Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.
Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.
To investigate arenavirus in Zambia, we characterized virus from the kidneys of 5 arenavirus RNA–positive rodents (Mastomys natalensis) among 263 captured. Full-genome sequences of the viruses suggested that they were new strains similar to Lassa virus–related arenaviruses. Analyzing samples from additional rodents and other species can elucidate epizootiologic aspects of arenaviruses.
Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPV GFP ). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the gene (HMPV A2b 180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b 180nt-dup strains, and six recombinant HMPV GFP strains, including the newly generated recombinant HMPV A2b 180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPV GFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.
ABSTRACT. The present study was designed to determine the effects of physiological stress on milk-somatic cell counts (SCC) and function of bovine peripheral blood leukocytes (PBL). Nine healthy lactating cows were used in the examination. Five cows were transported 100 km for 4 hr (transported group; TG), and 4 cows were penned (non-transported group; NTG). Blood and milk samples were collected at 0, 2, and 4 hr after loading, and at 2 hr, and 1, 2, 3, and 6 days after unloading. The following activities were mea sured: adhesion receptor (CD 18 and L-selectin) expression of neutrophils and monocytes, migration capacity and percentage of apoptotic cells of neutrophils, serum soluble L-selectin (sL-selectin), plasma cortisol, and SCC. A significant increase in plasma cortisol and milk SCC was observed in TG. Leukocytosis, derived from neutrophils was recorded in TG, and was indicated by apoptotic measurement as an increase of young cells from the marginal pool. Increased migration and decreased surface expression of both L-selectin and CD 18 in neutrophils were observed after transportation. Elevated serum sL-selectin was also noted as a result of transportation. The present study indicated that transport stress modulates peripheral blood neutrophil function, particularly enhancing migration capacity, and c auses diapedesis across the mammary epithelium. Increased milk SCC in transported cattle might be due to these phenomena, and severe physiological stress may bring about an increase in SCC in milk.
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