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

Timmermans A, Melendrez MC, Se Y, Chuang I, Samon N, Uthaimongkol N, Klungthong C, Manasatienkij W, Thaisomboonsuk B, Tyner SD, Rith S, Horm VS, Jarman RG, Bethell D, Chanarat N, Pavlin J, Wongstitwilairoong T, Saingam P, El BS, Fukuda MM, Touch S, Sovann L, Fernandez S, Buchy P, Chanthap L, Saunders D - PLoS ONE (2016)

Bottom Line: Most genes showed signatures of purifying selection.A single specimen of EV71 was found.Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, US Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

ABSTRACT
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.

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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.
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pone.0152529.g005: 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.

Mentions: All sequences were highly related with no more than 1.67% divergence between the strains detected in the study and respective seed viruses included in the vaccine composition (A/California/7/2009, A/Brisbane/10/2010, and A/Christchurch/16/2010; S5 Table, Fig 5) for any of the segments analyzed. When compared to the A/California/7/2009 vaccine strain, all Cambodian viruses on average had 11 amino acid changes within the HA segment. Amino acid changes found in the HA gene can be found in Table 4. No evidence for amino acid substitutions leading to changes in glycosylation was seen in the HA dataset; however, several changes among sequenced samples occurred in antigenic sites, polymorphic sites or had a recorded effect in in vitro analyses per the literature (S6 Table). For the NA gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or were found in polymorphic or antigenic sites (epitopes) (S6 Table). For the MP gene, all samples contained the S31N mutation that may confer amantadine resistance as determined by the Genbank Influenza Viral Resource Annotation tool [32]. No features or changes of interest were seen within the NP or NS segment. The HA and NA segments contained the most amino acid substitutions when compared to the reference (A/California/7/2009) as well as to the yearly defined groups (2011 and 2012). The MP and NP segments showed the fewest substitutions (Table 4). There were no samples within the HA amino acid analysis that had unique substitutions relative to the global sequences. However, the NA gene had unique amino acid substitutions not found with the global references; the function of these substitutions is unknown (S7 Table). Selection pressure was determined using the dN/dS statistic. Values of dN/dS > 1 are indicative of positive or adaptive selection, values <1 are indicative of purifying or negative selection and a value = 1 indicates neutral or no selection ongoing in the dataset. All samples were under purifying selection for all segments with exception to the NS segment of the 2011 Cambodian samples, which had a dN/dS value of 1.0 suggesting neutral selection (S8 Table).


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

Timmermans A, Melendrez MC, Se Y, Chuang I, Samon N, Uthaimongkol N, Klungthong C, Manasatienkij W, Thaisomboonsuk B, Tyner SD, Rith S, Horm VS, Jarman RG, Bethell D, Chanarat N, Pavlin J, Wongstitwilairoong T, Saingam P, El BS, Fukuda MM, Touch S, Sovann L, Fernandez S, Buchy P, Chanthap L, Saunders D - PLoS ONE (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814059&req=5

pone.0152529.g005: 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.
Mentions: All sequences were highly related with no more than 1.67% divergence between the strains detected in the study and respective seed viruses included in the vaccine composition (A/California/7/2009, A/Brisbane/10/2010, and A/Christchurch/16/2010; S5 Table, Fig 5) for any of the segments analyzed. When compared to the A/California/7/2009 vaccine strain, all Cambodian viruses on average had 11 amino acid changes within the HA segment. Amino acid changes found in the HA gene can be found in Table 4. No evidence for amino acid substitutions leading to changes in glycosylation was seen in the HA dataset; however, several changes among sequenced samples occurred in antigenic sites, polymorphic sites or had a recorded effect in in vitro analyses per the literature (S6 Table). For the NA gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or were found in polymorphic or antigenic sites (epitopes) (S6 Table). For the MP gene, all samples contained the S31N mutation that may confer amantadine resistance as determined by the Genbank Influenza Viral Resource Annotation tool [32]. No features or changes of interest were seen within the NP or NS segment. The HA and NA segments contained the most amino acid substitutions when compared to the reference (A/California/7/2009) as well as to the yearly defined groups (2011 and 2012). The MP and NP segments showed the fewest substitutions (Table 4). There were no samples within the HA amino acid analysis that had unique substitutions relative to the global sequences. However, the NA gene had unique amino acid substitutions not found with the global references; the function of these substitutions is unknown (S7 Table). Selection pressure was determined using the dN/dS statistic. Values of dN/dS > 1 are indicative of positive or adaptive selection, values <1 are indicative of purifying or negative selection and a value = 1 indicates neutral or no selection ongoing in the dataset. All samples were under purifying selection for all segments with exception to the NS segment of the 2011 Cambodian samples, which had a dN/dS value of 1.0 suggesting neutral selection (S8 Table).

Bottom Line: Most genes showed signatures of purifying selection.A single specimen of EV71 was found.Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, US Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus