Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus

Background: Despite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny. Study design: We document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09. Results: Complete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID50 of 104/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia. Conclusions: It is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities

Intense circulation of A/H5N1 and other avian influenza viruses in Cambodian live-bird markets with serological evidence of sub-clinical human infections

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

Quantifying within-host diversity of H5N1 influenza viruses in humans and poultry in Cambodia

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 (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

Transmission experiments support clade-level differences in the transmission and pathogenicity of Cambodian influenza A/H5N1 viruses

Influenza A/H5N1 has circulated in Asia since 2003 and is now enzootic in many countries in that region. In Cambodia, the virus has circulated since 2004 and has intermittently infected humans. During this period, we have noted differences in the rate of infections in humans, potentially associated with the circulation of different viral clades. In particular, a reassortant clade 1.1.2 virus emerged in early 2013 and was associated with a dramatic increase in infections of humans (34 cases) until it was replaced by a clade 2.3.2.1c virus in early 2014. In contrast, only one infection of a human has been reported in the 6 years since the clade 2.3.2.1c virus became the dominant circulating virus. We selected three viruses to represent the main viral clades that have circulated in Cambodia (clade 1.1.2, clade 1.1.2 reassortant, and clade 2.3.2.1c), and we conducted experiments to assess the virulence and transmissibility of these viruses in avian (chicken, duck) and mammalian (ferret) models. Our results suggest that the clade 2.3.2.1c virus is more “avian-like,” with high virulence in both ducks and chickens, but there is no evidence of aerosol transmission of the virus from ducks to ferrets. In contrast, the two clade 1 viruses were less virulent in experimentally infected and contact ducks. However, evidence of chicken-to-ferret aerosol transmission was observed for both clade 1 viruses. The transmission experiments provide insights into clade-level differences that might explain the variation in A/H5N1 infections of humans observed in Cambodia and other settings

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats

A(H5N1) Virus Evolution in South East Asia

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is an ongoing public health and socio-economic challenge, particularly in South East Asia. H5N1 is now endemic in poultry in many countries, and represents a major pandemic threat. Here, we describe the evolution of H5N1 virus in South East Asia, the reassortment events leading to high genetic diversity in the region, and factors responsible for virus spread. The virus has evolved with genetic variations affecting virulence, drug-resistance, and adaptation to new host species. The constant surveillance of these changes is of primary importance in the global efforts of the scientific community

Influenza A(H5N1) viruses with A(H9N2) single gene (matrix or PB1) reassortment isolated from Cambodian live bird markets

Live bird market surveillance for avian influenza viruses in Cambodia in 2015 has led to the detection of two 7:1 reassortant influenza A(H5N1) clade 2.3.2.1c viruses. These reassortant strains, designated A/duck/Cambodia/Z564W35M1/2015 and A/chicken/Cambodia/Z850W49M1/2015, both contained a single gene (PB1 and matrix gene, respectively) from concurrently circulating A(H9N2) influenza viruses. All other viral genes from both isolates clustered with A(H5N1) clade 2.3.2.1 viruses. Continued and prolonged co-circulation of influenza A(H5N1) and A(H9N2) viruses in Cambodian live bird markets may present a risk for the emergence of novel influenza reassortant viruses with negative agricultural and/or public health implications. © 201

Diversity of A(H5N1) clade 2.3.2.1c avian Influenza viruses with evidence of reassortment in Cambodia, 2014-2016

In Cambodia, highly pathogenic avian influenza A(H5N1) subtype viruses circulate endemically causing poultry outbreaks and zoonotic human cases. To investigate the genomic diversity and development of endemicity of the predominantly circulating clade 2.3.2.1c A(H5N1) viruses, we characterised 68 AIVs detected in poultry, the environment and from a single human A(H5N1) case from January 2014 to December 2016. Full genomes were generated for 42 A(H5N1) viruses. Phylogenetic analysis shows that five clade 2.3.2.1c genotypes, designated KH1 to KH5, were circulating in Cambodia during this period. The genotypes arose through multiple reassortment events with the neuraminidase (NA) and internal genes belonging to H5N1 clade 2.3.2.1a, clade 2.3.2.1b or A(H9N2) lineages. Phylogenies suggest that the Cambodian AIVs were derived from viruses circulating between Cambodian and Vietnamese poultry. Molecular analyses show that these viruses contained the hemagglutinin (HA) gene substitutions D94N, S133A, S155N, T156A, T188I and K189R known to increase binding to the human-type α2,6-linked sialic acid receptors. Two A(H5N1) viruses displayed the M2 gene S31N or A30T substitutions indicative of adamantane resistance, however, susceptibility testing towards neuraminidase inhibitors (oseltamivir, zanamivir, lananmivir and peramivir) of a subset of thirty clade 2.3.2.1c viruses showed susceptibility to all four drugs. This study shows that A(H5N1) viruses continue to reassort with other A(H5N1) and A(H9N2) viruses that are endemic in the region, highlighting the risk of introduction and emergence of novel A(H5N1) genotypes in Cambodia

The evolution and genetic diversity of avian influenza A(H9N2) viruses in Cambodia, 2015 – 2016

Low pathogenic A(H9N2) subtype avian influenza viruses (AIVs) were originally detected in Cambodian poultry in 2013, and now circulate endemically. We sequenced and characterised 64 A(H9N2) AIVs detected in Cambodian poultry (chickens and ducks) from January 2015 to May 2016. All A(H9) viruses collected in 2015 and 2016 belonged to a new BJ/94like h9-4.2.5 sub-lineage that emerged in the region during or after 2013, and was distinct to previously detected Cambodian viruses. Overall, there was a reduction of genetic diversity of H9N2 since 2013, however two genotypes were detected in circulation, P and V, with extensive reassortment between the viruses. Phylogenetic analysis showed a close relationship between A(H9N2) AIVs detected in Cambodian and Vietnamese poultry, highlighting cross-border trade/movement of live, domestic poultry between the countries. Wild birds may also play a role in A(H9N2) transmission in the region. Some genes of the Cambodian isolates frequently clustered with zoonotic A(H7N9), A(H9N2) and A(H10N8) viruses, suggesting a common ecology. Molecular analysis showed 100% of viruses contained the hemagglutinin (HA) Q226L substitution, which favours mammalian receptor type binding. All viruses were susceptible to the neuraminidase inhibitor antivirals; however, 41% contained the matrix (M2) S31N substitution associated with resistance to adamantanes. Overall, Cambodian A(H9N2) viruses possessed factors known to increase zoonotic potential, and therefore their evolution should be continually monitored