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• W1966650780 abstract "SC35M is a mouse-adapted variant of the highly pathogenic avian influenza virus SC35. We have previously shown that interspecies adaptation is mediated by mutations in the viral polymerase and that it is paralleled by the acquisition of high pathogenicity for mice. In the present study, we have compared virus spread and organ tropism of SC35 and SC35M in mice. We show that SC35 virus causes mild bronchiolitis in these animals, whereas infection with the mouse-adapted SC35M virus leads to severe hemorrhagic pneumonia with dissemination to other organs, including the brain. In SC35M-infected animals, viral RNA and viral antigen were detected in monocytes and macrophages, and SC35M, unlike SC35, replicated in lymphocyte and macrophage cultures in vitro. SC35M did not induce an adequate cytokine response but, unlike SC35, caused severe lymphopenia in mice. These observations suggest that the high efficiency of the SC35M polymerase is responsible for infection and depletion of lymphocytes and other white blood cells, which results in immune suppression and systemic virus spread. SC35M is a mouse-adapted variant of the highly pathogenic avian influenza virus SC35. We have previously shown that interspecies adaptation is mediated by mutations in the viral polymerase and that it is paralleled by the acquisition of high pathogenicity for mice. In the present study, we have compared virus spread and organ tropism of SC35 and SC35M in mice. We show that SC35 virus causes mild bronchiolitis in these animals, whereas infection with the mouse-adapted SC35M virus leads to severe hemorrhagic pneumonia with dissemination to other organs, including the brain. In SC35M-infected animals, viral RNA and viral antigen were detected in monocytes and macrophages, and SC35M, unlike SC35, replicated in lymphocyte and macrophage cultures in vitro. SC35M did not induce an adequate cytokine response but, unlike SC35, caused severe lymphopenia in mice. These observations suggest that the high efficiency of the SC35M polymerase is responsible for infection and depletion of lymphocytes and other white blood cells, which results in immune suppression and systemic virus spread. Highly pathogenic avian influenza viruses (HPAIV) have raised concern in recent years as potential human pathogens. Since 1997, H5N1 viruses have spread over large parts of Asia, Europe, and Africa and have occasionally caused disease in humans with an excessive case-fatality rate.1Claas EC Osterhaus AD van Beek R De Jong JC Rimmelzwaan GF Senne DA Krauss S Shortridge KF Webster RG Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.Lancet. 1998; 351: 472-477Abstract Full Text Full Text PDF PubMed Scopus (1198) Google Scholar, 2Subbarao K Klimov A Katz J Regnery H Lim W Hall H Perdue M Swayne D Bender C Huang J Hemphill M Rowe T Shaw M Xu X Fukuda K Cox N Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.Science. 1998; 279: 393-396Crossref PubMed Scopus (1210) Google Scholar Human infection with HPAIV of the H5N1 subtype initially seemed to be restricted to the lung,3Peiris JS Yu WC Leung CW Cheung CY Ng WF Nicholls JM Ng TK Chan KH Lai ST Lim WL Yuen KY Guan Y Re-emergence of fatal human influenza A subtype H5N1 disease.Lancet. 2004; 363: 617-619Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 4To KF Chan PK Chan KF Lee WK Lam WY Wong KF Tang NL Tsang DN Sung RY Buckley TA Tam JS Cheng AF Pathology of fatal human infection associated with avian influenza A H5N1 virus.J Med Virol. 2001; 63: 242-246Crossref PubMed Scopus (390) Google Scholar but recent reports5de Jong MD Simmons CP Thanh TT Hien VM Smith GJ Chau TN Hoang DM Chau NV Khanh TH Dong VC Qui PT Cam BV Ha do Q Guan Y Peiris JS Chinh NT Hien TT Farrar J Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia.Nat Med. 2006; 12: 1203-1207Crossref PubMed Scopus (1534) Google Scholar, 6Gu J Xie Z Gao Z Liu J Korteweg C Ye J Lau LT Lu J Gao Z Zhang B McNutt MA Lu M Anderson VM Gong E Yu AC Lipkin WI H5N1 infection of the respiratory tract and beyond: a molecular pathology study.Lancet. 