Evolutionary analysis of influenza A(H1N1)pdm09 during the pandemic and post-pandemic period in Pakistan.

BACKGROUND: The first case of influenza A(H1N1)pdm09 was detected in Pakistan in June 2009. Since then, it has continued to circulate causing considerable morbidity and mortality. The purpose of this study was to evaluate the evolutionary changes in influenza A(H1N1)pdm09 viruses from 2009 to 2016 and their relevance to the current vaccine viruses. METHODS: Respiratory specimens (throat or nasopharyngeal swabs) were collected from patients with influenza-like illness and severe acute respiratory illness. Samples were processed following the protocol of the US Centers for Disease Control and Prevention. Sequencing and phylogenetic analysis of Haemagglutinin and neuraminidase genes were carried out on representative isolates of Pakistan viruses. RESULTS: Between January 2009 and February 2016, out of 16,024 samples analysed, 1950 (12%) were positive for influenza A. During the pandemic period (2009-2010), influenza A(H1N1)pdm09 was the dominant strain with 366 out of 808 (45%) total influenza positive cases. In the post-pandemic period (2011-2016), a total of 1078 out of 1911 (56%) cases were positive for influenza A(H1N1)pdm09 with co-circulation of different influenza A subtypes. The Pakistan A(H1N1)pdm09 viruses belonged to two genetic clades: clade 7 in the pandemic period, and clade 7 (2011) and clade 6B (2015) in the post-pandemic period. Sequence analysis of genes coding for surface glycoprotein's of Haemagglutinin and neuraminidase had a high degree of sequence similarity with corresponding genes of regional viruses circulating in South-East Asia. CONCLUSION: Influenza A(H1N1)pdm09 viruses from Pakistan clustered into two genetic clades, with co-circulation of some variants. Key substitutions in the receptor binding site and a few changes indicative of virulence were also detected in the post-pandemic strains. Continued monitoring of the viruses is essential for early identification of potential variants of high virulence and their relevance to current vaccine strains.

Rotavirus surveillance in Pakistan during 2015-2016 reveals high prevalence of G12P[6].

The G12 rotavirus genotype has emerged globally since their first detection in 1987 from the Philippines; however it remains a rare cause of gastroenteritis in Pakistan. Rotavirus surveillance conducted during 2015-2016, assessed 3446 children <5 years hospitalized for gastroenteritis and found 802 (23.2%) positive on ELISA. Genotyping of a subset of positive samples (n = 319) revealed G12P[6] (11.28%) as the third most common G/P combination following G3P[8] (28.5%) and G1P[8] (12.5%); G2P[4] (10.65%) and G3P[6] (8.15%) were other frequently detected strains. Phylogenetic analysis of G12 strains from Pakistan revealed high genetic similarity to G12 strains from Italy, Thailand, Korea, and Great Britain as well as local strains within G12 lineage III. In conclusion, G12P[6] was a major contributor of RVA gastroenteritis in Pakistani children. Robust surveillance after the introduction of rotavirus vaccines will help determine the evolution of G12 and other circulating genotypes in the country.

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.

Zoonotic potential of highly pathogenic avian H7N3 influenza viruses from Pakistan.

H5 and H7 avian influenza viruses can become highly pathogenic in chickens after interspecies transmission. These viruses have transmitted directly to humans from birds in Eurasia and Africa (H5N1), the Netherlands (H7N7), and Canada (H7N3). Here we report antigenic, sequence, and phylogenetic analyses of H7N3 viruses isolated from chickens in Pakistan from 1995 to 2002. We compared the pathogenic and zoonotic potential of the Pakistani viruses in avian and mammalian hosts. In chickens, all of the isolates showed high pathogenicity with poor transmissibility to contact birds. Viral shedding from the trachea and cloaca was equivalent, but cloacal shedding occurred longer; dissemination of virus into the tissues was widespread. In contrast, the viruses replicated poorly in 6-week-old mallard ducks. In mammalian hosts, of the two Pakistani H7N3/02 viruses that caused weight loss, one also caused 40% mortality in mice without prior adaptation, and preliminary experiments in ferrets showed significant virus multiplication in the lungs, intestine, and conjunctiva. We conclude that the H7N3/02 isolates from Pakistan show limited antigenic drift and have evolved slowly during their 8-year circulation in chickens; however, these viruses have the potential to infect mammals.