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Immunocytochemical and Ultrastructural Evidence Supporting That Andes Hantavirus (ANDV) Is Transmitted Person-to-Person Through the Respiratory and/or Salivary Pathways

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Immunocytochemical and Ultrastructural Evidence Supporting That Andes Hantavirus (ANDV) Is Transmitted Person-to-Person Through the Respiratory and/or Salivary Pathways

Enrique Pizarro et al. Front Microbiol.

Abstract

In South America Andes hantavirus (ANDV) is hosted by the rodent Oligoryzomys longicaudatus (also known as pygmy rice rat). In humans, ANDV causes Hantavirus Pulmonary Syndrome (HPS), with a fatality rate of about 40%. Epidemiologic and molecular evidence has shown that ANDV can be transmitted from person to person. Sin Nombre hantavirus, occurring in North America, and ANDV are genetically related, and both cause HPS with similar clinical evolution and mortality rate. However, only ANDV is transmitted from person to person. A recent hantavirus outbreak in a small village in Southern Argentine, with 29 HPS cases and 11 deaths has brought to mind that person-to-person transmission continues to be a public health emergency. The present investigation was aimed to understand how does ANDV actually spread between persons. Tissue samples of lung and salivary glands from infected Oligoryzomys longicaudatus and lethal cases of human HPS were investigated by bright field immunocytochemistry, multichannel immunofluorescence, and transmission electron microscopy. The findings are consistent with ANDV infection and replication in the lung alveolar epithelium and macrophages, and in the secretory cells of the submandibular salivary glands. In the lung of infected Oligoryzomys longicaudatus and human cases HPS, the bulk of immunoreactive hantavirus antigens was localized in epithelial cells of the alveolar walls and macrophages. The ultrastructural study supports that in the lung of HPS patients the virus replicates in the alveolar epithelial cells with virus particles being discharged into the alveolar lumen. Virus-like particles were seen within vacuoles of the lung macrophages. Considering that these macrophages can reach the conductive segments of the airways, their expectoration becomes a deadly bullet for ANDV transmission. In the submandibular glands of infected rodents and HPS cases, ANDV antigens were in capillary endothelium, the secretory cells and filling the lumen of the excretory pathway. It is proposed that in patients with HPS caused by ANDV the alveolar epithelium and macrophages would be the gate for the airway spreading of the virus, while the salivary glands are a target for virus replication and an exit pathway through saliva.

Keywords: ANDV; alveolar epithelium; hantavirus; macrophages; person-to-person transmission; salivary glands.