2007; 370: 1137-1145Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 7Uiprasertkul M Puthavathana P Sangsiriwut K Pooruk P Srisook K Peiris M Nicholls JM Chokephaibulkit K Vanprapar N Auewarakul P Influenza A H5N1 replication sites in humans.Emerg Infect Dis. 2005; 11: 1036-1041Crossref PubMed Scopus (232) Google Scholar show that it can disseminate to organs beyond the respiratory tract with occasional involvement of the central nervous system. Cytokine dysfunction and high viral loads observed in H5N1-infected patients have been reported to contribute to H5N1 pathogenesis in humans.3Peiris JS Yu WC Leung CW Cheung CY Ng WF Nicholls JM Ng TK Chan KH Lai ST Lim WL Yuen KY Guan Y Re-emergence of fatal human influenza A subtype H5N1 disease.Lancet. 2004; 363: 617-619Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 5de Jong MD Simmons CP Thanh TT Hien VM Smith GJ Chau TN Hoang DM Chau NV Khanh TH Dong VC Qui PT Cam BV Ha do Q Guan Y Peiris JS Chinh NT Hien TT Farrar J Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia.Nat Med. 2006; 12: 1203-1207Crossref PubMed Scopus (1534) Google Scholar In 2004, a HPAIV of subtype H7N7 has caused a human outbreak. In most instances disease was mild, but one case was fatal.8Fouchier RA Schneeberger PM Rozendaal FW Broekman JM Kemink SA Munster V Kuiken T Rimmelzwaan GF Schutten M Van Doornum GJ Koch G Bosman A Koopmans M Osterhaus AD Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome.Proc Natl Acad Sci USA. 2004; 101: 1356-1361Crossref PubMed Scopus (925) Google Scholar Although, all of the H5N1 and H7N7 isolates obtained up to now are still avian viruses, mutations have been observed that are thought to promote adaptation to humans. Most of these adaptive mutations are located in the hemagglutinin,1Claas EC Osterhaus AD van Beek R De Jong JC Rimmelzwaan GF Senne DA Krauss S Shortridge KF Webster RG Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.Lancet. 1998; 351: 472-477Abstract Full Text Full Text PDF PubMed Scopus (1198) Google Scholar, 2Subbarao K Klimov A Katz J Regnery H Lim W Hall H Perdue M Swayne D Bender C Huang J Hemphill M Rowe T Shaw M Xu X Fukuda K Cox N Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.Science. 1998; 279: 393-396Crossref PubMed Scopus (1210) Google Scholar, 8Fouchier RA Schneeberger PM Rozendaal FW Broekman JM Kemink SA Munster V Kuiken T Rimmelzwaan GF Schutten M Van Doornum GJ Koch G Bosman A Koopmans M Osterhaus AD Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome.Proc Natl Acad Sci USA. 2004; 101: 1356-1361Crossref PubMed Scopus (925) Google Scholar, 9Hatta M Gao P Halfmann P Kawaoka Y Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses.Science. 2001; 293: 1840-1842Crossref PubMed Scopus (1131) Google Scholar, 10Matrosovich MN Matrosovich TY Gray T Roberts NA Klenk HD Human and avian influenza viruses target different cell types in cultures of human airway epithelium.Proc Natl Acad Sci USA. 2004; 101: 4620-4624Crossref PubMed Scopus (622) Google Scholar, 11Shinya K Ebina M Yamada S Ono M Kasai N Kawaoka Y Avian flu: influenza virus receptors in the human airway.Nature. 2006; 440: 435-436Crossref PubMed Scopus (1113) Google Scholar the NS1 protein,12Garcia-Sastre A Antiviral response in pandemic influenza viruses.Emerg Infect Dis. 2006; 12: 44-47Crossref PubMed Scopus (65) Google Scholar, 13Geiss GK Salvatore M Tumpey TM Carter VS Wang X Basler CF Taubenberger JK Bumgarner RE Palese P Katze MG Garcia-Sastre A Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza.Proc Natl Acad Sci USA. 2002; 99: 10736-10741Crossref PubMed Scopus (308) Google Scholar, 14Jiao P Tian G Li Y Deng G Jiang Y Liu C Liu W Bu Z Kawaoka Y Chen H A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice.J Virol. 2008; 82: 1146-1154Crossref PubMed Scopus (365) Google Scholar, 15Krug RM Yuan W Noah DL Latham AG Intracellular warfare between human influenza viruses and human cells: the roles of the viral NS1 protein.Virology. 