1. Hantavirus Christian A. García Sepúlveda MD PhD Laboratorio de Biología Molecular Facultad de Medicina Universidad Autónoma de San Luis Potosí

2. 2 Hantavirus Introduction Hantaviruses are rodent-borne viruses which may be transmitted to humans in aerosolized urine, feces, or saliva, and occasionally by bite. Hantaviruses cause three diseases in humans: Hemorrhagic Fever with Renal Syndrome (HFRS) Hantavirus Pulmonary Syndrome (HPS)‏ Nephropatia epidémica (NE)

3. 3 Hantavirus History While HPS has only been identified since 1993, HFRS was known to Russian doctors since 1913. HFRS recognized in China as early as 1000 years ago (Liao Dynasty 907-1125). Japanese troops in Manchuria (1934).‏ Russian physicians first to implicate mice in transmission (1939). Japanese physicians compile clinical and pathological description of “Epidemic Hemorrhagic Fever” (1940).

4. 4 Hantavirus All in a name In 1951 hemorrhagic fever outbreak in Korea during world war II affected 3,200 soldiers and resulted in a 7-15% mortality rate. Outbreak occurred along Hantaan River (tributary of the Imjin). Korean hemorrhagic fever (KHF) drew widespread international attention and was reclassified as HFRS in 1983 by the WHO along with “Epidemic Hemorrhagic Fever” and Russian Hemorrhagic fever outbreaks.

5. 5 DNA Virus RT ssDNA dsDNA Virus ssRNA+ dsRNA ssRNA- RNA Virus Hantavirus Virology - Species Rabies Ebola Flu Lassa

6. 6 Hantavirus Virology - Species Bunyaviridae family 5 genera, 250 species Genus Human disease BunyavirusLaCrosse encephalitis, others PhlebovirusRift Valley fever, sandfly fever NairovirusCrimean-Congo hemorrhagic fever TospovirusPlant virus, no known human disease HantavirusHemorrhagic fever with renal syndrome Hantavirus pulmonary syndrome More than 20 types are human pathogens.

7. 7 Hantavirus Virology - Species Prospect Hill virus (PHV) Puumala virus (PUUV) Rio Mamore virus (RIOMV) Rio Segundo virus (RIOSV) Seoul virus (SEOV) Sin Nombre virus (SNV) Thailand virus (THAIV) Thottapalayam virus (TPMV) Topografov virus (TOPV) Tula virus (TULV) Akabane virus Bakau virus Bunyamwera virus Andes virus (ANDV) Bayou virus (BAYV) Black Creek Canal virus (BCCV) Cano Delgadito virus (CADV) Choclo virus (CHOV) Dobrava-Belgrade virus (DOBV) Hantaan virus (HTNV) Isla Vista virus (ISLAV) Khabarovsk virus (KHAV) Laguna Negra virus (LANV) Muleshoe virus (MULV) New York virus (NYV)

8. 8 Hantavirus Virology - Species Hantavirus are similar to other Bunyaviridae members in: –Negative sense RNA genome (ssRNA-) –Lipid membrane –Tri-segmented genome Hantavirus are different to other Bunyaviridae members in: –Hantavirus are transmitted through aerosolized rodent urine, feces and saliva. –Others genera transmitted through arthropod vectors. ARBOVIRUS = Arthropod Borne Virus

9. 9 Hantavirus Virology – Physical properties Spherical or oval-shaped. 80-120 nm diameter. Grid-like surface pattern. Lipid bilayer envelope Granulofilamentous interior Survive 12 hours at 4°C, high salt concentration and non-physiological pH and 1-3 days after drying. Exposure to lipid solvents and nonionic detergents destroys viral envelope

10. 10 Hantavirus Virology – Structure & morphology Membrane Glycoproteins G1: 64-67kDa G2: 54 kDa, highly conserved Integral membrane proteins G1-G2 heterodimers form 7-8 nm projections on virion surface Cysteine-rich Contain asparagine-linked sugar groups Important in cell entry and pathogenesis

11. 11 Hantavirus Virology – Structure & morphology Nucleocapsid protein (N) 48 kDA Complexes with genomic vRNA in virus, as well as with cRNA after infection, but not with mRNA Necessary for virus replication and packaging

