Complete Genome Sequence of a Duck Astrovirus Discovered in Eastern China

We report here the complete genome sequence of a duck astrovirus (DAstV) isolated from a dead duckling in eastern China. Sequence analyses indicated that the genome of the astrovirus possessed a typical astrovirus organization. Comparison of the partial polymerase gene sequences of DAstV-1 and DAstV-2 showed that the astrovirus shared 94.4% and 64.2% nucleotide identity, respectively. The whole nucleotide sequence of the astrovirus had the highest homology with the sequence of DAstV-1 strain C-NGB (98.7%). Therefore, the strain we describe here is a DAstV-1 isolate.     

Crystal Structure of the Avian Astrovirus Capsid Spike

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes.

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Novel Coronavirus and Astrovirus in Delaware Bay Shorebirds

Background

Wild birds are an important but to some extent under-studied reservoir for emerging pathogens. We used unbiased sequencing methods for virus discovery in shorebird samples from the Delaware Bay, USA; an important feeding ground for thousands of migratory birds.

Findings

Analysis of shorebird fecal samples indicated the presence of a novel astrovirus and coronavirus. A sanderling sample yielded sequences with distant homology to avian nephritis virus 1, an astrovirus associated with acute nephritis in poultry. A ruddy turnstone sample yielded sequences with homology to deltacoronaviruses.

Conclusions

Our findings highlight shorebirds as a virus reservoir and the need to closely monitor wild bird populations for the emergence of novel virus variants.     

Vaccinia virus recombinant expressing an 87-kilodalton polyprotein that is sufficient to form astrovirus-like particles

Human astrovirus is an important cause of acute gastroenteritis. We have generated, for the first time, a vaccinia virus recombinant expressing the astrovirus 87-kDa structural polyprotein. The results demonstrate that this expression results in the formation of virus-like particles in the absence of other astrovirus proteins and genomic RNA. The purified trypsin-activated virus-like particles strongly resemble the complete astrovirus particles.     

Astrovirus as a possible cause of congenital tremor type AII in piglets?

Background

Congenital tremor is associated with demyelination of the brain and spinal cord and is clinically noted as outbreaks of trembling and shaking in newborn piglets during a limited time-period. Six forms of the disease have been described, where form AII may be caused by an, as yet, unidentified viral infection. This study aimed to investigate the presence of astrovirus and circovirus by sequencing and polymerase chain reaction (PCR) analysis and by relating the findings to the occurrence of disease and lesions in the brain, in 4–6 days-old piglets obtained from a clinical outbreak of congenital tremor.

Results

In piglets with congenital tremor, there were mild to moderate vacuolar changes of the white matter in the cerebrum, brain stem and cerebellum. In healthy piglets, less conspicuous vacuolar changes were detected. One healthy and one diseased piglet were positive for porcine circovirus type 2. The nested pan-PCR showed the presence of astrovirus in at least one brain region in all piglets and by sequencing, two different porcine astrovirus lineages were identified.

Conclusions

The results do not support previous studies identifying porcine circovirus type 2 as the cause of congenital tremor. The demonstration of astrovirus in the brain of piglets suffering from congenital tremor is interesting. However, astrovirus was demonstrated in both healthy and diseased individuals and therefore, further studies are warranted to determine the possible involvement of astrovirus in the pathogenesis of congenital tremor in pigs.

     

Detection of Antibodies against Turkey Astrovirus in Humans

Astroviruses are a leading cause of gastroenteritis in mammals and birds worldwide. Although historically thought to be species-specific, increasing evidence suggests that astroviruses may cross species barriers. In this report, we used enzyme-linked immunosorbent assays to screen sera from three distinct human cohorts involved in influenza studies in Memphis, TN or Chapel Hill, NC, and Midwestern poultry abattoir workers for antibodies to turkey astrovirus type 2 (TAstV-2). Surprisingly, 26% of one cohort’s population was TAstV-2 positive as compared to 0 and 8.9% in the other cohorts. This cohort was composed of people with exposure to turkeys in the Midwestern United States including abattoir workers, turkey growers, and non-occupationally exposed participants. The odds of testing positive for antibodies against turkey astrovirus among abattoir workers were approximately 3 times higher than the other groups. These studies suggest that people with contact to turkeys can develop serological responses to turkey astrovirus. Further work is needed to determine if these exposures result in virus replication and/or clinical disease.     

