A 27-Amino-Acid Deletion in the Neuraminidase Stalk Supports Replication of an Avian H2N2 Influenza A Virus in the Respiratory Tract of Chickens

The events and mechanisms that lead to interspecies transmission of, and host adaptation to, influenza A virus are unknown; however, both surface and internal proteins have been implicated. Our previous report highlighted the role that Japanese quail play as an intermediate host, expanding the host range of a mallard H2N2 virus, A/mallard/Potsdam/178-4/83 (H2N2), through viral adaptation. This quail-adapted virus supported transmission in quail and increased its host range to replicate and be transmitted efficiently in chickens. Here we report that of the six amino acid changes in the quail-adapted virus, a single change in the hemagglutinin (HA) was crucial for transmission in quail, while the changes in the polymerase genes favored replication at lower temperatures than those for the wild-type mallard virus. Reverse genetic analysis indicated that all adaptive mutations were necessary for transmission in chickens, further implicating quail in extending this virus to terrestrial poultry. Adaptation of the quail-adapted virus in chickens resulted in the alteration of viral tropism from intestinal shedding to shedding and transmission via the respiratory tract. Sequence analysis indicated that this chicken-adapted virus maintained all quail-adaptive mutations, as well as an additional change in the HA and, most notably, a 27-amino-acid deletion in the stalk region of neuraminidase (NA), a genotypic marker of influenza virus adaptation to chickens. This stalk deletion was shown to be responsible for the change in virus tropism from the intestine to the respiratory tract.Of the 16 known hemagglutinin (HA) subtypes, only 3 (H1, H2, and H3) have established stable lineages in humans. The H2N2 virus caused a pandemic in 1957 and circulated in the human population until reassortment of the H2N2 virus with an avian H3 virus resulted in the H3N2 pandemic of 1968 (36). Since then, H2N2 viruses have been absent from the human population; however, the H2 subtype has been repeatedly isolated in wild-bird surveillance, and its HA has been found to be antigenically similar to the H2 pandemic virus HA (23, 25, 36). An H2 influenza virus containing human-like receptor specificity was recently isolated as an H2N3 avian-swine reassortant. This virus caused disease and was transmitted in swine and ferrets (24), indicating that this subtype continues to circulate and mutate and can cross the species barrier to mammals. The repeat introduction of a novel H1N1 pandemic this past year (12, 37) highlights the need to understand the mechanisms of introduction, adaptation, and transmission of avian H2N2 influenza viruses in terrestrial birds and potentially mammalian species.Our previous study built on reports that Japanese quail (Coturnix coturnix) play an important role as an intermediate host in the adaptation of avian influenza viruses to land-based birds (38). Japanese quail are typically more susceptible to aquatic influenza viruses than other terrestrial poultry. These viruses establish infection in the respiratory tract, and shedding occurs via aerosol (2, 19, 26, 34, 38, 43). Quail have been implicated in the transmission of avian influenza viruses, such as H5N1 and H9N2 viruses, which have crossed the species barrier to infect humans (9, 14, 15, 22, 28). The susceptibility of quail to multiple subtypes and their role in interspecies transmission led to their removal from live-bird markets in Hong Kong in 2000; however, they continue to be an integral part of live-bird markets throughout the world. Their role as potential intermediate hosts requires further study to identify important molecular markers in the adaptation via quail of avian viruses to other terrestrial poultry, and possibly to humans.The molecular determinants of the host range and pathogenesis of influenza A viruses have been linked to multiple regions of the 11 genes, most notably those encoding the viral surface glycoproteins (HA and neuraminidase [NA]) and the polymerase proteins (PB2, PB1, PA, and NP). However, a comprehensive map of the various determinants remains incomplete, and the molecular mechanisms involved are unclear. In our previous report, we demonstrated that through the use of quail as an intermediate host, a mallard H2N2 influenza virus, A/mallard/Potsdam/178-4/83 (mall/178), which in its wild-type (wt) form was unable to be transmitted in quail or to establish an efficient infection in chickens, was able, in its adapted form (qa-mall/178), not only to be transmitted to sentinel quail but also to replicate to efficient levels in the chicken intestinal tract and to be transmitted to sentinel cagemates via the fecal-oral route. This adaptation was the result of six serial passages of lung homogenates in quail that led to six amino acid changes in four genes (38). Here we present data confirming the role that Japanese quail play in the transmission of this mall/178 H2N2 virus in land-based birds. Reverse genetics studies confirmed that the amino acid changes produced during the adaptation in quail were necessary for the infection of chickens with this virus and for its transmission in chickens. Further adaptation of the qa-mall/178 H2N2 virus in chickens, aimed at establishing replication in the respiratory tract, resulted in a deletion in the stalk region of the NA, which supported replication in the chicken trachea and lung. This 27-amino-acid deletion in the stalk region of the N2 NA is characteristic of the adaptation of aquatic influenza viruses to domestic poultry, particularly chickens (3, 5, 29). Our work indicates that through the use of quail as an intermediate host, this mallard H2N2 virus is able to further adapt within an additional terrestrial poultry species, potentially improving its chances of expanding its host range further.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Characterization of Polymeric Solutions as Injectable Vehicles for Hydroxyapatite Microspheres

