collected from sheep, goats, cattle and buffalo of different livestock farms of ten districts of Punjab province of Pakistan. Detection of CCHFV was done using enzyme link immunosorbent assay (ELISA) after proper identification of tick samples. The partial S-segment of CCHFV from ELISA positive tick samples was amplified by PCR and sequenced to determine the genotype of CCHFV. Out of2183 collected tick samples, 1913 ticks belonged to 5 species of genus Hyalomma as H. antolicum (48%), H. marginatum (30.2%), H. rufipes (10.82%), H. impressum (5.43%) and H. dromedarii (5.27%). While 270 ticks belonged to 3 species of genus Rhipicephalus as R. microplus (44.8%), R. sanguineus (32.22%) and R. turanicus (24.8%). The overall antigenic prevalence of CCHFV was found to be 12.13% in collected tick samples and 21 tick pools were sequenced for partial S-segment of CCHFV. All of the 21 tick pools were clustered in genotype IV (Asia-1). The highest prevalence of CCHFV was found in district Chakwal (24.13%) followed by Mianwali (23.68%), Rawalpindi (23.07%), Attock (20.0%), Rajanpur (10.52%) and Lahore (8.33%). In positive tick pools, the highest prevalence of CCHFV antigen was found in H. antolicum (39.6%) followed by H. marginatum (30.18%), H. rufipes (13.2%), H. impressum (3.77%), H. dromedarii (1.88%), R. microplus (5.66%) and R. sanguineus (5.66%). The current study confirms the presence of CCHFV in the ticks population of Punjab. The CCHF virus present in Punjab belongs to Asia-1 genotype. It is important to control the tick infestation of the animals present in these areas. So that the transmission cycle of CCHF can be inhibited.

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.

Avian Influenza A(H9N2) Virus in Poultry Worker, Pakistan, 2015.

Avian influenza A(H9N2) virus isolated from a poultry worker in Pakistan in 2015 was closely related to viruses detected in poultry farms. Observed mutations in the hemagglutinin related to receptor-binding affinity and antigenicity could affect cross-reactivity with prepandemic H9N2 vaccine strains.

Phylogenomics and Infectious Potential of Avian Avulaviruses Species-Type 1 Isolated from Healthy Green-Winged Teal (Anas carolinensis) from a Wetland Sanctuary of Indus River.

Given the importance of Avian avulaviruses (AAvVs) in commercial poultry, continuous monitoring and surveillance in natural reservoirs (waterfowls) is imperative. Here, we report full genomic and biologic characterization of two virulent AAvVs isolated from apparently asymptomatic green-winged teal (Anas carolinensis). Genetic characterization (genome length, coding potential, and presence of typical cleave motif [112RRQKR| F117]) and biologic assessment (HA, log 29; mean death time, 49.2-50 hr; 10-6.51 50% egg infective dose [EID50]/0.1 mL; and 1.5 intracerebral pathogenicity index [ICPI] value) revealed virulence of both isolates. Phylogenetic analysis of the complete genome and hypervariable region of the fusion (F) gene revealed clustering of both isolates within class II strains in close association with domestic poultry-origin AAvVs representing genotype VII and subgenotype VIIi. The inferred residue analysis of F and hemagglutinin-neuraminidase genes showed a number of substitutions in critical domains compared with reference strains of each genotype (I-XVIII). The isolates showed a high nucleotide resemblance (99%) with strain isolated previously from backyard poultry; however, they also showed a variable similarity (16.1% to 19.3%) with the most commonly used vaccine strains, Mukteswar (EF201805) and LaSota (AF077761). In accordance with pathogenicity assessment and horizontal transmission, the clinical and histopathologic observations in experimental chickens indicated the velogenic viscerotropic nature of AAvV 1 isolates. Taken together, this study confirms the evolutionary nature of AAvVs and their potential role in disease occurrence, necessitating continuous surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and potential impacts on commercial poultry.

Análisis filogenético y potencial infeccioso de avulavirus aviares de tipo 1 aislados de cercetas americanas (Anas carolinensis) de un santuario en los humedales del río Indo Dada la importancia de los avulavirus aviares en la avicultura, es imperativo tanto el monitoreo como la vigilancia continuos en los reservorios naturales (aves acuáticas). En este artículo se describe la caracterización genética completa y las características biológicas de dos avulavirus aviares virulentos aislados de cercetas americanas (Anas carolinensis) aparentemente asintomáticas. La caracterización genética (longitud del genoma, potencial de codificación y presencia del motivo típico de disociación [112RRQKR| F117]) y la evaluación biológica (ensayo de hemaglutinación [HA], log 29; tiempo promedio de mortalidad, 49.2­50 horas; 10­6.51 dosis infectantes50% [EID50] /0.1mL y el índice de patogenicidad intracerebral [ICPI] de 1.5, revelaron la virulencia de ambos aislamientos. El análisis filogenético del genoma completo y la región hipervariable del gene de fusión (F) revelaron la agrupación de ambos aislamientos con cepas de la clase II en estrecha asociación con los avulavirus de origen avícola que representan el genotipo VII y el subgenotipo VIIi. El análisis de residuos deducidos de los genes F y de la hemaglutininaneuraminidasa mostró varias sustituciones en los dominios críticos en comparación con las cepas de referencia de cada genotipo (IXVIII). Los aislamientos mostraron una gran semejanza en la secuencia de nucléotidos (99%) con una cepa aislada previamente de aves de traspatio; sin embargo, también mostraron similitudes variables (de 16.1% a 19.3%) con las cepas de vacunas más utilizadas, Mukteswar (EF201805) y LaSota (AF077761). De acuerdo con la evaluación de patogenicidad y la transmisión horizontal, las observaciones clínicas e histopatológicas en los pollos experimentales indicaron la naturaleza velogénica viscerotrópica de estos aislamientos de avulavirus del tipo 1. En conjunto, este estudio confirma la naturaleza evolutiva de los avulavirus aviares y su posible papel en la aparición de enfermedades, lo que requiere una vigilancia continua de las aves migratorias acuáticas para dilucidar mejor la epidemiología de la infección y el posible impacto en las aves comerciales.

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.