12. 12 Hantavirus Virology – Structure & morphology Polymerase (L) 247 kDA RNA-dependent RNA polymerase (RdRp)‏ Complexed with ribonucleocapsids in virion Endonuclease activity to cleave host mRNA Transcriptase activity for making cRNA and mRNA from vRNA Helicase activity to unwind vRNA during transcription

13. 13 Hantavirus Virology – Genome Bunyavirus-like ssRNA (-) Trisegmented Large (L) segment, 6.5 kb, codes for polymerase Medium (M) segment, 3.6-3.7kb, codes for G1 & G2 glycoproteins Small (S) segment: 1.7-2.1kb, codes for nucleocapsid protein (NP)

14. 14 Hantavirus Virology – Genome Panhandle structure Conserved repeated complementary sequences at 5’ and 3’ ends form panhandle structures

15. 15 Hantavirus Virology – Genome REPLICATION Viral polymerase transcribes negative-strand vRNA to sense cRNA cRNA is used as template to make more negative-strand vRNA pppG is used to prime cRNA and vRNA synthesis Same “prime and realign” strategy

16. 16 Hantavirus Virology – Life cycle Attachment G1 & G2 glycoproteins interact with cell surface receptors Pathogenic hantaviruses bind β3 integrins Non-pathogenic hantaviruses bind β1 receptors

17. 17 Hantavirus Virology – Life cycle Integrins Heterodimeric receptors composed of α and β subunits. Involved in endothelial cell adhesion, platelet aggregation, cell migration, Ca ++ channel activation & extracellular matrix interactions. Expressed by endothelial cells, macrophages & platelets (cells infected by Hantavirus) Attachment of G1/G2 proteins of virion to integrin initiates endocytosis & also activates the receptor.

18. 18 Hantavirus Virology – Life cycle Entry and uncoating Virus particles bound to integrin receptors are taken in by receptor mediated endocytosis Newly formed vesicles are acidified Acidic environment changes conformation of G1 & G2 Viral and cell membranes fuse vRNA & polymerase are released into cytoplasm

19. 19 Hantavirus Virology – Life cycle Primary transcription Viral polymerase transcribes nucleoprotein-coated negative- sense vRNA to mRNA. Polymerase acts as endonuclease and cleaves host mRNAs 7-18 nt from the 5’ cap. Capped oligonucleotides from cell’s own mRNA are used to prime transcription.

20. 20 Hantavirus Virology – Life cycle Secondary transcription Extra vRNA synthesized during replication is used as template to make mRNA. Since more template is present after vRNA is replicated, more mRNA can be transcribed and more viral proteins can be made

21. 21 Hantavirus Virology – Life cycle Translation L & S segment mRNA is translated on free ribosomes. M segment mRNA translated on ER-bound ribosomes. G1 & G2 peptides produced from M are cleaved cotranslationally Separate signal sequences for G1 and G2 cause ER attachment and embed the peptides in ER membrane.

22. 22 Hantavirus Virology – Life cycle Replication Viral polymerase transcribes negative-strand vRNA to sense cRNA. cRNA is used as a template to make negative-strand vRNA More genetic material means more virions produced pppG is used to prime cRNA and vRNA synthesis “Prime and realign” strategy.

23. 23 Hantavirus Virology – Life cycle Assembly Membrane-bound G1 & G2 peptides are transported to Golgi Carbohydrates are attached by N-linked glycosylation vRNA complexes with nucleopcapsid. vRNA forms panhandle structure. vRNA complexes with polymerase

24. 24 Hantavirus Virology – Life cycle Virion release By either Golgi budding or membrane budding

25. 25 Hantavirus Virology – Life cycle Virion release By either Golgi budding or membrane budding Virion particle is formed inside the Golgi Virions are transported to cell membrane by vesicles and released by exocytosis, just like in secretion

26. 26 Hantavirus Virology – Life cycle Virion release By either Golgi budding or membrane budding Sin Nombre & Black Creek Canal G1 & G2 embedded into cell membrane through Golgi vesicles Virions bud from cell membrane, not through Golgi

27. 27 Hantavirus Virology – Pathogenesis Virus replication typically halts host macromolecule synthesis. Hantavirus release does not require host cell lysis. Hantavirus is able to establish a persistent infection in rodent host cells. Hantavirus replication does not affect reservoir cell’s natural functions.