A polymeric solution and a reinforcement phase can work as an injectable material to fill up bone defects. However, the properties of the solution should be suitable to enable the transport of that extra phase. Additionally, the use of biocompatible materials is a requirement for tissue regeneration. Thus, we intended to optimize a biocompatible polymeric solution able to carry hydroxyapatite microspheres into bone defects using an orthopedic injectable device. To achieve that goal, polymers usually regarded as biocompatible were selected, namely sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and Na-alginate (ALG). The rheological properties of the polymeric solutions at different concentrations were assessed by viscosimetry before and after moist heat sterilization. In order to correlate rheological properties with injectability, solutions were tested using an orthopedic device applied for minimal invasive surgeries. Among the three polymers, ALG solutions presented the most suitable properties for our goal and a non-sterile ALG 6% solution was successfully used to perform preliminary injection tests of hydroxyapatite microspheres. Sterile ALG 7.25% solution was found to closely match non-sterile ALG 6% properties and it was selected as the optimal vehicle. Finally, sterile ALG 7.25% physical stability was studied at different temperatures over a 3-month period. It was observed that its rheological properties presented minor changes when stored at 25°C or at 4°C.     

Relationship of the methylenetetrahydrofolate reductase C677T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer

The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with the expression of a thermolabile enzyme with decreased activity that influences the pool of methyl-donor molecules. Several studies have reported an association between C677T polymorphism and susceptibility to colorectal cancer (CRC). Considering that methylation abnormalities appear to be important for the pathogenesis of CRC, we examined the correlation between the genotype of the MTHFR C677T polymorphism, hypermethylation of the promoter region of five relevant genes (DAPK, MGMT, hMLH1, p16(INK4a), and p14(ARF)), and microsatellite instability, in 106 patients with primary CRCs in Brazil. We did not find significant differences in the genotypic frequencies of the MTHFR C677T polymorphism when one or more loci were hypermethylated. However, we did find a significant excess of 677TT individuals among patients with CRC who had microsatellite instability. This strong association was independent of the methylation status of hMLH1 and of the biogeographical genomic ancestry of the patients. Although the mechanism responsible for the link between the C677T polymorphism and microsatellite instability was not apparent, this finding may provide a clue towards a better understanding of the pathogenesis of microsatellite instability in human colorectal cancer.     

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.     

Y-chromosome haplotypes in azoospermic Israeli men

Among azoospermic and severely oligozoospermic men, 7-15% present microdeletions of a region on the long arm of the Y chromosome that has been called AZF (azoospermia factor). Because these deletions present varying relative frequencies in different populations, we decided to ascertain whether their presence was correlated with specific Y-chromosome haplotypes. For that, we evaluated 51 infertile Israeli men, 9 of whom had microdeletions in AZF. Haplotypes were identified using a hierarchical system with eight biallelic DNA markers. We also checked for the presence of the deletion marker 50f2/C, which was absent in all seven patients with isolated AZFc deletion and also in the one patient with isolated AZFb deletion, suggesting that these microdeletions overlap. As expected, haplogroup J was the most common (47%), followed by equal frequencies of haplogroups Y* (xDE, J, K), P* (xR1a, R1b8), K* (xP), and E. In six patients with AZFc deficiencies of comparable size, three belonged to haplogroup J, two belonged to haplogroup P* (xR1a, R1b8), and one belonged to haplogroup R1a. Also, there were no significant differences in the haplotype frequencies between the groups with and without microdeletions. Thus we did not identify any association of a specific haplogroup with predisposition to de novo deletion of the AZF region in the Israeli population.     

Trypanosoma cruzi: role of host genetic background in the differential tissue distribution of parasite clonal populations

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, has quite a variable clinical presentation, ranging from asymptomatic to severe chronic cardiac and/or gastrointestinal disease. The reason for that is not completely understood, but both parasite and host genetic traits are certainly involved. Recently, we have demonstrated clinically and experimentally that the genetic variability of T. cruzi is one of the determinants of the pattern of tissue involvement in Chagas' disease. We then decided to turn our attention to the role of host genetic background. To study this, we compared the infection of four lineages of mice [three inbred (BALB/c, DBA-2, and c57Black/6) and one outbred (Swiss)] with two T. cruzi clonal populations, the Col1.7G2 clone and the JG monoclonal strain. The tissue distribution of T. cruzi strains was identical for BALB/c and DBA-2 mice, but very different in C57BL/6 (H-2^b) and outbred Swiss mice. This result clearly demonstrates the importance of host genetic aspects in the process. Since BALB/c and DBA-2 have the same H-2 haplotype (H-2^d) and C57BL/6 does not (H-2^b), it is possible that MHC variability may be involved in influencing the tissue distribution of involvement in experimental Chagas' disease of the mouse.Abbreviations: PCR, polymerase chain reaction; LSSP-PCR, low-stringency single specific primer PCR; kDNA, kinetoplast DNA; MHC, major histocompatibility complex; dNTP, 2^′-deoxynucleotide 5^′-triphosphate