2003; 309: 181-189Crossref PubMed Scopus (222) Google Scholar, 16Seo SH Hoffmann E Webster RG Lethal H5N1 influenza viruses escape host antiviral cytokine responses.Nat Med. 2002; 8: 950-954Crossref PubMed Scopus (604) Google Scholar and the polymerase proteins.17Gabriel G Dauber B Wolff T Planz O Klenk HD Stech J The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc Natl Acad Sci USA. 2005; 102: 18590-18595Crossref PubMed Scopus (560) Google Scholar, 18Salomon R Franks J Govorkova EA Ilyushina NA Yen HL Hulse-Post DJ Humberd J Trichet M Rehg JE Webby RJ Webster RG Hoffmann E The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04.J Exp Med. 2006; 203: 689-697Crossref PubMed Scopus (307) Google Scholar Recent studies from our laboratory on strain SC35, an HPAIV of subtype H7N7, and its mouse-adapted variant SC35M have highlighted the prominent role of the viral polymerase in interspecies transmission. We have identified seven mutations in the polymerase complex that were responsible for enhanced transcription and replication efficiency of SC35M in mammalian cells and thus for adaptation to mice,17Gabriel G Dauber B Wolff T Planz O Klenk HD Stech J The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc Natl Acad Sci USA. 2005; 102: 18590-18595Crossref PubMed Scopus (560) Google Scholar and we found that adaptation of the polymerase proteins to the nuclear import machinery was a crucial mechanism in this process.19Gabriel G Herwig A Klenk HD Interaction of polymerase subunit PB2 and NP with importin α1 is a determinant of host range of influenza A virus.PLoS Pathog. 2008; 4: e11Crossref PubMed Scopus (306) Google Scholar It was also clear from these studies that the mutations in the polymerase were not only responsible for mouse adaptation but also for increased mouse pathogenicity of SC35M. In the present study, we have compared SC35 and SC35M for spread of infection and tissue tropism in mice. We found that infection with SC35 is restricted to the lung with mild symptoms of disease. In contrast, infection with SC35M is characterized by an always lethal outcome with severe pneumonia and systemic spread that is presumably mediated by virus replication in T cells and monocytes and a concominant lymphocyte depletion. EL-4 (murine T lymphocyte) and J774 (murine macrophage) cell lines were provided by H. Garn (Philipps University Marburg, Marburg, Germany). Cells were grown in RPMI 1640 medium (PAA) supplemented with 10% fetal calf serum (Invitrogen, San Diego, CA). In addition, J774 cells were supplemented with 1% nonessential amino acids (PAA). Murine lung cells (LA-4) were grown in Ham’s F-12 (Invitrogen) supplemented with 15% fetal calf serum (Invitrogen) and 1% nonessential amino acids (PAA). Influenza A viruses were propagated in 11-day-old embryonated chicken eggs. The recombinant viruses SC35 and SC35M were described before.17Gabriel G Dauber B Wolff T Planz O Klenk HD Stech J The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc Natl Acad Sci USA. 2005; 102: 18590-18595Crossref PubMed Scopus (560) Google Scholar We ascertained the identity of the recombinant viruses by sequencing amplicons of each viral gene segment by using RT-PCR as described previously.17Gabriel G Dauber B Wolff T Planz O Klenk HD Stech J The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc Natl Acad Sci USA. 2005; 102: 18590-18595Crossref PubMed Scopus (560) Google Scholar Murine lung cells (LA-4) were infected using multiplicity of infection 2 with recombinant virus. At 6 hours p.i., cells were harvested, and total RNA was isolated using TRIzol reagent (Invitrogen). After spectrophotometric quantification and normalization of total RNA, primer extensions were performed as described elsewhere.20Gabriel G Abram M Keiner B Wagner R Klenk HD Stech J Differential polymerase activity in avian and mammalian cells determines host range of influenza virus.J Virol. 2007; 81: 9601-9604Crossref PubMed Scopus (101) Google Scholar, 21Gabriel G Nordmann A Stein DA Iversen PL Klenk HD Morpholino oligomers targeting the PB1 and NP genes enhance the survival of mice infected with highly pathogenic influenza A H7N7 virus.J Gen Virol. 