ABSTRACT. Babesia microti, the erythroparasitic cause of human babesiosis, has long been taken to be a single species because classification by parasite morphology and host spectrum blurred distinctions between the parasites. Phylogenetic analyses of the 18S ribosomal RNA gene (18S rDNA) and, more recently, the β-tubulin gene have suggested inter-group heterogeneity. Intra-group relationships, however, remain unknown. This study was conducted to clarify the intra-and inter-group phylogenetic features of the B. microti-group parasites with the η subunit of the chaperonin-containing t-complex polypeptide l (CCTη) gene as a candidate genetic marker for defining the B. microti group. We prepared complete sequences of the CCTη gene from 36 piroplasms and compared the phylogenetic trees. The B. microti-group parasites clustered in a monophyletic assemblage separate from the Babesia sensu stricto and Theileria genera and subdivided predominantly into 4 clades (U.S., Kobe, Hobetsu, Munich) with highly significant evolutionary distances between the clades. B. rodhaini branched at the base of the B. microti-group parasites. In addition, a unique intron presence/absence matrix not observable in 18S rDNA or β-tubulin set the B. microti group entirely apart from either Babesia sensu stricto or Theileria. These results have strong implications for public health, suggesting that the B. microti-group parasites are a full-fledged genus comprising, for now, four core species, i.e., U.S., Kobe, Hobetsu, and Munich species nova. Furthermore, the CCTη gene is an instructive and definitive genetic marker for analyzing B. microti and related parasites.

BackgroundRift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, while in livestock it causes fever and high abortion rates.Methodology/Principal FindingsSequence analysis showed that a wild-type RVFV ZH501 preparation consisted of two major viral subpopulations, with a single nucleotide heterogeneity at nucleotide 847 of M segment (M847); one had a G residue at M847 encoding glycine in a major viral envelope Gn protein, while the other carried A residue encoding glutamic acid at the corresponding site. Two ZH501-derived viruses, rZH501-M847-G and rZH501-M847-A, carried identical genomic sequences, except that the former and the latter had G and A, respectively, at M847 were recovered by using a reverse genetics system. Intraperitoneal inoculation of rZH501-M847-A into mice caused a rapid and efficient viral accumulation in the sera, livers, spleens, kidneys and brains, and killed most of the mice within 8 days, whereas rZH501-M847-G caused low viremia titers, did not replicate as efficiently as did rZH501-M847-A in these organs, and had attenuated virulence to mice. Remarkably, as early as 2 days postinfection with rZH501-M847-G, the viruses carrying A at M847 emerged and became the major virus population thereafter, while replicating viruses retained the input A residue at M847 in rZH501-M847-A-infected mice.Conclusions/SignificanceThese data demonstrated that the single nucleotide substitution in the Gn protein substantially affected the RVFV mouse virulence and that a virus population carrying the virulent viral genotype quickly emerged and became the major viral population within a few days in mice that were inoculated with the attenuated virus.

ABSTRACTThe speciesBabesia microti, commonly found in rodents, demonstrates a high degree of genetic diversity. Three lineages, U.S., Kobe, and Hobetsu, are known to have zoonotic potential, but their tick vector(s) in Japan remains to be elucidated. We conducted a field investigation at Nemuro on Hokkaido Island and at Sumoto on Awaji Island, where up to two of the three lineages occur with similar frequencies in reservoirs. By flagging vegetation at these spots and surrounding areas, 4,010 ticks, comprising six species, were collected. A nested PCR that detects the 18S rRNA gene ofBabesiaspecies revealed thatIxodes ovatusandI. persulcatusalone were positive. Lineage-specific PCR for rRNA-positive samples demonstrated thatI. ovatusandI. persulcatuscarried, respectively, the Hobetsu and U.S. parasites. No Kobe-specific DNA was detected. InfectedI. ovatusticks were found at multiple sites, including Nemuro and Sumoto, with minimum infection rates (MIR) of ∼12.3%. However, allI. persulcatusticks collected within the same regions, a total of 535, were negative for the Hobetsu lineage, indicating thatI. ovatus, but notI. persulcatus, was the vector for the lineage. At Nemuro, U.S. lineage was detected in 2 of 139 adultI. persulcatusticks (MIR, 1.4%), for the first time, while 48 ofI. ovatusticks were negative for that lineage. Laboratory experiments confirmed the transmission of Hobetsu and U.S. parasites to hamsters viaI. ovatusandI. persulcatus, respectively. Differences in vector capacity shown by MIRs at Nemuro, where the two species were equally likely to acquire either lineage of parasite, may explain the difference in distribution of Hobetsu throughout Japan and U.S. taxa in Nemuro. These findings are of importance in the assessment of the regional risk for babesiosis in humans.