Genetic Analysis of the Capsid Region of Human Astrovirus Serotype 3 Isolated in Japan

The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.     

多重RT-PCR用于临床检测三种胃肠炎病毒的研究

轮状病毒、诺瓦克病毒和星状病毒是引起病毒性胃肠炎的主要病原因子。研究采用JV12/JV13、P1/P2和Mon340/Mon348三对引物,建立了同时检测这3种病毒的多重RT-PCR技术,并应用于128份临床粪便样本的检测,检出轮状病毒62份(48.44%),诺瓦克病毒8份(6.25%),星状病毒11份(8.59%)。在灵敏度试验中,轮状病毒的检测灵敏度为5pg/mL、诺瓦克病毒和星状病毒的检测灵敏度均为50pg/mL。该研究所建立3种常见胃肠炎病毒的多重RT-PCR方法具有特异性强、灵敏度高的特点,可用于临床病原诊断和溯源。     

Molecular analysis of a 348 base-pair segment of open reading frame 2 of human astrovirus. A characterization of Colombian isolates

We are reporting computational studies of several genotyped strains of astrovirus isolated in Colombia which we have genotyped using the 348-bp segment located between nucleotides 258 and 606 close to the amino terminal region of the complete ORF 2. By biocomputational techniques this 348-bp segment from the different strains was translated into an amino acid sequence. The sequences were aligned and compared in order to build a dendrogram. Our results show that the 348 bp and the 116 amino acid peptides cluster in a very conservative way, showing slight genetic variations between them. This slight sequence variability does not allow us to identify common amino acid substitution patterns shared by all members of each HAstV specific type, thus suggesting that antigenic epitopes are probably located outside the 116 peptide fragment of this capsid protein. These results show that there is little recombination among different regions of the ORF2, thus suggesting that this genotyping method should continue to be useful for serotyping HAstV isolates.     

Sequence analysis of the human virome in febrile and afebrile children

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF. We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.     

Cloning and characterization of human astrovirus immunoreactive epitopes.

We report the cloning of antigenic, protein-coding regions of human astrovirus serotype 1 that appear to be common to most, if not all, serotypes of human astrovirus. Screening of lambda gt11 libraries identified three different but overlapping clones (A43, A35, and A1) and one independent clone (A14) that reacted with serum from a rabbit repeatedly immunized with purified astrovirus particles but not with its preimmunization serum. These clones were shown to be astrovirus specific. Of note, a radiolabeled probe representing the immunoreactive clones A43-A35-A1 hybridized exclusively to the 7.2-kb astrovirus genomic RNA, while a clone A14-specific probe hybridized with both the genomic and the 2.8-kb astrovirus subgenomic RNAs. This suggests that the immunoreactive epitopes, selected by antiserum to purified astrovirus particles, are encoded by the subgenomic RNA as well as other regions of the genomic RNA.     

Functional crosstalk between exon enhancers, polypyrimidine tracts and branchpoint sequences.

We recently identified enhancer elements that activate the weak 3' splice site of alpha-tropomyosin exon 2 as well as a variety of heterologous weak 3' splice sites. To understand their mechanism of action, we devised an iterative selection strategy to identify functional pyrimidine tracts and branchpoint sequences in the presence or absence of enhancer elements. Surprisingly, we found that strong pyrimidine tracts were selected regardless of the presence of enhancer elements. However, the presence of enhancer elements resulted in the selection of multiple, non-consensus branchpoint sequences. Thus, enhancer elements apparently activate weak 3' splice sites primarily by increasing the efficiency of splicing of introns containing branchpoint sequences with less than optimal U2-branchpoint pairing arrangements. Comparison of consensus sequences from both our selection strategy and compilations of published intron sequences suggests that exon enhancer elements could be widespread and play an important role in the selection of 3' splice sites.     

De novo assembly of mitochondrial genomes provides insights into genetic diversity and molecular evolution in wild boars and domestic pigs

Up to date, the scarcity of publicly available complete mitochondrial sequences for European wild pigs hampers deeper understanding about the genetic changes following domestication. Here, we have assembled 26 de novo mtDNA sequences of European wild boars from next generation sequencing (NGS) data and downloaded 174 complete mtDNA sequences to assess the genetic relationship, nucleotide diversity, and selection. The Bayesian consensus tree reveals the clear divergence between the European and Asian clade and a very small portion (10 out of 200 samples) of maternal introgression. The overall nucleotides diversities of the mtDNA sequences have been reduced following domestication. Interestingly, the selection efficiencies in both European and Asian domestic pigs are reduced, probably caused by changes in both selection constraints and maternal population size following domestication. This study suggests that de novo assembled mitogenomes can be a great boon to uncover the genetic turnover following domestication. Further investigation is warranted to include more samples from the ever-increasing amounts of NGS data to help us to better understand the process of domestication.     