2008; 89: 939-948Crossref PubMed Scopus (54) Google Scholar Briefly, 1 μg of total RNA was mixed with 1 pmol DNA primer labeled at its 5′-end with [γ-32P]ATP and T4 polynucleotide kinase in 6 μl of water and denatured at 99°C for 5 minutes. The mixture was then cooled on ice, and after addition of the reverse trancriptase SuperScriptII (Invitrogen) to the reaction buffer, primer extensions were performed at 45°C for 1 hour. Two nucleoprotein specific primers were used in the same reverse transcription reaction: 5′-GATGTGTCTTTCCAGGGGCG-3′ (for detection of vRNA), 5′-GCCTCCCTTCATAGTCGCTG-3′ (for detection of cRNA and mRNA), and 5′-TCCCAGGCGGTCTCCCATCC-3′ (for detection of 5S rRNA) for standardization. Transcription products were analyzed on 6% polyacrylamide gels containing 7 M urea in Tris-borate EDTA buffer and detected by autoradiography. Transcription products of two independent experiments were quantified using Aida Image Analyzer 3.27 software. EL-4 and J774 cells were inoculated with virus at a multiplicity of infection of 0.01. Virus inoculum was removed after 30 minutes of incubation at 37°C, and cells were washed two times with PBS. Cells were then incubated in the appropriate medium containing 0.2% bovine serum albumin at 37°C. At time points 0, 24, 48, 72, and 120 hours postinfection (p.i.) supernatants were collected and plaque forming units (PFU) determined on MDCK cells as described previously.17Gabriel G Dauber B Wolff T Planz O Klenk HD Stech J The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc Natl Acad Sci USA. 2005; 102: 18590-18595Crossref PubMed Scopus (560) Google Scholar The growth curves shown are the average result of two independent experiments. The animal experiments were performed according to the guidelines of the German animal protection law. All animal protocols were approved by the relevant German authority, the Regierungspräsidium Gießen. We anesthetized 16 female BALB/c mice per group, 4–6 weeks old (purchased from Harlan Winkelmann, Borchen, Germany) with ketamine-xylazine (100 and 10 mg/kg, respectively) and inoculated intranasally with 106 PFU of SC35 or SC35M, respectively, in 50 μl diluted in PBS. For cytokine assays, three mice per group for each time point were sacrificed at 3 and 6 days p.i. Virus titers were determined in whole organ homogenates of lung, brain, and heart tissues on day 3 p.i. of three animals by plaque assay. To exclude any additional unwanted mutations in the viral genome, we have sequenced the whole cDNA from infected organ homogenisates as described before.17Gabriel G Dauber B Wolff T Planz O Klenk HD Stech J The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc Natl Acad Sci USA. 2005; 102: 18590-18595Crossref PubMed Scopus (560) Google Scholar The remaining animals were observed for 14 days after infection for signs of disease, and their weight was monitored daily. For in situ hybridization and immunohistochemistry, 10 animals were infected with 106 PFU of SC35 or SC35M, respectively, as described above. Several organs, including lung, heart, brain, liver, kidney, spleen and gut of five animals per group, were removed on days 3 and 6 p.i., fixed over night in 4% buffered paraformaldehyde, and embedded in paraffin. Tissue sections (5-μm thick) were used for staining. Influenza RNA in tissues was detected using single-stranded 35S-labeled RNA probes that were synthesized from a pBluescript KS+ vector containing a fragment of the NP gene (nt 1077 to 1442) of A/FPV/Rostock/34 (H7N1).22Feldmann A Schafer MK Garten W Klenk HD Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34 (H7N1) in chicken embryos.J Virol. 2000; 74: 8018-8027Crossref PubMed Scopus (89) Google Scholar Linearization of this plasmid with HindIII and subsequent T7 RNA polymerase transcription produced an antisense RNA probe suited to detect NP-specific RNA. Control RNA probes were obtained from the vector containing the dual-promoter plasmid of coxsackievirus B3 (pCVB3-R1).23Klingel K Hohenadl C Canu A Albrecht M Seemann M Mall G Kandolf R Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation.Proc Natl Acad Sci USA. 1992; 89: 314-318Crossref PubMed Scopus (388) Google Scholar Pretreatment, hybridization, and washing conditions of dewaxed 5-μm paraffin tissue sections were