Longitudinal surveillance of 2 live bird markets in Cambodia revealed year-round, high co-circulation of H5, H7, and H9 influenza viruses. We detected influenza A viruses in 51.3% of ducks and 39.6% ...of chickens, and co-infections, mainly by H5 and H9 viruses, in 0.8% of ducks and 4.5% of chickens.
Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations ...during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift, and half of the variants detected within-host are never detected on the H5N1 virus phylogeny. However, we do detect a subset of mutations linked to human receptor binding and replication (PB2 E627K, HA A150V, and HA Q238L) that arose in multiple, independent humans. PB2 E627K and HA A150V were also enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adaptive. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection, many of which are detected at >5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses.
Thirty-five human influenza A(H5N1) cases were reported in Cambodia during 2013-2014 after emergence of a clade 1.1.2 reassortant virus. We tested 881 villagers and found 2 cases of pauci- or ...asymptomatic infection. Seroprevalence after emergence of the reassortant strain (0.2%) was lower than the aggregate seroprevalence of 1.3% reported in earlier studies.
The Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011.
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.
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.
Highly pathogenic avian influenza A (H5N1) virus has been of public health concern since 2003. Probable risk factors for A(H5N1) transmission to human have been demonstrated in several studies or ...epidemiological reports. However, transmission patterns may differ according to demographic characteristics of the population and local practices. This article aggregates these data from three studies with data collected in the previous surveys in 2006 and 2007 to further examine the risks factors associated with presence of anti-A(H5) antibodies among villagers residing within outbreak areas.
We aggregated 5-year data (2006-2010) from serology survey and matched case-control studies in Cambodia to further examine the risks factors associated with A(H5N1) infection among villagers in the outbreak areas.
Serotesting among villagers detected 35 (1.5 % 0-2.6) positive cases suggesting recent exposure to A(H5N1) virus. Practices associated with A(H5N1) infection among all ages were: having poultry cage or nesting area under or adjacent to the house (OR: 6.7 1.6-28.3; p = 0.010) and transporting poultry to market (OR: 17.6 1.6-193.7; p = 0.019). Practices found as risk factors for the infection among age under 20 years were swimming/bathing in ponds also accessed by domestic poultry (OR: 4.6 1.1-19.1; p = 0.038). Association with consuming wild birds reached borderline significance (p = 0.066).
Our results suggest that swimming/bathing in contaminated pond water and close contact with poultry may present a risk of A(H5N1) transmission to human.
Surveillance for avian influenza viruses (AIVs) in poultry and environmental samples was conducted in four live-bird markets in Cambodia from January through November 2013. Through real-time RT-PCR ...testing, AIVs were detected in 45% of 1048 samples collected throughout the year. Detection rates ranged from 32% and 18% in duck and chicken swabs, respectively, to 75% in carcass wash water samples. Influenza A/H5N1 virus was detected in 79% of samples positive for influenza A virus and 35% of all samples collected. Sequence analysis of full-length haemagglutinin (HA) and neuraminidase (NA) genes from A/H5N1 viruses, and full-genome analysis of six representative isolates, revealed that the clade 1.1.2 reassortant virus associated with Cambodian human cases during 2013 was the only A/H5N1 virus detected during the year. However, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HA and NA genes revealed co-circulation of at least nine low pathogenic AIVs from HA1, HA2, HA3, HA4, HA6, HA7, HA9, HA10 and HA11 subtypes. Four repeated serological surveys were conducted throughout the year in a cohort of 125 poultry workers. Serological testing found an overall prevalence of 4.5% and 1.8% for antibodies to A/H5N1 and A/H9N2, respectively. Seroconversion rates of 3.7 and 0.9 cases per 1000 person-months participation were detected for A/H5N1 and A/H9N2, respectively. Peak AIV circulation was associated with the Lunar New Year festival. Knowledge of periods of increased circulation of avian influenza in markets should inform intervention measures such as market cleaning and closures to reduce risk of human infections and emergence of novel AIVs.
Summary Background Human infection with the avian influenza A H5N1 virus results in disease with a high fatality rate, against which antiviral treatments have limited efficacy. We aimed to ...investigate the safety, pharmacokinetics, and therapeutic potential of specific polyclonal immunoglobulin equine F(ab')2 fragments raised against influenza A/Vietnam/1194/2004 virus (H5N1 subtype) in healthy volunteers. Methods We did a randomised, double-blind, placebo-controlled, single-centre phase 1 study. In stage 1 (one infusion) and stage 2 (five infusions) of the trial, we randomly assigned healthy male volunteers to receive once-daily intravenous infusions of 0·85 U/kg body weight of F(ab')2 or once-daily saline placebo. Randomisation was done centrally, with one block of four patients and one block for substitutes (three actives, one placebo) in stage 1, and two blocks of six patients (five actives and one placebo) and the same block for substitutes in stage 2. The primary objective was assessment of the clinical and laboratory safety of F(ab')2 , which was monitored for 22 days in the group that received one dose (assessments on days 0–2, 4, 8, 15, and 22) and 33 days in the group that received five doses (days 0–6, 8, 10, 12, 19, 26, and 33). A final post-study safety assessment was done at 120 days. We also assessed pharmacokinetic outcomes, and assayed haemagglutination and seroneutralisation activity. Analysis was done according to intention-to-treat. This trial is registered with ClinicalTrials.gov , number NCT02295813. Findings We enrolled 16 healthy Asian men between Sept 28 and Dec 28, 2012, and randomly assigned 13 to one or five doses of F(ab')2 and three to placebo. F(ab')2 was well tolerated, and no deaths or serious adverse events occurred. Three patients had mild adverse events (one each of blepharospasm, sinusitis, and pyrexia). The pyrexia (38°C) was regarded as probably related to the infusion, and resolved after 37 min. Our laboratory assessments of blood and urine samples and physical examinations of heart rate, electrocardiogram readings, and weight showed no clinically significant safety issues. Mean peak plasma concentrations were 19·3 μg/mL (SD 3·5) with the one dose schedule and 23·0 μg/mL (4·5) with the five-dose schedule. F(ab')2 were still detectable in plasma on average up to 5 days after five doses. Haemagglutination inhibition was only increased after the third dose, but in-vitro seroneutralisation activity was transiently increased after each of the five doses to concentrations regarded as clinically beneficial in infected patients. Interpretation F(ab')2 showed good safety, tolerability, and therapeutic potential for managing of H5N1 exposed patients. Funding Fab'entech.
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•Five novel genotypes are identified based on VP1 sequences.•Co-infections with other viruses is demonstrated.•The genome of rhinoviruses is saturated.•A novel classification system ...considering amino acid sequences should be developed.
Acute respiratory viral infections are a major cause of morbidity during early childhood in developing countries. Human rhinoviruses are the most frequent cause of upper respiratory tract infections in humans, which can range in severity from asymptomatic to clinically severe disease. In this study we collected 4170 nasopharyngeal swabs from patients hospitalised with influenza-like illness in two Cambodian provincial hospitals between 2007 and 2010. Samples were screened for 18 respiratory viruses using 5 multiplex PCRs. A total of 11.2% of samples tested positive for human rhinoviruses (HRV). VP4/2 and VP1 regions were amplified and sequenced to study the distribution of rhinoviruses genotypes and species in Cambodia during this three-year period. Five novel genotypes, 2 species A, 2 species B and 1 species C were identified based on VP1 sequences. Co-infections with other viruses were demonstrated.
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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