28. 28 Hantavirus Virology – Pathogenesis Systemic Inflammatory Response Immune system activated against Hantavirus epitopes Virus epitopes expressed on surface of host cells triggers cytotoxic T-cell attack on host tissues Symptoms are consistent with inflammatory response

29. 29 Hantavirus Virology – Host specificity Each strain of hantavirus has a specific rodent host Hantavirus species appear to have co-evolved with host rodent species Rodents carrying hantavirus are asymptomatic 3 main groups: –sigmodontinae – new world mice and rats –murinae – old world mice and rats –arvicolinae – voles and lemmings

30. 30 Hantavirus Epidemiology – Reservoirs Host is an organism that harbors a virus or parasite, or commensal symbiont providing nourishment and shelter. A primary host or definitive host is a host in which the parasite reaches maturity and, if applicable, reproduces sexually.

31. 31 Hantavirus Epidemiology – Reservoirs Host is an organism that harbors a virus or parasite, or commensal symbiont providing nourishment and shelter. A secondary host or intermediate host is a host that harbors the parasite only for a short period, during which (usually) some developmental stage is completed.

32. 32 Hantavirus Epidemiology – Reservoirs Host is an organism that harbors a virus or parasite, or commensal symbiont providing nourishment and shelter. A paratenic host is similar to a secondary host, only that it is not needed for the parasite's development cycle to progress.

33. 33 Hantavirus Epidemiology – Reservoirs Host is an organism that harbors a virus or parasite, or commensal symbiont providing nourishment and shelter. A reservoir host is a primary host that can harbor a pathogen indefinitely with no ill effects, may be reinfected several times.

34. 34 Hantavirus Epidemiology – Reservoirs Host is an organism that harbors a virus or parasite, or commensal symbiont providing nourishment and shelter. A dead-end host is an intermediate host that does not allow transmission to the definite (primary) host.

35. 35 Hantavirus Epidemiology – Reservoir distribution Striped field mouse (Apodemus agrarius) Hantaan Virus Brown or Norway rat (Rattus norvegicus) Seoul virus Bank vole (Clethrionomys glareolus) Puumala virus Yellow-necked field mouse (Apodemus flavicollis) Dobrava virus.

36. 36 Hantavirus Epidemiology – Geographical distribution New World Hantaviruses Sin Nombre Peromyscus maniculatus Rio Segundo Reithrodontomys mexicanus El Moro Canyon Reithrodontomys megalotis Andes Oligoryzomys longicaudatus Bayou Oryzomys palustris Black Creek Canal Sigmodon hispidus Rio Mamore Oligoryzomys microtis Laguna Negra Calomys laucha Muleshoe Sigmodon hispidus New York Peromyscus leucopus Juquitiba Unknown Host Maciel Necromys benefactus Hu39694 Unknown Host Lechiguanas Oligoryzomys flavescens Pergamino Akodon azarae Orán Oligoryzomys longicaudatus Caño Delgadito Sigmodon alstoni Isla Vista Isla Vista Microtus californicus Bloodland Lake Microtus ochrogaster Prospect Hill Microtus pennsylvanicus Bermejo Oligoryzomys chacoensis Calabazo Zygodontomys brevicauda Choclo Oligoryzomys fulvescens

37. 37 Hantavirus Epidemiology – Reservoirs The Deer Mouse is a deceptively cute animal, with big eyes and big ears. Its head and body are normally about 2 - 3 in long and the tail adds another 2 - 3 in in length. The underbelly is always white and the tail has sharply defined white sides. The deer mouse is found almost everywhere in North America. Usually, the deer mouse likes woodlands, but also turns up in desert areas. Peromyscus maniculatus (Sin Nombre Virus)

38. 38 Hantavirus Four Corners Disease and HPS –Hantavirus Pulmonary Syndrome

39. 39 Hantavirus HPS & Four Corners Outbreak May 1993 –First clinical case –Abrupt fever, myalgia, pulmonary edema –Otherwise healthy individuals who enjoyed outdoor activities

40. 40 Hantavirus HPS & Four Corners Outbreak May 1993 N = 465 100% Gender Male 292 64% Female 173 37% RaceWhite 355 78% American indian 87 19% Black 72% Asian 31% EthnicityHispanic65 14% Case fatality16535% Age (years)Mean 38 (10-83)

41. 41 Hantavirus HPS & Four Corners Outbreak June 1993 –12 fatalities –Unexplained Adult Respiratory Distress Syndrome (ARDS) –Sera cross-reacted with Hantaan, Seoul, Puumala virus (CDC) –Rodents trapped - deer mouse main reservoir