performed as described previously.23Klingel K Hohenadl C Canu A Albrecht M Seemann M Mall G Kandolf R Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation.Proc Natl Acad Sci USA. 1992; 89: 314-318Crossref PubMed Scopus (388) Google Scholar Slide preparations were subjected to autoradiography, exposed for 3 weeks at 4°C, and counterstained with H&E. Before immunohistochemical stainings of deparaffinated lung tissue with a monoclonal ferret antibody recognizing influenza A/Vic/3/75 (H3N2) (provided by the World Health Organization, Geneva, Switzerland) (1:500) slides were pretreated with 0.1 M citrate buffer, pH 6.0. A biotinylated anti-ferret antibody (1:200; Rockland) was used followed by the Zytochem-Plus horseradish peroxidase (Zytomed) kit and AEC as substrate under the conditions described by the manufacturer. Controls using normal rat serum were run to exclude nonspecific staining. Blood samples of animals infected with 106 PFU of SC35 or SC35M, respectively, were collected on days 3 and 6 p.i. in K3EDTA tubes. For the automated blood cells count, 500 μl of blood was analyzed with the Animal Blood Counter ABC Vet scil (Vet Novations). Frequencies of white blood cells, red blood cells, platelets, as well as hemoglobin and hematocrit, were measured. Recommended settings and calibrations for mouse hematology were set according to the manufacturer’s operation manual. The data presented are average results from three to five infected mice for each time point. Blood (100 μl) from either naive C57BL/6 mice or C57BL/6 mice infected with influenza virus (see above) for 3 days was diluted in 5 ml of FACS buffer (PBS 2% fetal calf serum, EDTA) and centrifuged at 300 × g for 5 minutes at 4°C. The pellet was incubated with a 1:100 dilution of anti-CD3 FITC, anti-CD8 CyChrome, and anti-CD11b PE (BD Biosciences) in 100 μl of FACS buffer for 30 minutes at 4°C. Thereafter the cells were washed once. Before FACS analysis, erythrocytes were lysed, and cells were fixed with FACS lysing solution (BD Biosciences). Flow cytometry was performed on a dual-laser FACSCalibur and analyzed with CellQuest software (BD Biosciences). A mouse cytokine antibody array (RayBiotech, Inc.) was used according to the manufacturer’s protocols to detect a panel of cytokines and chemokines from the lung and spleen homogenates of influenza infected mice. Organs were homogenized in 1 ml of PBS, centrifuged at 1000 rpm for 10 minutes, and the supernatant was aliquoted and frozen at −80°C. Samples were not thawed more than once for this assay. The array tests for the following cytokines and chemokines: granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL12-p40p70, IL12-p70, IL-13, IL-17, interferon-γ, monocyte chemoattractant protein (MCP)-1, MCP-5, regulated on activation normal T cell expressed and secreted, stem cell factor, soluble tumor necrosis factor α receptor I (sTNFRI), TNF-α, and vascular endothelial growth factor. Briefly, membranes with bound cytokine antibodies were incubated with 200 μg of lung or spleen homogenate. After incubation, each membrane was washed three times and incubated with biotin-conjugated anti-cytokine antibody and then with horseradish peroxidase-conjugated streptavidin. Cytokine levels were then detected by chemiluminescence and revealed on X-ray film. Intensity of signals was quantified by TINA 2.0 software. Background signals comparable with PBS infected animals were set 1. Each column represents three independent assays. Groups of 16 mice were infected with either SC35 or SC35M virus as described in Materials and Methods. SC35-infected mice underwent a temporary weight loss of 20 to 30%, but all infected animals survived infection (log10MLD50 > 6.0 PFU). In contrast, mice infected with SC35M started to die on day 4 p.i., and all infected animals succumbed to infection by day 7 p.i. (log10MLD50 = 2.8 PFU) (Figure 1A). It has to be pointed out that SC35 and SC35M used throughout this study were recombinant viruses generated by reverse genetics.17Gabriel G Dauber B Wolff T Planz O Klenk HD Stech J The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.Proc Natl Acad Sci USA. 2005; 102: 18590-18595Crossref PubMed Scopus (560) Google Scholar Nonrecombinant SC35 differed from SC35 used here by a higher pathogenicity (10 to 25% lethality at the same inoculation dose).24Scheiblauer H Kendal AP Rott R Pathogenicity of influenza A/Seal/Mass/1/80 virus mutants for mammalian species.Arch Virol. 