Astrovirus-induced synthesis of nitric oxide contributes to virus control during infection

Astrovirus is one of the major causes of infant and childhood diarrhea worldwide. Our understanding of astrovirus pathogenesis trails behind our knowledge of its molecular and epidemiologic properties. Using a recently developed small-animal model, we investigated the mechanisms by which astrovirus induces diarrhea and the role of both the adaptive and innate immune responses to turkey astrovirus type-2 (TAstV-2) infection. Astrovirus-infected animals were analyzed for changes in total lymphocyte populations, alterations in CD4(+)/CD8(+) ratios, production of virus-specific antibodies (Abs), and macrophage activation. There were no changes in the numbers of circulating or splenic lymphocytes or in CD4(+)/CD8(+) ratios compared to controls. Additionally, there was only a modest production of virus-specific Abs. However, adherent spleen cells from infected animals produced more nitric oxide (NO) in response to ex vivo stimulation with lipopolysaccharide. In vitro analysis demonstrated that TAstV-2 induced macrophage production of inducible nitric oxide synthase. Studies using NO donors and inhibitors in vivo demonstrated, for the first time, that NO inhibited astrovirus replication. These studies suggest that NO is important in limiting astrovirus replication and are the first, to our knowledge, to describe the potential role of innate immunity in astrovirus infection.     

Population Genetics and Molecular Evolution of DNA Sequences in Transposable Elements. I. A Simulation Framework

A population genetic simulation framework is developed to understand the behavior and molecular evolution of DNA sequences of transposable elements. Our model incorporates random transposition and excision of transposable element (TE) copies, two modes of selection against TEs, and degeneration of transpositional activity by point mutations. We first investigated the relationships between the behavior of the copy number of TEs and these parameters. Our results show that when selection is weak, the genome can maintain a relatively large number of TEs, but most of them are less active. In contrast, with strong selection, the genome can maintain only a limited number of TEs but the proportion of active copies is large. In such a case, there could be substantial fluctuations of the copy number over generations. We also explored how DNA sequences of TEs evolve through the simulations. In general, active copies form clusters around the original sequence, while less active copies have long branches specific to themselves, exhibiting a star-shaped phylogeny. It is demonstrated that the phylogeny of TE sequences could be informative to understand the dynamics of TE evolution.     

Temperature and salt concentration alter base-sequence selectivity of a duplex DNA-binding protein

A structural polymorphism of nucleic acids, which depends on the concentration of cations and the conditions of hydration, are strongly involved with interactions between DNA and proteins. In this paper, we report that different DNA sequences bound to hyperthermostable TATA-box-binding protein (PhoTBP) at different combinations of temperature and salt concentration in in vitro selection experiments. As a result of the interaction of-these selected DNAs with PhoTBP, characteristic changes in the numbers of water molecules and ions occurred under each condition of the selection experiment. This finding could help us to understand the solvent environment-dependent preference for base sequences in protein–DNA interactions.     

On the Origin of Macromolecular Sequences

The origin of the degree and type of order found in biological macromolecules is not adequately explained solely as an accumulation of genetic restrictions acquired through natural selection from otherwise unrestricted primeval sequences capable of self-replication, since the biological process of replication is itself dependent on the pre-existence of such order, and since the number of sequences that could have ever been tested by selection on the earth is an insignificant fraction of the number of unrestricted sequences which would be possible. Therefore the hypothesis is considered that replication and selection began from well ordered sequences, rather than random sequences. It is shown how the Turing concept of computation in fed-back, discrete-state automata can lead to the generation of order withour pre-existing instructions, and how this computation can result in self-repeating, random-like, but well ordered sequences of great length. Macromolecular models of such computers are suggested on the basis of mechanisms proposed for the growth of eutactic polymers. Such self-replicating, mutable sequences may then evolve genetic control which is sufficient to accommodate the information accumulated by natural selection. The structure and function of enzymes and structural proteins is related to this model, and statistical evidence from known amino acid sequences is shown to be consistent with some degree of non-genetic ordering.