Nadia Mukhtar

Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds

Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we identified a novel genotype of H9N2 viruses in widespread circulation in poultry in Pakistan that contained polymerases (PB2, PB1 and PA) and non-structural (NS) gene segments identical to highly pathogenic H7N3 viruses. Here, we investigated the potential of these viruses to cause disease and assessed the transmission capability of the virus within and between poultry and wild terrestrial avian species. Groups of broilers, layers, jungle fowl, quail, sparrows or crows were infected with a representative strain (A/chicken/UDL-01/08) of this H9N2 virus and then mixed with naïve birds of the same breed or species, or different species to examine transmission. With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with quail showing the most pronounced clinical signs. Virus shedding was detected in all infected birds, with quail showing the greatest levels of virus secretion, but only very low levels of virus were found in directly infected crow samples. Efficient virus intra-species transmission was observed within each group with the exception of crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens and sparrows and vice versa and efficient transmission was seen in either direction. These results highlight the ease of spread of this group of H9N2 viruses between domesticated poultry and sparrows and show that sparrows need to be considered as a high risk species for transmitting H9N2 viruses between premises.

Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan.

ABSTRACT One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite the existence of high-average NDV-specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV.

Genetic diversity of Newcastle disease virus in Pakistan: a countrywide perspective

BackgroundNewcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease is endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic nature of strains circulating throughout Pakistan is currently lacking.Material and methodsTo ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks, originating from all the provinces of Pakistan, showing high mortality (30–80%). The samples represented domesticated poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to amplification of full fusion gene and subsequent sequencing and phylogenetic analysis.ResultsThe deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif (112RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion gene indicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK) province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strains to 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province.ConclusionsTaken together, data indicated the prevalence of multiple lineages of NDV in different poultry population including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their potential for interspecies transmission and disease diagnosis and control strategies.

Complete Genome Sequence of a Velogenic Neurotropic Avian Paramyxovirus 1 Isolated from Peacocks (Pavo cristatus) in a Wildlife Park in Pakistan

ABSTRACT Avian paramyxovirus serotype 1 (APMV-1) was isolated from an acute and highly contagious outbreak in peacocks (Pavo cristatus) in a wildlife park in Pakistan. A velogenic neurotropic form of APMV-1 caused a 100% case fatality rate and killed 190 peacocks within a week. Biological and serological characterizations showed features of a velogenic strain of APMV-1, and these results were further confirmed by sequence analysis of the cleavage site in the fusion protein. The complete genome of one of the isolates was sequenced, and phylogenetic analysis was conducted. The analysis showed that this isolate belonged to genotype VII, specifically, to subgenotype VIIa, and clustered closely with isolates characterized from Indonesia in the 1990s. Interestingly, the isolate showed significant differences from previously characterized APMV-1 isolates from commercial and rural chickens in Pakistan. The work presented here is the first complete genome sequence of any APMV-1 isolate from wild birds in the region and therefore highlights the need for increased awareness and surveillance in such bird species.

An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA

BackgroundFew serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the “mycoides cluster” frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study.ResultsThe strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the “mycoides cluster”. Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals.ConclusionsThis newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.

Detection of selected arboviral infections in patients with history of persistent fever in Pakistan

Surveillance is a valuable tool for understanding prevailing and previously undiagnosed infections in a geographic area. We examined 480 archived serum samples from patients with history of persistent fever (>40°C, 60-72h) who were referred to hospitals in Rawalpindi/Islamabad, Lahore, and Faisalabad districts for dengue antibody detection in 2014-15. Each sample was processed for detection of antigens and seroconversion, using real-time polymerase chain reaction and enzyme linked immunosorbent assay, respectively, against dengue haemorrhagic fever (DHF) virus serotypes 1-4, West Nile virus fever (WNVF), Crimean-Congo haemorrhagic fever (CCHF), and Chikungunya virus (CGV). The presence of antigens and antibodies to at least one of the studied viral haemorrhagic fevers (VHFs) was detected in 465 (96.8%, 95% CI: 94.9-98.1) and 442 samples (92.1%, 95% CI: 89.3-94.2), respectively. No sera were found positive to CCHF. There was a significant association between gender and positivity to at least one of the VHFs (χ2=8.12, df=1, p<0.005). Except for DHF serotype 2 and 3 (ττ=0.41), Goodman and Kruskals Tau statistic revealed no significant association for occurrence of different viruses within the studied population (ττ=0-0.06). Cosinor analysis confirmed significant seasonality, with a higher number of cases of persistent fever in August through November, peaking in October. The study suggests circulation of multiple arthropod-borne viral infections and, in addition to DHF, ascertain the needs for screening patients for CGV and WNVF too. It also demonstrates the necessity of well-integrated disease surveillance in several geographic regions and at-risk populations in Pakistan to develop appropriate disease and vector control strategies.