1995; 140: 341-348Crossref PubMed Scopus (41) Google Scholar Although not completely understood, this phenomenon may reflect differences in quasispecies equilibria. We have also compared the polymerase activities of SC35 and SC35M in mouse lung cells by primer extension assay. As shown in Figure 1B, the transcription and replication activity of SC35M was 3 to 10 times higher in these cells than that of SC35. These results confirm our previous observations indicating that the enhanced pathogenicity of SC35M for mice is the result of increased polymerase efficiency in murine cells.20Gabriel G Abram M Keiner B Wagner R Klenk HD Stech J Differential polymerase activity in avian and mammalian cells determines host range of influenza virus.J Virol. 2007; 81: 9601-9604Crossref PubMed Scopus (101) Google Scholar We have determined virus titers in organ homogenates of mice sacrificed at various times after infection. At 3 days p.i., SC35M was recovered from lung, brain, heart, liver, kidney, spleen, and gut (Figure 2A). In contrast, significant amounts of SC35 were only observed in the lung, but titers were about 100 times lower when compared with SC35M infection (Figure 2A). Virus was also detected at low titers (<102 PFU/ml) in the serum 3 and 6 days after infection with SC35M but not in SC35-infected animals (Table 1).Table 1Virus Titers in Serum of Infected MiceVirusVirus titer 3 days p.i. (PFU/ml)Virus titer 6 days p.i. (PFU/ml)PBS00SC3500SC35M3060Mice were infected with 106 pfu of SC35 and SC35M. As a control, PBS-inoculated mice were used. On days 3 and 6 p.i., blood was collected from three infected mice at each time point by retroocular puncture. Virus titers in serum were detected by plaque assay. Open table in a new tab Mice were infected with 106 pfu of SC35 and SC35M. As a control, PBS-inoculated mice were used. On days 3 and 6 p.i., blood was collected from three infected mice at each time point by retroocular puncture. Virus titers in serum were detected by plaque assay. Organ tropism was also analyzed by radioactive in situ hybridization. At day 3 p.i., SC35 was only observed in single cells of the lung. Also, at day 6 p.i., a few alveolar cells were found to contain viral RNA; however, further development of the infection was not observed in the lung. Pulmonary damage and inflammation was only marginal, both at days 3 and 6 p.i. SC35 RNA, inflammation, or necrosis/apoptosis was neither detected in brain nor in heart liver, kidney, spleen, and gut tissues (Figure 2B). In contrast to mice infected with SC35, in situ hybridization revealed numerous alveolar and bronchial epithelial cells positive for viral RNA in SC35M-infected animals at days 3 (Figures 2B and 3) and 6 p.i. (Figure 2B). Furthermore, at day 6 p.i., the lungs of SC35M-infected mice revealed typical pulmonary lesions with desquamation and destruction of epithelial cells and inflammation with hemorrhages (Figure 2B). Viral RNA was not only detected in the lung but also in the brain where it was specifically located in the temporal lobes. Neither inflammatory and other histological lesions nor viral RNA could be detected in heart, liver, kidney, spleen, and gut (Figure 2B). These observations suggest that SC35M infects parenchymal cells only in the brain and that infectious virus recovered from other organs (Figure 2A) originates from blood present in these tissues. The data obtained by in situ hybridization were complemented by immunohistochemical analysis of viral proteins in lung tissue. The patterns of SC35M-infected cells that were obtained with both procedures in alveolar and bronchial epithelia were similar (Figure 3, A–D). Interestingly, the viral RNA (Figure 3A, arrow) and protein (Figure 3C, arrow) were also present in mononuclear inflammatory cells in the lung. In