Origins of the 2009 H1N1 influenza pandemic in swine in Mexico.

Asia is considered an important source of influenza A virus (IAV) pandemics, owing to large, diverse viral reservoirs in poultry and swine. However, the zoonotic origins of the 2009 A/H1N1 influenza pandemic virus (pdmH1N1) remain unclear, due to conflicting evidence from swine and humans. There is strong evidence that the first human outbreak of pdmH1N1 occurred in Mexico in early 2009. However, no related swine viruses have been detected in Mexico or any part of the Americas, and to date the most closely related ancestor viruses were identified in Asian swine. Here, we use 58 new whole-genome sequences from IAVs collected in Mexican swine to establish that the swine virus responsible for the 2009 pandemic evolved in central Mexico. This finding highlights how the 2009 pandemic arose from a region not considered a pandemic risk, owing to an expansion of IAV diversity in swine resulting from long-distance live swine trade.

Tumor genetic analyses of patients with metastatic renal cell carcinoma and extended benefit from mTOR inhibitor therapy.

PURPOSE: Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity. EXPERIMENTAL DESIGN: We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list. RESULTS: Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions. CONCLUSIONS: Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents.

HOXB1 founder mutation in humans recapitulates the phenotype of Hoxb1-/- mice.

Members of the highly conserved homeobox (HOX) gene family encode transcription factors that confer cellular and tissue identities along the antero-posterior axis of mice and humans. We have identified a founder homozygous missense mutation in HOXB1 in two families from a conservative German American population. The resulting phenotype includes bilateral facial palsy, hearing loss, and strabismus and correlates extensively with the previously reported Hoxb1(-/-) mouse phenotype. The missense variant is predicted to result in the substitution of a cysteine for an arginine at amino acid residue 207 (Arg207Cys), which corresponds to the highly conserved Arg5 of the homeodomain. Arg5 interacts with thymine in the minor groove of DNA through hydrogen bonding and electrostatic attraction. Molecular modeling and an in vitro DNA-protein binding assay predict that the mutation would disrupt these interactions, destabilize the HOXB1:PBX1:DNA complex, and alter HOXB1 transcriptional activity.

Adaptation of human parainfluenza virus to airway epithelium reveals fusion properties required for growth in host tissue.

UNLABELLED: Paramyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). We have previously shown that viruses with an HN/F pair that is highly fusogenic in monolayers of immortalized cells due to mutations in HN's secondary sialic acid binding site are growth impaired in differentiated human airway epithelium (HAE) cultures and in vivo. Here we have shown that adaptation of HPIV3 to growth in the lung is determined by specific features of HN and F that are different from those required for growth in cultured immortalized cells. An HPIV3 virus bearing a mutated HN (H552Q), which is fit and fusogenic in immortalized cells but unfit for growth in the lung, evolved into a less-fusogenic but viable virus in differentiated human airway epithelium. Stepwise evolution led to a progressive decrease in efficiency of fusion activation by the HN/F pair, with a mutation in F first decreasing the activation of F by HN and a mutation in HN's secondary sialic acid binding site decreasing fusion activation further and producing a stable virus. Adaptation of HPIV3 to successful growth in HAE is determined by specific features of HN and F that lead to a less easily activated fusion mechanism. IMPORTANCE: Human parainfluenza viruses (HPIVs) are paramyxoviruses that cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide, but there are currently no vaccines or antivirals available for treatment. Enveloped viruses must fuse their membrane with the target cell membrane in order to initiate infection. Parainfluenza virus fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. The receptor-binding hemagglutinin-neuraminidase (HN), upon receptor engagement, activates the fusion protein (F) to penetrate the target cell and mediate viral entry. In this study, we show that the precise balance of fusion activation properties of these two glycoproteins during entry is key for infection. In clinically relevant tissues, viruses evolve to acquire a set of fusion features that provide key clues about requirements for infection in human beings.

Whole blood transfusion in small animals: indications and effects.