Figures

FIGURE 1
FIGURE 1
Lung samples from infected Oligoryzomys longicaudatus. Paraffin sections of rodents fixed by vascular perfusion with Bouin fluid. Immunostaining with anti-ANDV. (A) Low magnification view of a 7 μm thick section showing the uneven distribution of ANDV + material throughout the lung. Macrophages loaded with immunoreactive granules are seen in the lung parenchyma and attached to the luminal wall of bronchioles (arrow, small square). Area framed by rectangle is shown in (B). Inset. Detailed magnification of area framed in (A). (B) High power view of area framed in (B). Macrophages containing ANDV + masses and granules are seen in the alveolar septa and traversing the alveolar wall. The epithelial cells (ep) lining the alveolar wall contain ANDV + granules of varying size (arrows). Inset. Immunoreactive macrophages are seen in the lumen of alveolar sacs and ducts. (C) 2 μm thick section immunostained for ANDV and visualized with Nomarski optic. ANDV + granules are seen in the cells lining the alveolar walls (arrows). Asterisks indicate blood capillaries. (D) 2 μm thick section immunostained for ANDV and background staining with Toluidine Blue. 1 and 2 point to immunoreactive macrophages at the alveolar wall and reaching the alveolar lumen, respectively. Arrow points the ANDV material in an epithelial cell. (E) Combined use of silver methenamine staining and ANDV immunostaining. Low magnification view of a 7 μm thick section showing the well-preserved lung histology. An area similar to that framed is shown in (F). (F) Basement membranes of the alveolar and capillary (c) walls are stained in black by silver methenamine. Arrows point to ANDV + granules in cells located between basement membrane and alveolar lumen. (G) 7 μm thick section showing the ANDV + material in alveolar epithelial cells (ep. arrow) and in blood capillaries (bc). ad, alveolar duct; al, alveolar lumen; br, bronchiole; rb, respiratory bronchiole; Tb, toluidine blue; SM, silver methenamine. Scale bars. (A) 70 μm; inset 8 μm; (B) 7 μm; inset 7 μm; (C) 15 μm; (D) 14 μm; (E) 170 μm; (F) 12 μm, (G) 8 μm.
FIGURE 2
FIGURE 2
Human lung from lethal Hantavirus pulmonary syndrome (HPS) cases. (A) Paraffin section immunostained with anti-ANDV. Immunoreactive Hantavirus antigens are present in epithelial cells (ep) lining the alveoli, septal and luminal macrophages (M) and in cells located within blood vessels (bv) that might correspond to monocytes (Mo?). Broken and full blue arrows point to small and large immunoreactive inclusions in epithelial cells, respectively. (B) Double immunofluorescence for ANDV (green) and CF68 (red). Particulate ANDV antigens are seen in alveolar septa (arrow). There are luminal macrophages that contain virus antigens (M+) and others that do not (M–). Inset. M+ macrophage visualized only with the channel for ANDV. (C) A large blood vessel (bv) located close to an alveolar septum is shown. The alveolar epithelium (ep) contains viral granules (arrow). Cells 1 to 4 likely correspond to circulating ANDV + monocytes at different stages of migration into the alveolar lumen (al). (D) 7 μm thick paraffin section stained first with silver methenamine (SM) and then immunostained with anti-N-ANDV. The basal lamina of the epithelial and endothelial cells appears black (full black arrow). Cell located between the basal lamina and the alveolar lumen (al) contains masses (full blue arrow) and granules (broken blue arrow) of immunoreactive Hantavirus antigens (broken arrows). (E) 7 μm thick paraffin section immunostained with anti-N-ANDV and background stained with Toluidine blue (Tb). Nomarski optic. Viral antigens are seen in alveolar epithelial cells (ep) and in endothelial cells (en). Broken and full blue arrows point to small and large immunoreactive inclusions in epithelial cells, respectively. al, alveolar lumen; bv, blood vessel; ep, alveolar epithelium; en, endothelium; Mo, monocyte?; M, macrophage; Tb, toluidine blue; SM, silver methenamine. Scale bars. (A) 20 μm; (B) 14 μm; (C) 16 μm; (D) 10 μm; (E) 6 μm.
FIGURE 3
FIGURE 3
Human lung from lethal Hantavirus pulmonary syndrome (HPS) cases. (A) 7 μm thick paraffin section stained with silver methenamine (SM) and then immunostained with anti-N-ANDV. Detailed magnification of an alveolar duct showing viral material in epithelial cells (ep) and in luminal macrophages. The latter are loaded with antigen granules (Ag), N-ANDV + masses (NP) and carbon inclusions (cp) of varying size. (A’) Luminal macrophages display a varying degree of N-ANDV antigens (Ag) and carbon particles (cp) load (1–3). (B,B’) Double immunofluorescence for ANDV (green) and CF68 (red) of luminal macrophages. There are CD68+, ANDV + macrophages (1, 2) and CD68+ cells devoid of viral granules (3). Scale bars. (A) 10 μm; (A’) 20 μm; (B,B’) 10 μm. (C,D) Schematic representation of the immunocytochemical tracking of virus antigens in the lung of patients with Hantavirus pulmonary syndrome (HPS). (C) The walls of an alveolus (al) and a bronchiole (br) are represented. Red, endothelium, yellow basement membrane, blue alveolar epithelium, light brown, ciliated epithelium. Viral antigens are shown in epithelium (ep), circulatory monocytes (Mo), migratory macrophages (migr. M) and macrophages in the respiratory pathway (br). Resident microphages are shown to contain viral (red) and carbon (black) particles. (D) Detailed drawing showing viral-like particles (small red dots), viral masses (NP, nucleocapsid protein?) (large red dots) in endothelium, epithelium, monocytes and migratory macrophages. Resident macrophages containing carbon particles (black dots) also contain virus antigens. The possibility that resident macrophages incorporate those Hantaviruses that have reached the alveolar lumen either from the epithelial cells or from the airway, is represented. The large masses of virus antigens, likely corresponding to nucleocapsid protein would suggest viral replication in endothelium, epithelium and macrophages (see also Figure 5B).
