Epidemiological and virological characteristics of influenza viruses circulating in Cambodia from 2009 to 2011

Background: The Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011. Methodology/Principal Findings: Sentinel and study sites collected nasopharyngeal specimens for diagnostic detection, virus isolation, antigenic characterization, sequencing and antiviral susceptibility analysis from patients who fulfilled case definitions for influenza-like illness, acute lower respiratory infections and event-based surveillance. Each year in Cambodia, influenza viruses were detected mainly from June to November, during the rainy season. Antigenic analysis show that A/H1N1pdm09 isolates belonged to the A/California/7/2009-like group. Circulating A/H3N2 strains were A/Brisbane/10/2007-like in 2009 before drifting to A/Perth/16/2009-like in 2010 and 2011. The Cambodian influenza B isolates from 2009 to 2011 all belonged to the B/Victoria lineage represented by the vaccine strains B/Brisbane/60/2008 and B/Malaysia/2506/2004. Sequences of the M2 gene obtained from representative 2009–2011 A/H3N2 and A/H1N1pdm09 strains all contained the S31N mutation associated with adamantanes resistance except for one A/H1N1pdm09 strain isolated in 2011 that lacked this mutation. No reduction in the susceptibility to neuraminidase inhibitors was observed among the influenza viruses circulating from 2009 to 2011. Phylogenetic analysis revealed that A/H3N2 strains clustered each year to a distinct group while most A/H1N1pdm09 isolates belonged to the S203T clade. Conclusions/Significance: In Cambodia, from 2009 to 2011, influenza activity occurred throughout the year with peak seasonality during the rainy season from June to November. Seasonal influenza epidemics were due to multiple genetically distinct viruses, even though all of the isolates were antigenically similar to the reference vaccine strains. The drug susceptibility profile of Cambodian influenza strains revealed that neuraminidase inhibitors would be the drug of choice for influenza treatment and chemoprophylaxis in Cambodia, as adamantanes are no longer expected to be effective

Monthly distribution of influenza virus detected from 2009 to 2011.

<p>Monthly distribution of influenza virus detected from 2009 to 2011.</p

Human Sentinel Surveillance of Influenza and Other Respiratory Viral Pathogens in Border Areas of Western Cambodia

<div><p>Little is known about circulation of influenza and other respiratory viruses in remote populations along the Thai-Cambodia border in western Cambodia. We screened 586 outpatients (median age 5, range 1–77) presenting with influenza-like-illness (ILI) at 4 sentinel sites in western Cambodia between May 2010 and December 2012. Real-time reverse transcriptase (rRT) PCR for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ILI-specimens negative for influenza were cultured, followed by rRT-PCR for enterovirus and rhinovirus (EV/RV) and EV71. Influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from June to November. Isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in Cambodia. Influenza vaccination coverage was low (<20%). Western Cambodian H1N1(2009) isolate genomes were more closely related to 10 earlier Cambodia isolates (94.4% genome conservation) than to 13 Thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the Thai references. Most genes showed signatures of purifying selection. Viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus A4, A6, A8, A9, A12, B3, B4 and echovirus E6 and E9 using nested RT-PCR methods. A single specimen of EV71 was found. Despite proximity to Thailand, influenza epidemiology of these western Cambodian isolates followed patterns observed elsewhere in Cambodia, continuing to support current vaccine and treatment recommendations from the Cambodian National Influenza Center. Amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.</p></div

Human Sentinel Surveillance of Influenza and Other Respiratory Viral Pathogens in Border Areas of Western Cambodia - Fig 5

pH1N1 maximum likelihood phylogenetic trees (aLRT support >70 for all vaccine clades and major nodes) for the (A) HA and (B) NA segments for 14 samples from Cambodia and references in Genbank (grey) collected in 2011 and 2012. Vaccine strains are highlighted in red. Samples are colored coded by sampling site.</p

Phylogenetic analysis of the HA genes (1685nt (nt1–1685)) of influenza A/H1N1pdm09 virus isolates collected in Cambodia from 2009 to 2011.

<p>The phylogenetic analysis was conducted as a distance-based neighbour-joining phylogenetic tree of influenza using the HKY model and generated using PAUP software with 1,000 bootstrap replicates (values ≥60 shown on branch) and rooted to A/California/07/2009. Major amino acid changes are shown in block letter at the appropriate nodes. The vaccine strain is boxed. Scale bar indicates number of nucleotide substitution per site.</p

Influenza and other respiratory viruses detected between 2010–2012.

<p>(A) Number of ILI and influenza subtypes, May 2010-Dec 2012. (B) Number of other respiratory viruses among influenza-negative ILI specimens May 2010-Dec 2012.</p

Neuraminidase inhibitors resistance in influenza viruses, 2009–2011.

<p>Neuraminidase inhibitors resistance in influenza viruses, 2009–2011.</p

Dataset descriptions and models used in maximum likelihood phylogenetic analysis.

<p>Dataset descriptions and models used in maximum likelihood phylogenetic analysis.</p

Influenza isolates collected in Cambodia by type and subtype in 2009-2011.

a<p>Specimens analyzed by the WHO Collaborating Center in Melbourne from 2009 to 2011.</p>b<p>H1N1pdm09.</p>c<p>Influenza B.</p><p>Influenza isolates collected in Cambodia by type and subtype in 2009-2011.</p