ANTIGENIC AND MOLECULAR CHARACTERIZATION OF INFLUENZA VIRUSES ISOLATED FROM PATIENTS WITH RESPIRATORY TRACT INFECTIONS IN NAIROBI II

Influenza virus infection is a highly contagious respiratory disease that can spread easily and that is responsible for considerable morbidity and mortality each year. There are three types of influenza viruses, designated A, Band C all which cause the disease characteristically recognized as influenza. Types A and B influenza viruses can cause a wide spectrum of illness, including lower respiratory tract disease, pneumonia and even, in the case of type A, encephalopathy and encephalitis. On the other hand type C infections are limited to the upper respiratory tract. While antivirals are available against the virus, vaccination remains the most important measure for reducing this sizeable public health burden. However, as a result of the antigenic variation of its surface glycoproteins, the influenza virus is an ever-changing target. Influenza vaccines therefore present special challenges because each year the vaccine strains must be updated in order to retain optimal protective efficiency. In the face of a looming pandemic, there is need to step up surveillance and characterize local strains inorder to identify vaccine strain variants and a potential pandemic strain. This study is the first attempt to characterize local influenza strains. Between January and August 2005, 660 throat swab samples were collected from patients in eleven sentinel sites around Nairobi that are involved in influenza surveillance. The samples were processed for immunofluorescence, stained using SimulfluorTM Flu AlFlu B reagent and examined under a fluorescent microscope. 252 (38.2%) samples were found to be influenza B while none was positive for influenza A. The positive samples were inoculated and cultured onto MDCK cell mono layers in tissue culture tubes. A 'blind' passage was performed and the samples frozen at -85°C. Haemagglutination (HA) using 1% guinea pig red blood cells was performed on recovered isolates to determine haemagglutinating isolates and 135 (54.4%) samples gave a positive HA result. These were confirmed to be influenza viruses and subtyped by the Haemagglutination inhibition test (HAl) using the 2005 reference reagents for influenza virus diagnosis prepared by the WHO Collaborating Center for Reference and Research on Influenza, Melbourne, Australia. 134 (99.25%) isolates were characterized as influenza B/Shanghai/361/2002-like strains while 1 (0.75%) isolate was identified as an influenza B/Hong Kong/330/2001-like strain. RT-PCR was performed on the HAl positive isolates that had different titres from the vaccine strain and some which had similar titres with the vaccine strain. This was followed by a sequencing PCR and sequencing of the HA 1 domain of the HA gene carried out in an automated sequencer ABI 310. 8 isolates were successfully sequenced on the basis of the HAl titres observed in HAl and a BLAST analysis used to identify similar strains. All the sequences that were generated in this study were analyzed phylogenetically. Multiple sequence alignments and phylogenetic analysis of the nucleotide sequences obtained was carried out by ClustalW software inorder to generate phylogenetic trees. Influenza A was not isolated in this study, it's presence in tropical African countries is usually associated with epidemics and none was experienced in the study period. At least 3 variants of the vaccine strain were identified indicating that the local strains are drifting away from the vaccine strain. The nucleotide sequences were translated using the EMBOSS software and the resultant amino acid sequences used to generate phylogenetic trees. Based Oi1 the protein sequences, all the isolates were found to be closely related showing that antigenic drift observed is slow. Findings in this study and others globally have led to the WHO changing the influenza B vaccine component for the 2006 season. This study reveals that local strains are progressively undergoing antigenic drift and recommends wider continued active surveillance.