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

Seroepidemiology of Human Enterovirus 71 Infection among Children, Cambodia.

Enterovirus 71 is reported to have emerged in Cambodia in 2012; at least 54 children with severe encephalitis died during that outbreak. We used serum samples collected during 2000-2011 to show that the virus had been widespread in the country for at least a decade before the 2012 outbreak

Relation between cannabis use and subcortical volumes in people at clinical high risk of psychosis

Among people at genetic risk of schizophrenia, those who use cannabis show smaller thalamic and hippocampal volumes. We evaluated this relationship in people at clinical high risk (CHR) of psychosis. The Alcohol and Drug Use Scale was used to identify 132 CHR cannabis users, the majority of whom were non-dependent cannabis users, 387 CHR non-users, and 204 healthy control non-users, and all participants completed magnetic resonance imaging scans. Volumes of the thalamus, hippocampus and amygdala were extracted with FreeSurfer, and compared across groups. Comparing all CHR participants with healthy control participants revealed no significant differences in volumes of any ROI. However, when comparing CHR users to CHR non-users, a significant ROI × Cannabis group effect emerged: CHR users showed significantly smaller amygdala compared to CHR non-users. However, when limiting analysis to CHR subjects who reported using alcohol at a ‘use without impairment’ severity level, the amygdala effect was non-significant; rather, smaller hippocampal volumes were seen in CHR cannabis users compared to non-users. Controlling statistically for effects of alcohol and tobacco use rendered all results non-significant. These results highlight the importance of controlling for residual confounding effects of other substance use when examining the relationship between cannabis use and neural structure

Traumatic brain injury in individuals at clinical high risk for psychosis

Recent research suggests that a traumatic brain injury (TBI) can significantly increase the risk of later development of psychosis. However, it is unknown whether people at clinical high risk (CHR) of psychosis have experienced TBI at higher rates, compared to otherwise healthy individuals. This study evaluated the prevalence of mild TBI, whether it was related to past trauma and the relationship of mild TBI to later transition to psychosis

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

New ophthalmosaurid ichthyosaurs from the European lower cretaceous demonstrate extensive ichthyosaur survival across the Jurassic–Cretaceous boundary

Background Ichthyosauria is a diverse clade of marine amniotes that spanned most of the Mesozoic. Until recently, most authors interpreted the fossil record as showing that three major extinction events affected this group during its history: one during the latest Triassic, one at the Jurassic–Cretaceous boundary (JCB), and one (resulting in total extinction) at the Cenomanian-Turonian boundary. The JCB was believed to eradicate most of the peculiar morphotypes found in the Late Jurassic, in favor of apparently less specialized forms in the Cretaceous. However, the record of ichthyosaurs from the Berriasian–Barremian interval is extremely limited, and the effects of the end-Jurassic extinction event on ichthyosaurs remains poorly understood. Methodology/Principal Findings Based on new material from the Hauterivian of England and Germany and on abundant material from the Cambridge Greensand Formation, we name a new ophthalmosaurid, Acamptonectes densus gen. et sp. nov. This taxon shares numerous features with Ophthalmosaurus, a genus now restricted to the Callovian–Berriasian interval. Our phylogenetic analysis indicates that Ophthalmosauridae diverged early in its history into two markedly distinct clades, Ophthalmosaurinae and Platypterygiinae, both of which cross the JCB and persist to the late Albian at least. To evaluate the effect of the JCB extinction event on ichthyosaurs, we calculated cladogenesis, extinction, and survival rates for each stage of the Oxfordian–Barremian interval, under different scenarios. The extinction rate during the JCB never surpasses the background extinction rate for the Oxfordian–Barremian interval and the JCB records one of the highest survival rates of the interval. Conclusions/Significance There is currently no evidence that ichthyosaurs were affected by the JCB extinction event, in contrast to many other marine groups. Ophthalmosaurid ichthyosaurs remained diverse from their rapid radiation in the Middle Jurassic to their total extinction at the beginning of the Late Cretaceous

Circulation and characterization of seasonal influenza viruses in Cambodia, 2012‐2015

Background: Influenza virus circulation is monitored through the Cambodian influenza‐like illness (ILI) sentinel surveillance system and isolates are characterized by the National Influenza Centre (NIC). Seasonal influenza circulation has previously been characterized by year‐round activity and a peak during the rainy season (June‐November). Objectives: We documented the circulation of seasonal influenza in Cambodia for 2012‐2015 and investigated genetic, antigenic, and antiviral resistance characteristics of influenza isolates. Patients/Methods Respiratory samples were collected from patients presenting with influenza‐like illness (ILI) at 11 hospitals throughout Cambodia. First‐line screening was conducted by the National Institute of Public Health and the Armed Forces Research Institute of Medical Sciences. Confirmation of testing and genetic, antigenic and antiviral resistance characterization was conducted by Institute Pasteur in Cambodia, the NIC. Additional virus characterization was conducted by the WHO Collaborating Centre for Reference and Research on Influenza (Melbourne, Australia). Results: Between 2012 and 2015, 1,238 influenza‐positive samples were submitted to the NIC. Influenza A(H3N2) (55.3%) was the dominant subtype, followed by influenza B (30.9%; predominantly B/Yamagata‐lineage) and A(H1N1)pdm09 (13.9%). Circulation of influenza viruses began earlier in 2014 and 2015 than previously described, coincident with the emergence of A(H3N2) clades 3C.2a and 3C.3a, respectively. There was high diversity in the antigenicity of A(H3N2) viruses, and to a smaller extent influenza B viruses, during this period, with some mismatches with the northern and southern hemisphere vaccine formulations. All isolates tested were susceptible to the influenza antiviral drugs oseltamivir and zanamivir. Conclusions: Seasonal and year‐round co‐circulation of multiple influenza types/subtypes were detected in Cambodia during 2012‐2015