Molecular Evolutionary Analysis of the Influenza A(H1N1)pdm, May–September, 2009: Temporal and Spatial Spreading Profile of the Viruses in Japan

BACKGROUND: In March 2009, pandemic influenza A(H1N1) (A(H1N1)pdm) emerged in Mexico and the United States. In Japan, since the first outbreak of A(H1N1)pdm in Osaka and Hyogo Prefectures occurred in the middle of May 2009, the virus had spread over 16 of 47 prefectures as of June 4, 2009. METHODS/PRINCIPAL FINDINGS: We analyzed all-segment concatenated genome sequences of 75 isolates of A(H1N1)pdm viruses in Japan, and compared them with 163 full-genome sequences in the world. Two analyzing methods, distance-based and Bayesian coalescent MCMC inferences were adopted to elucidate an evolutionary relationship of the viruses in the world and Japan. Regardless of the method, the viruses in the world were classified into four distinct clusters with a few exceptions. Cluster 1 was originated earlier than cluster 2, while cluster 2 was more widely spread around the world. The other two clusters (clusters 1.2 and 1.3) were suggested to be distinct reassortants with different types of segment assortments. The viruses in Japan seemed to be a multiple origin, which were derived from approximately 28 transported cases. Twelve cases were associated with monophyletic groups consisting of Japanese viruses, which were referred to as micro-clade. While most of the micro-clades belonged to the cluster 2, the clade of the first cases of infection in Japan originated from cluster 1.2. Micro-clades of Osaka/Kobe and the Fukuoka cases, both of which were school-wide outbreaks, were eradicated. Time of most recent common ancestor (tMRCA) for each micro-clade demonstrated that some distinct viruses were transmitted in Japan between late May and early June, 2009, and appeared to spread nation-wide throughout summer. CONCLUSIONS: Our results suggest that many viruses were transmitted from abroad in late May 2009 irrespective of preventive actions against the pandemic influenza, and that the influenza A(H1N1)pdm had become a pandemic stage in June 2009 in Japan

Monitoring and Characterization of Oseltamivir-Resistant Pandemic (H1N1) 2009 Virus, Japan, 2009–2010

No evidence of sustained spread was found, but 2 incidents of human-to-human transmission were suspected

Gcm1 is involved in cell proliferation and fibrosis during kidney regeneration after ischemia–reperfusion injury

AbstractIn acute kidney injury (AKI), the S3 segment of the proximal tubule is particularly damaged, as it is most vulnerable to ischemia. However, this region is also involved in renal tubular regeneration. To deeply understand the mechanism of the repair process after ischemic injury in AKI, we focused on glial cells missing 1 (Gcm1), which is one of the genes expressed in the S3 segment. Gcm1 is essential for the development of the placenta, and Gcm1 knockout (KO) is embryonically lethal. Thus, the function of Gcm1 in the kidney has not been analyzed yet. We analyzed the function of Gcm1 in the kidney by specifically knocking out Gcm1 in the kidney. We created an ischemia–reperfusion injury (IRI) model to observe the repair process after AKI. We found that Gcm1 expression was transiently increased during the recovery phase of IRI. In Gcm1 conditional KO mice, during the recovery phase of IRI, tubular cell proliferation reduced and transforming growth factor-β1 expression was downregulated resulting in a reduction in fibrosis. In vitro, Gcm1 overexpression promoted cell proliferation and upregulated TGF-β1 expression. These findings indicate that Gcm1 is involved in the mechanisms of fibrosis and cell proliferation after ischemic injury of the kidney.</jats:p

Gcm2 regulates the maintenance of parathyroid cells in adult mice.

Glial cells missing homolog 2 (GCM2), a zinc finger-type transcription factor, is essential for the development of parathyroid glands. It is considered to be a master regulator because the glands do not form when Gcm2 is deficient. Remarkably, Gcm2 expression is maintained throughout the fetal stage and after birth. Considering the Gcm2 function in embryonic stages, it is predicted that Gcm2 maintains parathyroid cell differentiation and survival in adults. However, there is a lack of research regarding the function of Gcm2 in adulthood. Therefore, we analyzed Gcm2 function in adult tamoxifen-inducible Gcm2 conditional knockout mice. One month after tamoxifen injection, Gcm2-knockout mice showed no significant difference in serum calcium, phosphate, and PTH levels and in the expressions of calcium-sensing receptor (Casr) and parathyroid hormone (Pth), whereas Ki-67 positive cells were decreased and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) positive cell number did not change, as compared with those of controls. Seven months after tamoxifen injection, Gcm2-knockout mice showed shrinkage of the parathyroid glands and fewer parathyroid cells. A significant decrease was noted in Casr- and Pth-expressing cells and serum PTH and Ca levels, whereas serum phosphate levels increased, as compared with those of controls. All our results concluded that a reduction of Gcm2 expression leads to a reduction of parathyroid cell proliferation, an increase in cell death, and an attenuation of parathyroid function. Therefore, we indicate that Gcm2 plays a prominent role in adult parathyroid cell proliferation and maintenance

Biochemical and histological analyses in the 7MP.I mice.

