Isolation and characterization of influenza viruses circulating among avian, swine and human populations in Sri Lanka

Abstract

Ecological and phylogenic studies suggest that wild waterfowl are the natural reservoirs of influenza A viruses. All HA and NA subtypes are found in avian species especially wild aquatic birds, whereas viral subtypes of mammalian influenza viruses are limited. Restricted growth of avian influenza viruses in mammalian species is thought to be a barrier to the emergence of new pandemic strains with direct avian to human transmission. In Sri Lanka, although epidemiological and virological studies on outbreaks of influenza on humans have been carried out, there are no reported data on influenza subtypes circulating among human, swine and avian populations during any specified periods. In this study 300 nasopharyngeal aspirates (NPA) obtained from human patients with acute respiratory tract infection, 750 cloacal samples, 750 tracheal samples and 750 blood samples from birds , and 300 tracheal and 300 blood samples from pigs were analyzed to identify the influenza subtypes circulating among these different populations in the country. Of the 300 NPA samples, 138 were screened by the indirect immunofluorescence test followed by the direct immunofluorescence test (DFA) to identify the viral etiological agents responsible for the infection: Influenza A (20/138), para influenza group (2/138), adeno (3/138) and respiratory syncitial virus (3/138) were identified. All 300 NPA samples were inoculated on to MDCK cell cultures and haemagglutination and DFA test were performed on the cultures that showed CPE: 24 Influenza A and 9 Influenza B viruses were isolated and were characterized as Influenza A Panama/2000/99 (H3N2) and Influenza B/Sichuan/379/99-like virus, using reference antisera supplied by the World Health Organization. One virus (Influenza A/swine/Sri lanka/48/04) was isolated from a pig brought to the Government slaughter house in Dematagoda and was sub-typed as H3N2. Genetic studies carried out on this isolate showed that this virus was identical to the H3N2 viruses isolated in humans during the study period. Serological analysis revealed that 61.66 percent of the sampled population also had antibodies to this virus and that none had antibodies to the common subtypes of swine influenza A viruses namely, A/HK/4167/1999 H1N1, A/HK/2422/1998/H3N2 Sydney-like and A/HK/1774/1999 Europian swine-like control viruses. Influenza viruses could not be isolated from the tracheal and cloacal samples collected from poultry when the samples were inoculated into 9-11 day old embryonated eggs. However, the serum collected from this avian population when tested in a HAI assay using H6 (1720) and H9 (Y280) reference antigens, showed evidence of infection with both these subtypes: 12.8 percent (96/750) and 5.2 percent (39/750) of birds had antibodies against A/quail/HK 1721-30/99 (H6N2) and H9 A/duck/ Hong Kong /Y280 (H9N2) subtypes respectively and 3.6 percent had sero-converted to both subtypes of viruses indicating dual infection.