FIGURE 4
FIGURE 4
Transmission electron microscopy of human lung from a lethal HPS case. (A) Low magnification view of the walls of an alveolum (al). The arrows point to the plasma membrane of neighbor epithelial cells (Ep). BM, basement membrane. Areas of this field are shown at higher magnification in the following figures. (B) Alveolar walls of the alveolum shown in (A). The cytoplasm of epithelial cells contains filamentous (fil) and granular (asterisks) inclusions. Virus-like particles (VP) and tubular structures appear lined along the apical domain of the plasma membrane. The arrows point to the plasma membrane of neighbor epithelial cells. (C) Detailed magnification of area framed by rectangle in B showing filamentous (fil, arrow) and granular (asterisk) inclusions. (D) Detailed magnification of area framed by square in B showing virus-like particles (VP) and granular inclusions (asterisk) that are in continuity with the content of tubular formations projecting to alveolar lumen (arrows). (E) Paranuclear region of an epithelial cell (Ep) showing the fine structure of the filamentous inclusions (broken arrows). (F) Alveolar epithelial cells (Ep) with isolated or clustered virus-like particles associated to the luminal cell surface. BM, basement membrane; IS, intercellular space filled with densely packed granulo-filamentous material. Left inset. Detailed view of virus-like particles (VP). Right inset. High magnification of electron dense material occupying a dilated intercellular space (IS) with a virus-like particle located at the luminal end (arrow). Scale bars. (A) 460 nm; (B) 175 nm; (C) 140 nm; (D) 105 nm; (E) 200 nm; (F) 370 nm, left inset 75 nm, right inset 180 nm.
FIGURE 5
FIGURE 5
Transmission electron microscopy of human lung from a lethal HPS case. (A) Low magnification of an area including a portion of an alveolar epithelial cells (Ep) with its basement membrane (BM), a blood capillary (bc) and an interstitial macrophage displaying large vacuoles (asterisk). (B) Detailed view of the area framed by bottom rectangle in D. Filamentous inclusions (broken arrow) occupying the cytoplasm between large phago-lysosomes (ph/l) are seen. (C) Area shown in top rectangle of (D) showing granulous inclusions near the wall of a vacuole and virus-like particles (VP) protruding into the vacuole lumen (arrow). (D) Large area of cytoplasm of a macrophage displaying numerous phago-lysosomes (ph/l) and large vacuoles with virus-like particles inside. Inset. Detailed view of a vacuole wall showing virus-like particles (VP) protruding into the lumen or lying free. (E) Area shown in right rectangle of (D) showing filamentous (broken arrow) and granulous (asterisks) inclusions. Virus-like particles protrude into the vacuole lumen (full arrow) or lie free inside the vacuole (VP). Scale bars. (A) 2 μm; (B) 130 nm; (C) 120 nm; (D) 230 nm, inset 90 nm; (E) 80 nm.
FIGURE 6
FIGURE 6
Submandibular salivary gland from infected Oligoryzomys longicaudatus. (A) Fixation by vascular perfusion with Bouin fluid. Paraffin section immunostained for ANDV antigens. There are lobules strongly immunoreactive while others are completely devoid of virus antigens. X125. (B) Section adjacent to previous one immunostained for ANDV and background stained with Toluidine blue. The supranuclear region of cells forming serous acini contain viral granules (arrows). Mucous acini are devoid of virus antigens (asterisks). (C) Paraffin section of a serous acinus. Triple channel for phase contrast (pc) and double immunofluorescence for amylase (red) and N-ANDV (green), Acinar cells contain amylase at the base and N-ANDV granules and masses in the supranuclear cytoplasm. n, cell nucleus; al, acinus lumen. (D) Section of a serous acinus immunostained for N-ANDV. Virus material is seen as large masses located at base of the cells (large white arrows), as supranuclear granules (white arrow), as fine apical granules (black arrow) and filling the acinus lumen (al, asterisks). n, cell nucleus. (E) Section of a serous acinus immunostained for ANDV. ANDV + material appears as numerous supranuclear granules (arrows) and filling the lumen of acinus (al, asterisks). The large masses at the cell base are not visualized. (F,F’) Sequential immunostaining of the same section of submaxillary gland from infected Oligoryzomys longicaudatus, using anti-ANDV labeled with FITC in the first sequence (F’) and anti-N-LNV in the second sequence revealed by the PAP method (F). Material located in the lumen of acinus reacts with both antibodies (asterisks). Anti-N-LNV reacts, in addition, with large intracellular masses located at the cell base (arrows) and granules located near the acinus lumen (arrowhead). (G) Blood capillary containing virus antigens. (H) Line drawing of a serous acinus of submandibular gland representing some of the findings shown in previous figures. Bm, basement membrane, GA, Golgi apparatus; ER, endoplasmic reticulum; n, cell nucleus; NP, nucleocapsid protein; vp, virus particles. The bent arrows indicate the probable passageway of virus from endothelium to secretory cells and to the acinar lumen. Scale bars. (A) 400 μm; (B) 20 μm; (C) 6 μm; (D) 5 μm; (E) 6 μm; (F,F’) 10 μm; (G) 8 μm.