42. 42 Hantavirus HPS & Four Corners Outbreak Winter and spring 1993 –Drought for several years followed by snow and rain –Vegetation blossomed and rodent population grew tenfold Virus isolated and named –Sin Nombre Virus (SNV) Newly emerging virus has been present since 1959 –38 year old Utah man –Events inspired 1995 film, Outbreak

43. 43 Hantavirus HPS & Four Corners Outbreak Sin Nombre virus caused approximately 200 confirmed cases of HPS during the six month outbreak and led to a 50% mortality rate.

44. 44 Hantavirus HPS & Non-SNV strains Black Creek Canal Virus -FL Bayou Virus - TX New York-1 Virus -NY Sin Nombre Peromyscus maniculatus El Moro Canyon Reithrodontomys megalotis Bayou Oryzomys palustris Black Creek Canal Sigmodon hispidus Muleshoe New York Peromyscus leucopus Isla Vista Isla Vista Microtus californicus Bloodland Lake Microtus ochrogaster Prospect Hill Microtus pennsylvanicus

45. 45 Hantavirus Epidemiology – Geographical distribution New World Hantaviruses Brazil (168)‏ Paraguay (74)‏ Uruguay (23)‏ Argentina (404)‏ United States (335)‏ Canada (36)‏ Panama (31)‏ Chile (273)‏ Bolivia (20)‏ Countries with reported cases of HPS Countries with no reported cases of HPS

46. 46 Hantavirus HPS– Seasonal variations Most cases reported during the late spring and early summer –1993 HPS outbreak followed a dramatic increase in precipitation (92-93 el niño). –Similar pattern of rainfall followed by drought during 95-96 Paraguay HPS outbreak.

47. 47 Hantavirus HPS– Seasonal variations Above normal precipitation leads to abundance in food resources for rodents (increase in rodent population). When food sources are depleted, rodents move into more permissive environments (human establishments).

48. 48 Hantavirus Epidemiology – Transmission Infectious virus is shed through host saliva, urine and fecal matter. Humans are infected by inhaling the virus from aerosols produced while host is excreting waste. Initially only HFRS strains known to transmit human to human. Human to human transmission of HPS only documented in argentina (andes virus)‏.

49. 49 Hantavirus Epidemiology – Transmission Peridomestic exposure Peridomestic & occupational Exposure Peridomestic & recreational exposure Occupational exposure Entering/cleaning rodent-infested structures 69% (48/70) 19% (13/70) 9% (6/70) 4% (3/70) 9% (6/70)

50. 50 Hantavirus Clinical characteristics Three different clinical manifestations of hantavirus infection caused by different viral strains: Hemorrhagic fever with renal syndrome (HFRS)‏ Found in Europe and Asia Nephropathia Epidemica (NE)‏ Found in Europe Hantavirus pulmonary syndrome (HPS)‏ Found in north and south America

51. 51 Hantavirus Hantavirus Pulmonary Syndrome (HPS) HPS has a 14 to 17 day incubation period. At onset patients will have a headache, fever, myalgias and a general feeling of discomfort. Most patients present with abdominal pain, nausea vomiting and a low platelet count. Patients seek medical attention until their cardiopulmonary system becomes compromised. Severe lung edema sets in 48 hours after cardiopulmonary involvement. Death usually occurs due to shock and cardiac complications (40%)

52. 52 Hantavirus Hantavirus Pulmonary Syndrome (HPS) The discovery of HPS in North America led to retrospective studies in South America. More than 140 cases of HPS confirmed in Argentina. The Andes hantavirus was discovered in long tailed pygmy rice rats in southern Argentina. Andes Virus is the only known hantavirus to be transmitted person-to- person. Prior to the HPS outbreak, the only known hantaviruses were those that caused HFRS

53. 53 Hantavirus Clinical characteristics - HPS Clinical stages: 1 - Incubation (4-30 d)‏ 2 - Febrile Phase ‏ 3 - Cardiopulmonary phase 4 - Diuretic phase 5 - Convalescent phase

54. 54 Hantavirus Clinical characteristics - HPS Clinical stages: 1 - Incubation (4-30 d)‏ 2 - Febrile Phase ‏ 3 - Cardiopulmonary phase 4 - Diuretic phase 5 - Convalescent phase 3-5 days Fever, myalgia, malaise Other symptoms: headache, dizziness, anorexia, nausea, vomiting, and diarrhea.

