Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom

The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to alpha-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. IMPORTANCE: Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection

Penetration of infectious prion protein in the intestine during the lactation period.

Prion diseases are fatal neurodegenerative zoonotic foodborne disorders, which are caused by an abnormal isoform of prion protein (PrPSc) derived from the cellular isoform of prion protein (PrPC). According to epidemiological surveillance and in vivo experiments, exposure to the PrPSc during the weaning period is fraught with risk, suggesting that, during development, the intestinal defenses and the immune system are involved in PrPSc infection susceptibility. Although it remains unclear how PrPSc passes through the natural biological barriers during its invasion of intestinal cells, the 37 kDa/67 kDa laminin receptor is suspected to be one of the receptors involved in PrPSc-incorporation. In addition, we have recently shown that the neonatal Fc receptor (nFcR), which contributes to the uptake of maternal antibodies into the intestine, may play an important role in PrPSc incorporation. In this review, recent studies on PrPSc uptake and models of PrPSc incorporation into the intestine via the laminin and Fc receptors are described

Altered vector competence in an experimental mosquito-mouse transmission model of Zika infection

<div><p>Few animal models of Zika virus (ZIKV) infection have incorporated arthropod-borne transmission. Here, we establish an <i>Aedes aegypti</i> mosquito model of ZIKV infection of mice, and demonstrate altered vector competency among three strains, (Orlando, ORL, Ho Chi Minh, HCM, and Patilas, PAT). All strains acquired ZIKV in their midguts after a blood meal from infected mice, but ZIKV transmission only occurred in mice fed upon by HCM, and to a lesser extent PAT, but not ORL, mosquitoes. This defect in transmission from ORL or PAT mosquitoes was overcome by intrathoracic injection of ZIKV into mosquito. Genetic analysis revealed significant diversity among these strains, suggesting a genetic basis for differences in ability for mosquito strains to transmit ZIKV. The intrathoracic injection mosquito-mouse transmission model is critical to understanding the influence of mosquitoes on ZIKV transmission, infectivity and pathogenesis in the vertebrate host, and represents a natural transmission route for testing vaccines and therapeutics.</p></div

Genes proximal to microsatellite clusters driving genetic variability in mosquito strains.

<p>Genes proximal to microsatellite clusters driving genetic variability in mosquito strains.</p

Intrathoracic injection of the Ho Chi Minh (HCM), Patilas (PAT) and Orlando (ORL) strains of <i>Ae</i>. <i>aegypti</i> with ZIKV results in transmission to mice.

<p>HCM, PAT and ORL strains of <i>Ae</i>. <i>aegypti</i> were infected by intrathoracic injection with 10³ PFU of ZIKV, and after 7 or 10 days allowed to feed on naïve mice. (A) Experimental workflow for the ZIKV injection experiments. (B) Blood was collected every other day for one week from naïve mice fed on by mosquitoes intrathoracically-infected with ZIKV for 7 or 10 days, and analyzed for ZIKV infection by qRT-PCR. ZIKV RNA levels were normalized to mouse <i>β</i> actin (ACTB) RNA levels. (C) Naïve mice fed on by mosquitoes intrathoracically-infected with ZIKV were monitored for survival for at least 32 days after infected mosquitoes-feeding. Data shown are pooled from at least two independent experiments. (n = 6/group for ORL-Day 7; n = 7/group for ORL-Day 10; n = 7/group for PAT-Day 7; n = 7/group for PAT-Day 10; n = 7/group for HCM-Day 7; n = 6/group for HCM-Day 10.) Significance was calculated using two-way ANOVA with a post-hoc Tukey test. ** P<0.01.</p

Ho Chi Minh (HCM), Patilas (PAT) and Orlando (ORL) strains of <i>Aedes aegypti</i> are genetically diverse.

<p>Six strains of <i>Ae</i>. <i>aegypti</i> (Liverpool, Rockefeller, Ho Chi Minh (HCM), Orlando (ORL), Amacuzac (Mexico) and Patillas (Puerto Rico) strains) were genotyped at 12 microsatellite loci and population genetic analyses were performed to compare these populations. (A) PCA analysis showing the extent of genetic diversity of various laboratory and field-collected strains of <i>Ae</i>. <i>aegypti</i>. The bar plot with eigenvalues shows the amount of variance represented by each principal component, black bars indicate the components illustrated in these PCA. The units of the grid are indicated at the top right corner. (B) Results of the Bayesian clustering analysis with STRUCTURE [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006350#pntd.0006350.ref024" target="_blank">24</a>]. Shown is the bar plot indicating the genetic groupings of the three strains used for ZIKV infections. Each vertical bar represents an individual. The height of each bar represents the probability of assignment to each of (upper panel) K = 2 clusters or (lower panel) K = 3 clusters. Each cluster is indicated by different colours: HCM: blue, PAT: pink and ORL: red. (C) Frequency of alleles highlighting the differences among <i>Aedes aegypti</i> laboratory populations from Ho Chi Minh, Orlando Strain, and Patilas, challenged with ZIKV. Two of the 12 microsatellite loci genotyped contributed to the population differentiation observed at a loading threshold of 0.10; AC1 and CT2. More specifically, alleles AC1:209, CT2:184, and CT2:188. Alleles are represented by numbers from 1–4 in the graph for simplification; AC1 (1: 195, 2: 197, 3: 209, 4: 201) and CT2 (1: 188, 2: 184, 3: 196). (D) Discriminant Analysis of Principal Components (DAPC) on microsatellite allele frequencies showing two clear genetic clusters with minimal overlap; colours are as in the upper panel of B.</p