Survival of infectious particles and persistence of virus RNA in simple biotopes (experiments A).

<p> A.1: only water of various origins maintained at 25°C and inoculated to a final concentration of 5×10⁴ EID50/mL with H5N1 virus of animal or avian origin. A.2:water and mud containing an estimated final concentration of virus of avian origin of 5×10⁴ EID50/mL water maintained at 25°C (A.2.1) or various concentrations of viruses of avian and human origins and maintained at various temperatures (A.2.2). A: Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. H: Human origin strain stands for the A/Cambodia/408008/2005 strain.*last day of the corresponding experiment at which samples could be collected and tested.</p

Design of experiments D and laboratory results to assess the role of <i>Betta splendens</i> fish in the transmission cycle of H5N1 virus in water.

<p> The 3 horizontal rectangles represent different types of water in which animals were immersed: contaminated vs non-contaminated. Tadpoles (T) were immersed along with the male fighting fish (F) in contaminated water. Black-filled lines represent the male fighting fish and the tadpoles immersed in contaminated water from day 0 (D0). At day 5, male contaminated fish were placed over-night in clean water before being exposed to non-contaminated females in clean water. White-filled lines represent naïve female fighting fish immersed in non-contaminated water from day 0. Stars indicate collection of fish and tadpoles (when present) for testing. The two experiments are discernable and identified as D.1 and D.2. White stars correspond to detection of infectious virus in fish. Black stars indicate that H5N1 virus was not recovered from fish's organs after inoculation into embryonated eggs. Numbers included in black boxes correspond to average viral loads measured in fish (F) and tadpoles (T) that were immersed in contaminated water. Numbers in white boxes correspond to average viral loads measured in female fighting fish that were immersed in clean water and exposed to contaminated male fish. Water samples were collected from contaminated water (experiment D.1) from day 0 (D0) up to day 20 (D20). Water samples were collected from non-contaminated water (experiment D.2) from day 0 (D0) to day 12 (D12). The values presented in italic correspond to the viral load measured in water samples. When the numbers in italic are displayed in white color, this indicates that infectious virus was also detected. When the numbers in italic are written in black color, this means that the virus was not recovered after inoculation into eggs.</p

Experimental conditions used for the study in simple (A) and complex (B) biotopes.

a<p>Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. Human origin strain stands for the A/Cambodia/408008/2005 strain.</p>b<p>T° = Temperature (°C).</p

Survival of infectious particles and persistence of virus RNA in complex biotopes (experiments B).

<p>B.1: complex biotopes inoculated with virus of avian or human origins at various final concentrations and maintained at 25°C. B.2: complex biotopes inoculated with virus of avian or human origins at various final concentrations and maintained at various temperatures. A: Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. H: Human origin strain stands for the A/Cambodia/408008/2005 strain.*last day of the corresponding experiment at which samples could be collected and tested.</p

Survival of infectious particles and persistence of virus RNA in water and fauna in experiments C and D.

<p> C: water inoculated with the virus of human origin, at a final estimated concentration of 5×10⁴ EID50/mL, maintained at 25°C and containing mussels. D: water inoculated with the virus of avian origin, at a final estimated concentration of 2×10² EID50/mL, maintained at 17°C and containing fighting fish and tadpoles.*last day of the corresponding experiment at which samples could be collected and tested.</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

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

Laboratory testing flowchart for influenza-like-illness (ILI).

<p>Laboratory testing flowchart for influenza-like-illness (ILI).</p

Changes in glycosylation, antigenic and polymorphic sites linked to amino acid (AA) substitutions and sub-antigenic/catalytic sites (epitopes) where AA substitutions were found for hemagglutinin (HA) and neuraminidase (NA) segments of H3N2.

<p>Changes in glycosylation, antigenic and polymorphic sites linked to amino acid (AA) substitutions and sub-antigenic/catalytic sites (epitopes) where AA substitutions were found for hemagglutinin (HA) and neuraminidase (NA) segments of H3N2.</p

General characteristics of the study population.

<p>General characteristics of the study population.</p