FIGURE 7
FIGURE 7
Submandibular salivary gland from infected Oligoryzomys longicaudatus. Fixation by vascular perfusion with Bouin fluid. (A) Section of serous acini processed for double immunofluorescence for amylase (red) and N-ANDV (green). Acinar lumen (al) is filled with a material reacting with both antibodies (orange). (A’,A”) Same acinus as in (A), visualized separately for the N-ANDV and the amylase channels. al, acinus lumen. (B–B”) Cross section of an intercalated duct containing a material reacting with antibodies against N-ANDV and amylase. (C) Adjacent section to that of (B), processed for the PAS reaction for glycoproteins. The content of the intercalated duct (id) and its basement membrane (arrows) are PAS+. (D) Double immunofluorescence of submandibular gland for N-ANDV (green) and β3 integrin (red). There are β3 integrin + acini devoid of virus antigens (arrows) and others that are β3 integrin+, N-ANDV + (asterisk). (D’) Detailed magnification of acinus shown in (D) (asterisk). β3 integrin + material is at the cell base (arrow) and N-ANDV + masses and granules occupy most of the cytoplasm of serous cells. (E,E’) Sequential immunostaining of the same section of a serous acinar cell using anti-ANDV labeled with FITC in the first sequence (E’) and anti-N-LNV in the second sequence revealed by the PAP method (E). Material located in the perinuclear region reacts with both antibodies (thin arrows). Large masses located at the cell base (thick arrows) only react with anti-N-LNV (thick arrows). (F) Schematic representation of the events that would occur in serous secreting cells of the submandibular gland of Oligoryzomys longicaudatus infected with ANDV. Key events of the Hantavirus life cycle largely substantiated in previous investigations (Jonsson et al., 2010; Muyangwa et al., 2015) have been included for the better understanding of the present immunocytochemical findings. Left part of the chart: (1) Attachment of Hantavirus to the basal domain of plasma membrane of acinar cell through interaction with receptors (β3 integrin; and a second receptor of unknown nature?). (2) Entry by endocytosis. (3) Uncoating. (4) Transcription of viral RNAs. (5) Translation of virus cRNA into nucleocapsid proteins (NP) using free ribosomes (5) and into virus glycoproteins using membrane-bound ribosomes (5’) at endoplasmic reticulum (ER). (6) Transport to the Golgi apparatus (GA). (7) Replication of virus RNA. (8, 9) Assembly of virus components on route to the plasma membrane. (10) Exit. Right part of the chart: biosynthesis of amylase (53 kDa glycoprotein) at the endoplasmic reticulum (ER), glycosylation and packaging at the Golgi apparatus (orange arrow). Secretory granules (SG) move to the apical plasma membrane and underdo exocytosis. Scale bars. (A) 7 μm; (A’,A”) 5 μm; (B,B’,B”,C) 3.5 μm; (D) 20 μm; (D’) 8 μm; (E,E’) 7 μm.
FIGURE 8
FIGURE 8
Submandibular salivary gland from lethal Hantavirus pulmonary syndrome cases. (A–H) Paraffin sections immunostained with anti-ANDV and background stained with Toluidine blue. (A) Low magnification showing that only mucous secreting cells contain immunoreactive hantavirus antigens. (B,B’) Adjacent sections processes for the complete immunocytochemical procedure (B) and a procedure in which incubation in the primary antibody has been omitted (B’). (C,C’) Adjacent sections of a mixed acinus processed for the complete (C) and the incomplete (control, C’) immunocytochemical procedure. ma, mucous acinus; sd, serous demilune. (D) The cells of mucous acini are filled with Hantavirus antigens. Al, acinus lumen. (E) Mixed acini. Al, acinus lumen; ma, mucous acinus; se, serous demilune. Insert. Cross section of a striated duct immunostained for ANDV and background stained whit hemathoxylin. Viral granules are seen in the supranuclear cytoplasm; at the cell base striations are recognizable (asterisk). (F) Line drawing of a mixed acinus and its excretory pathways of human submandibular gland. Virus granules (vp) localize in endothelium of capillaries (bc), mucous cells (ma) and in the lumen of the different segments of excretory pathway, namely, intercalated duct (id), striated duct (st), excretory duct (ed). Cells of striated ducts have numerous infoldings of the basal plasma membrane and numerous mitochondria. At this site, the primary secretion produced in the acinus is modified by inflow of K+ and the outflow of Na+. Green double-head arrow transcytosis of virus? (G,H) Viral antigens are in the lumen (asterisks) of intercalated ducts (id), striated ducts (st) and excretory ducts (ed), and in the cytoplasm of striated cells (arrows) and endothelial cells of capillaries. X1250. Scale bars. (A) 200 μm; (B,B’) 200 μm; (C,C’) 15 μm; (D) 15 μm; (E) 15 μm; inset 15 μm; (G) 12 μm; (H) 20 μm.

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