Comparative Efficacy of Stabilizers on the Thermostability of Peste des Petits Ruminants Vaccine

Peste des petits ruminants (PPR) is an acute and highly contagious fatal disease of small ruminants mainly affecting sheep and goats, and camels. PPR is endemic disease in many developing and tropical countries including Pakistan, and vaccine is currently the only means of disease control. However, lack of established infrastructures and high temperature, maintaining cold chain for effective delivery of the vaccine is a challenge. All vaccines that are currently being applied for the control of PPR are thermo-labile and require cold chain for stability. In this study, we assessed the efficacy of different stabilizers on the thermostability of live attenuated vaccine of PPR virus (PPRV). Five formulations of four thermo stabilizers including lactalbuminhydrolysate sucrose (LS), trehalose dihydrate (TD), weybridge medium (WBM) and buffered gelatin sorbitol (BUGS) were used to prepare vaccine using Nigeria 75/1 strain of PPR virus. Results revealed that vaccine having LS proved maximum stability as it reduced minimal viral infectivity (0.42 log10TCID50) during lyophilization as well as three time higher shelf life (3.69 days) as compared to commercial vaccine (1.23 days) at 37°C. Moreover, it was also revealed that PPR vaccine lyophilized with LS, WBM and TD, and was more stable than commercial vaccine having shelf life of 206-304 days at 4°C, 8-10 days at 25°C and 2.5-3.7 days at 37°C. Using regression analysis on the shelf life of vaccine preparations in different thermo stabilizers, we can demonstrate that BUGS provided minimal stability as well as maximum viral infectivity loss (1.49 log10TCID50) after lyophilization. Taken together, we provide foundation studies to improve the thermostability of PPRV to facilitate the immunization of susceptible animals in situations where meeting cold chain is not feasible. Efforts are required to genetically select strains that confer protection at higher temperature without compromised immunogenicity which is potentially feasible with the established reverse genetics.

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.

Cloning and expression of NS3 gene of Pakistani isolate type 2 dengue virus

Abstract Introduction Dengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines. Material and Methods Blood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors. Results The gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells. Conclusion Recombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.

Oral Health Assessment, an Epidemiological Survey among Dental Patients from Lahore, Pakistan

| objective:Present study was conducted to assess the oral health practices, oral hygiene habits and associated dental problems among subjects from Lahore, Pakistan. Methods: This cross sectional epidemiological study was consisted of sixty two, 11-56 years old dental subjects from both genders enrolled from Jinnah hospital, Lahore for oral examination. Questionnaire included the questions regarding basic demographic information, medical history, life style and behavior and dietary history was distributed among enrolled subjects and they were interviewed using the World Health Organization health protocols. results: From the dental enrolled subjects, 30.6% patients have chronic diseases which included kidney dysfunction, digestive problems, respiratory diseases, Diabetes, bone and joint problems and hypertension. The prevalence of cavities was high 82.3% among dental patients and remaining have swollen gums, abscess and pain and of them 75.8% were underwent through past treatment of dental filling, implant, extraction and root canal. Teeth sensitivity and chewing difficulty was experienced by 88.7% of subjects. For dental hygiene, tooth brushing was carried out by 85.5%, miswak by 9.7% and low percentage of rinsing practice that was 4.8%. 91.9% of the subjects have visited the dentist when needed and the remaining percentage have practice of dentist visit one month or two months. About dietary history of usage of artificial sweetener, candies, ice-cream, rusk, carbonated beverages, chewing gums, cookies, snakes, eggs, meat, milk, cheese, yogurt, fruits, vegetables etc. was varied among subjects with respect to daily, weekly and never use. conclusion: Awareness programs should be conducted to promote oral health awareness and its impact on systemic health amongst the general population of Lahore. Continuous implementation of oral health awareness programs in community can improve the oral health and better oral health practices results in better quality of life.