addition, infected monocytes were detected in peripheral blood (Figure 3, E and F). Taken together, these data show that SC35 causes a mild infection in mice restricted to the lung, whereas SC35M causes lethal hemorrhagic pneumonia with systemic virus spread and infection of the central nervous system. We have then compared blood parameters in infected mice and control animals. On days 3 and 6 p.i., blood was collected by retroocular puncture and analyzed for white blood cells, red blood cells, hemoglobin, hematocrit, platelets, lymphocytes, monocytes, and granulocytes (Table 2). In mice infected with SC35, levels of monocytes, lymphocytes, and granulocytes as well as total white blood cells increased over the period of 6 days, while platelet counts dropped transiently at day 3 p.i. On the other hand, in SC35M-infected mice, a substantial drop of hemoglobin and hematocrit were observed on day 6 p.i., which is most likely due to blood loss as the result of the hemorrhagic pneumonia (Figures 2B and 3, A–F). SC35M infection further led to a constant reduction of white blood cell numbers, which was already apparent at day 3 p.i. and further continued until day 6 p.i. The decrease was most prominent with lymphocytes. Their number dropped by >50% at day 3 p.i. and stayed at this low level throughout the period of observation. To shed light on the response of individual subsets of lymphocytes to infection with SC35 and SC35M, we performed FACS analysis of blood on day 3 p.i. using specific antibodies targeting CD3+, CD4+, and CD8+ T cells and CD11b monocytes as described in Materials and Methods. In SC35M-infected mice, about 50% of CD8+ T cells and >70% of CD4+ T cells were depleted 3 days p.i. (Figure 4, A and C), whereas SC35 infection resulted only in a moderate shift in T-cell proportions (Figure 4B). These data are compatible with the view that infection with SC35 that is confined to the lung elicits protective T-cell response in mice. In contrast, systemic infection with SC35M is characterized by a rapid and severe depletion of T cells.Table 2Blood Parameters of Infected MiceSC35SC35 mol/LBlood cell countPBS3 days p.i.6 days p.i.3 days p.i.6 days p.i.WBC (103/mm3)6.46.79.25.03.8±0.7±3.0±0.9±0.1±1.2RBC (106/mm3)7.99.18.510.45.8±0.8±1.3±0.9±0.3±1.1HGB (g/dl)14.116.314.517.69.6±1.1±1.8±1.7±0.3±1.7HCT (%)44.445.447.156.230.5±3.8±6.4±5.3±1.6±6.0PLT (103/mm3)11614477821348446±157±179±141±68±234LYM (103/mm3)4.83.15.92.11.9±0.6±0.5±0.4±0.1±0.7MO (103/mm3)0.010.20.10.10.05±0.0±0.3±0.0±0.0±0.1GRA (103/mm3)1.63.93.32.81.9±0.2±0.4±0.4±0.3±0.4Mice were infected with 106 PFU of SC35 and SC35M. Blood of three to five infected animals was collected 3 and 6 days p.i. by retroocular puncture and analyzed using an ABC blood counter for detection of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), platelets (PLT), lymphocytes (LYM), monocytes (MO), and granulocytes (GRA). As a control, PBS-inoculated mice were used. SDs (±) are indicated underneath the bold measurements. Open table in a new tab Mice were infected with 106 PFU of SC35 and SC35M. Blood of three to five infected animals was collected 3 and 6 days p.i. by retroocular puncture and analyzed using an ABC blood counter for detection of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), platelets (PLT), lymphocytes (LYM), monocytes (MO), and granulocytes (GRA). As a control, PBS-inoculated mice were used. SDs (±) are indicated underneath the bold measurements. To assess the cytokine and chemokine response in mice on infection with the two different influenza viruses, we have screened lungs and spleens of infected animals using a panel of antibodies specific for 22 d" @default.
• W1966650780 created "2016-06-24" @default.
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• W1966650780 creator A5024909409 @default.
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• W1966650780 date "2009-09-01" @default.
• W1966650780 modified "2023-09-27" @default.
• W1966650780 title "Spread of Infection and Lymphocyte Depletion in Mice Depends on Polymerase of Influenza Virus" @default.
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