The U.S. lineage, one of the major clades in the Babesia microti group, is known as a causal agent of human babesiosis mostly in the northeastern and upper midwestern United States. This lineage, however, also is distributed throughout the temperate zone of Eurasia with several reported human cases, although convincing evidence of the identity of the specific vector(s) in this area is lacking. Here, the goal was to demonstrate the presence of infectious parasites directly in salivary glands of Ixodes persulcatus, from which U.S. lineage genetic sequences have been detected in Asia, and to molecularly characterize the isolates. Five PCRpositive specimens were individually inoculated into hamsters, resulting in infections in four; consequently, four strains were newly established. Molecular characterization, including 18S rRNA, ␤-tubulin, and CCT7 gene sequences, as well as Western blot analysis and indirect fluorescent antibody assay, revealed that all four strains were identical to each other and to the U.S. lineage strains isolated from rodents captured in Japan. The 18S rRNA gene sequence from the isolates was identical to those from I. persulcatus in Russia and China, but the genetic and antigenic profiles of the Japanese parasites differ from those in the United States and Europe. Together with previous epidemiological and transmission studies, we conclude that I. persulcatus is likely the principal vector for the B. microti U.S. lineage in Japan and presumably in northeastern Eurasia. IMPORTANCEThe major cause of human babesiosis, the tick-borne blood parasite Babesia microti, U.S. lineage, is widely distributed in the temperate Northern Hemisphere. However, the specific tick vector(s) remains unidentified in Eurasia, where there are people with antibodies to the B. microti U.S. lineage and cases of human babesiosis. In this study, the first isolation of B. microti U.S. lineage from Ixodes persulcatus ticks, a principal vector for many tick-borne diseases, is described in Japan. Limited antigenic cross-reaction was found between the Japan and United States isolates. Thus, current serological tests based on U.S. isolates may underestimate B. microti occurrence outside the United States. This study and previous studies indicate that I. persulcatus is part of the B. microti U.S. lineage life cycle in Japan and, presumably, northeastern Eurasia. This report will be important for public health, especially since infection may occur through transfusion, and also to researchers in the field of parasitology.

Rift Valley fever virus (RVFV) (genus Rift Valley Fever virus (RVFV), which belongs to the genusPhlebovirus in the family Bunyaviridae, is endemic in subSaharan African countries and causes large outbreaks in endemic areas and countries outside of the endemic area, including Egypt, Saudi Arabia, and Yemen (2). RVFV is transmitted by mosquitoes, and the virus infection causes a high rate of abortions in pregnant ruminants and acute lethal hepatitis in newborn lambs (26). Most human patients show an acute febrile myalgic syndrome, with a small minority of patients, perhaps ϳ1%, experiencing severe hemorrhagic fever or encephalitis. Also, some patients show retinal vasculitis, which results in partial blindness for an undefined period (22).RVFV has three single-stranded genomic RNA segments, designated L, M, and S. The L and M RNA segments are of negative polarity. The L segment contains a 6,279-nucleotidelong open reading frame (ORF) encoding L protein and a viral RNA-dependent RNA polymerase (RdRp), while the M segment has a single ORF encoding NSm protein, a 78-kDa protein, and two major viral envelope proteins, Gn and Gc. The S segment is of ambisense polarity and encodes NSs protein and N protein; the former and the latter are encoded in anti-viralsense RNA and in viral-sense RNA, respectively. In bunyaviruses, both L protein and N protein are needed for viral RNA replication and viral mRNA synthesis, the latter of which uses a host mRNA-derived cap structure as a primer (3,16,29). N protein encapsidates viral RNAs to form helical nucleocapsids, which serve as a template for viral RNA synthesis (21, 23).RVFV L protein plays a central role in viral RNA synthesis, yet very little is known about how L protein exerts its functions; the properties of viral replication complexes, structures of viral RNA polymerases, and host factors needed for viral RNA synthesis have been characterized poorly for bunyaviruses. Past studies of other negative-stranded RNA viruses, including Sendai virus (36), parainfluenza virus 3 (37), and measles virus (4), all of which belong to the paramyxoviruses, and lymphocytic choriomeningitis virus (LCMV) (32), an arenavirus, suggested that their L proteins function as an oligomer to exert the RdRp function. In addition, the influenza A virus RdRp heterotrimer (18, 19) has been reported to form oligomers. Currently, it is unclear whether the L proteins of bunyaviruses also form an oligomer and exhibit RdRp activities in infected cells. We present here that RVFV L protein forms an oligomer. We further identified L protein regions that were involved in L oligomerization and explored the biological importance of the L protein oligomer for viral RNA synthesis. MATERIALS AND METHODSCells and viruses. BHK/T7-9 cells stably expressing T7 RNA polymerase (15) and Vero E6 cells were maintained as described previously (14). rMP12-rLuc (14), an RVFV MP-12 strain carrying the Renilla luciferase (rLuc) gene in place of the NSs gene in the S segment, was used.Plasmids. Plasmids encoding the mutant L protei...

No abstract

No abstract

Parasites of the Asia lineage, which are closely related to in Europe and sp. strain MO1 in the United States, were recently reported in sika deer () in eastern Japan. To identify the tick vector(s) for this parasite, we conducted a field survey in Hokkaido, Japan, where the infection rate in sika deer is the highest in the country. A specific PCR system which detects and discriminates between lineages within and between those lineages and showed that (11/822), but not sympatric (0/595) or sp. (0/163) ticks, carried Asia lineage. Genomic DNA was archived from salivary glands of partially engorged females and three isolates of Asia lineage were newly described. The 18S rRNA gene sequence of the isolates formed the Asia lineage cluster with those previously described in sika deer isolates. One salivary gland also contained parasites of U.S. lineage, which were subsequently isolated in a hamster (strain Etb5) was also detected in one tick. The 18S rRNA sequence of Etb5 was 99.7% identical to that of (AY046575) and was phylogenetically positioned in a taxon composed of isolates from Europe, China, and Russia. The geographical distribution of is consistent with that of in sika deer in Japan. These results suggest that is a principal vector for in Japan and Eurasia, where is predominantly distributed. The Asia lineage of parasites closely related to in Europe and sp. MO1 in the United States was recently reported in in eastern Japan. In this study, specific PCR for the Asia lineage identified 11 positives in 822 host-seeking ticks, a principal vector for many tick-borne disease agents. Gene sequences of three isolates obtained from DNA in salivary glands of female ticks were identical to each other and to those in We also demonstrate the coinfection of Asia lineage with U.S. lineage in a tick salivary gland and, furthermore, isolated the latter in a hamster. These results suggest that is the principal vector for as well as for , and both parasites may be occasionally cotransmitted by This report will be important for public health, since infection may occur through transfusion.