Transfusion therapy is a major resource that can improve the patient's capability to overcome the underlying disease. However, the effects of whole blood infusion, and how they affect the patient's outcome, are not yet clear. For this study, a protocol was developed in order to monitor a group of 15 animals (9 dogs, 6 cats) that received a total of 19 transfusions; 3 animals received more than one transfusion each. The most common indications for blood transfusion included acute blood loss (47%), coagulopathy (33%) and other anaemias (20%). The mean pre-transfusion packed cell volume (PCV) of animals with acute blood loss (18%) was higher than in the group of coagulopathy (15%) or other anaemias (15%). The survival rates at 6 days after transfusion were greater in the coagulopathy (80.0%) and other anaemias (66.7%) than in the group of acute blood loss (42.9%). After transfusion, pulse rate ( p <0.01) and platelet count ( p <0.05) decreased significantly, and there was a significant increase in body temperature of the animals that suffered from hypothermia before the transfusion ( p <0.05). Overall survival was predictable based upon posttransfusion body temperature, observed PCV change, the difference between the obtained and the calculated PCV, and administered transfusion volume ( p <0.05).

Whole blood transfusion in small animals: indications and effects

Transfusion therapy is a major resource that can improve the patient's capability to overcome the underlying disease. However, the effects of whole blood infusion, and how they affect the patient's outcome, are not yet clear. For this study, a protocol was developed in order to monitor a group of 15 animals (9 dogs, 6 cats) that received a total of 19 transfusions; 3 animals received more than one transfusion each. The most common indications for blood transfusion included acute blood loss (47 percent), coagulopathy (33 percent) and other anaemias (20 percent). The mean pre-transfusion packed cell volume (PCV) of animals with acute blood loss (18 percent) was higher than in the group of coagulopathy (15 percent) or other anaemias (15 percent). The survival rates at 6 days after transfusion were greater in the coagulopathy (80.0 percent) and other anaemias (66.7 percent) than in the group of acute blood loss (42.9 percent). After transfusion, pulse rate ( p <0.01) and platelet count ( p <0.05) decreased significantly, and there was a significant increase in body temperature of the animals that suffered from hypothermia before the transfusion ( p <0.05). Overall survival was predictable based upon posttransfusion body temperature, observed PCV change, the difference between the obtained and the calculated PCV, and administered transfusion volume ( p <0.05).

A terapia transfusional é um importante recurso que pode auxiliar na recuperação do doente face à patologia primária. No entanto, não estão totalmente esclarecidos os efeitos de uma transfusão sanguínea, e que consequências podem ter no desfecho clínico. Para este estudo, foi desenvolvido um protocolo de forma a monitorizar 15 animais (9 cães e 6 gatos) que receberam um total de 19 transfusões; 3 animais receberam mais do que uma unidade de sangue. As indicações mais comuns para realização da transfusão foram hemorragia aguda (47 por cento), coagulopatia (33 por cento) e outras anemias (20 por cento). O hematócrito pré-transfusional médio dos animais com hemorragia aguda (18 por cento) foi superior ao dos animais com coagulopatia (15 por cento) ou outras anemias (15 por cento). A taxa de sobrevivência ao sexto dia pós-transfusão foi maior no grupo da coagulopatia (80,0 por cento) e de outras anemias (66,7 por cento), do que no grupo da hemorragia aguda (42,9 por cento). Após a transfusão, a frequência de pulso ( p <0,01) e a contagem de plaquetas ( p <0,05) diminuíram significativamente, e houve um aumento significativo da temperatura corporal nos animais com hipotermia antes da transfusão ( p <0,05). A sobrevivência foi predizível com base na temperatura pós-transfusão, a variação do Ht, a diferença entre o Ht real e o Ht esperado, e o volume de transfusão aplicado ( p <0,05).

Congenital erythropoietic porphyria: identification and expression of eight novel mutations in the uroporphyrinogen III synthase gene.

Mutations in the uroporphyrinogen III synthase (URO-synthase) gene cause congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of haem biosynthesis. Molecular analysis of the URO-synthase gene in seven unrelated CEP patients revealed eight novel mutations. These included four missense mutations (A69T, E81D, G188W and I219S), a deletion (21delG), two insertions (398insG and 672ins28) and one complex mutation (627del6ins39), as well as three previously reported mutations, C73R, T228M, and -86C-->A. When the four novel missense mutations were expressed in Escherichia coli, only E81D expressed significant enzymatic activity (30% of expressed wild-type activity), which was thermolabile. In addition, reverse transcription polymerase chain reaction studies demonstrated that E81D, which altered the penultimate nucleotide in exon 4, impaired splicing and caused about 85% exon 4 skipping. The identification and expression of these mutations provided genotype-phenotype correlations and further evidence of the molecular heterogeneity underlying this erythropoietic porphyria.