(A–C) Graphs show the serum levels of Ca (A), P (B), and 1–84 PTH (C) in control (black bar, n = 7) and 7MP.I mice (white bar, n = 6) mice (U-test, *P In situ hybridization for parathyroid marker genes: Gcm2 (F, I), Pth (G, J), and Casr (H, K) in control (F–H) and in 7MP.I mice (I–K) (scale bars = 100 μm).</p

Analysis of cell proliferation and death in 1MP.I mice by Ki-67 and TUNEL immunostaining.

(A, B) Ki-67 immunostaining of the parathyroid gland tissues in control mice (A) and 1MP.I mice (B). Brown-colored cells are Ki-67-positive cells, and black arrowheads indicate Ki-67-positive parathyroid cells (Scale bar = 100 μm). (C) Control and 1MP.I tissues had Ki-67-positive cell ratios of 2.4% (n = 9) and 0.77% (n = 7), respectively (U-test, *P 0.05).</p

Construction of the <i>Gcm2</i> conditional knockout mouse model.

(A) Gcm2 has five exons: exon 2 and 3 contain the DNA binding site (black box). At the targeting vector construction, a loxP sequence was inserted in intron 1 (306 bp upstream of exon 2), and a loxP and FRT-flanked neo-cassette sequence was inserted in intron 3 (333 bp downstream of exon 3). The schema indicates the wild-type, targeting vector, targeted allele, floxed allele, and conditional knockout allele from the top to the bottom. The positions of the PCR primers are shown by the black thick arrows. (B) Genomic PCR in wild-type (C57BL/6N 846 bp) and floxed (Gcm2E2-3flNeor/ E2-3flNeor 922 bp) mice. (C) Genomic PCR in the wild-type (1454 bp), floxed (1522 bp), and Gcm2 knockout (1522 bp and 402 bp) alleles. The short band of the KO schema indicates the KO band (white arrow).</p

Biochemical and histological analyses of 1MP.I mice.

(A–C) The graphs indicate the biochemical results for serum Ca, P, and 1–84 PTH concentrations for control mice (white bar, n = 15) and 1MP.I mice (black bar, n = 14) (U-test, P > 0.05). (D, E) Histology of the parathyroid gland of control mice (D) and 1MP.I mice (E) stained with hematoxylin and eosin. Yellow arrowhead (E) indicates acinar structures surrounded by cells in the parathyroid glands. (F–K) In situ hybridization of Gcm2 (F and I), Pth (G and J), and Casr (H and K) in the parathyroid gland of control mice (F–H) and 1MP.I mice (I–K). All scale bars were 100 μm.</p

Analysis of cell proliferation by Ki-67 immunostaining in 7MP.I cells.

(A, B) Ki-67 immunostaining in the parathyroid glands of control (A) and 7MP.I (B) mice (black arrowheads indicate Ki-67-positive parathyroid cells, and yellow arrowheads indicate Ki-67-positive stromal cells; scale bar = 100 μm). (C) Control and 7MP.I tissues had Ki-67-positive cell ratios of 2.1% (black bar, n = 6) and 0.6% (white bar, n = 5), respectively (U-test, *P S2D–S2F Fig for PCNA). (D, E) TUNEL staining of the parathyroid gland tissues in control mice (D) and 7MP.I mice (E). TUNEL-positive cells are indicated in green and all cells were stained blue with DAPI. The white arrowheads indicate the TUNEL-positive parathyroid cells (scale bar = 10 μm). (F) Control and 7MP.I tissues had TUNEL-positive cell ratios of 0.09% (black bar, n = 6) and 1.27% (white bar, n = 5), respectively (U-test, *P > 0.05) (See also S4 Fig).</p

Expression patterns for marker genes in <i>Gcm2</i> conditional knockout mice.

In situ hybridization of parathyroid markers Gcm2 (B, F), Casr (C, G), and Pth (D, H) and thymus marker genes Foxn1 (A, E) in the primordial tissue (thymus and parathyroid) of E12.5 mice in control (A–D) and Gcm2 conditional knockout (E–H) groups. The dotted lines indicate the thymus–parathyroid primordium. The thymus (TH) and parathyroid (PT) regions are shown in A and E, respectively. Scale bars indicate 100 μm.</p