55. 55 Hantavirus Clinical characteristics - HPS Clinical stages: 1 - Incubation (4-30 d)‏ 2 - Febrile Phase ‏ 3 - Cardiopulmonary phase 4 - Diuretic phase 5 - Convalescent phase Lasts between 4 and 24 hrs Non-productive cough and tachypnea Rapid progression of shock Worsenning of pulmonary edema Hypovolemia (progressive leakage syndrome) Hypotension and oliguria due to hypovolemia Thrombocytopenia (especially during febrile phase)‏ Death within 24-48 hrs due to hypoxia and/or myocardial failure

56. 56 Hantavirus Clinical characteristics - HPS Clinical stages: 1 - Incubation (4-30 d)‏ 2 - Febrile Phase ‏ 3 - Cardiopulmonary phase 4 - Diuretic phase 5 - Convalescent phase Bilateral interstitial infiltrates Moderate to rapid progression Bilateral alveolar infiltrates Pleural effusion Normal heart size (not cardiogenic edema)

57. 57 Hantavirus Clinical characteristics - HPS Clinical stages: 1 - Incubation (4-30 d)‏ 2 - Febrile Phase ‏ 3 - Cardiopulmonary phase 4 - Diuretic phase 5 - Convalescent phase Several days to several weeks Beginning of recovery Rapid clearance of pulmonary edema Resolution of fever and shock Anorexia, fatigue due to dehydration

58. 58 Hantavirus Clinical characteristics - HPS Clinical stages: 1 - Incubation (4-30 d)‏ 2 - Febrile Phase ‏ 3 - Cardiopulmonary phase 4 - Diuretic phase 5 - Convalescent phase Up to 2 months Results in chronic decreased small-airway volume Diminished alveolar diffusing capacity

59. 59 Hantavirus Clinical characteristics - HFRS Occurs throughout Europe and Asia Formerly had other names, including Korean hemorrhagic fever, epidemic hemorrhagic fever & nephropathia epidemica ~15% fatality Worldwide 150,000–200,000 cases per year Outbreaks linked to contact with field rodents during planting and harvesting of crops

60. 60 Hantavirus Clinical characteristics - HFRS Clinical stages: 1 - Incubation (4-40 d)‏ 2 - Febrile Phase ‏ 3 - Hypotensive Phase 4 - Oliguric Phase 5 – Recovery: - Diuretic Phase - Convalescent Phase

61. 61 Hantavirus Clinical characteristics - HFRS 3-5 days Characterized by fever, chills Headache, severe myalgia, nausea Blurred vision, photophobia, eye pain by movement Flushing of face, V-area of the neck and back Petechiae (small red spots on skin)‏ Abdominal pain and back pain. Thirst, edema, hemoconcentration, postural hypotension Clinical stages: 1 - Incubation (4-40 d)‏ 2 - Febrile Phase ‏ 3 - Hypotensive Phase 4 - Oliguric Phase 5 – Recovery: - Diuretic Phase - Convalescent Phase

62. 62 Hantavirus Clinical characteristics - HFRS Hours to days Blood pressure decrease, hypovolemia, shock Worsening of bleeding manifestations: petechiae, epistaxis, gastrointestinal & intracranial bleeding ↑Urea & ↑Creatinine in blood. Proteinuria Leukocytosis, thrombocytopenia. Clinical stages: 1 - Incubation (4-40 d)‏ 2 - Febrile Phase ‏ 3 - Hypotensive Phase 4 - Oliguric Phase 5 – Recovery: - Diuretic Phase - Convalescent Phase

63. 63 Hantavirus Clinical characteristics - HFRS 3-7 days Oliguria due to renal dysfunction Hypervolemia leading to hypertension Blood electrolyte imbalance Hemorrhagic symptoms continue Severe complications: cardiac failure pulmonary edema, & cerebral bleeding Clinical stages: 1 - Incubation (4-40 d)‏ 2 - Febrile Phase ‏ 3 - Hypotensive Phase 4 - Oliguric Phase 5 – Recovery: - Diuretic Phase - Convalescent Phase

64. 64 Hantavirus Clinical characteristics - HFRS Several days to several weeks Beginning of recovery 3-6 liters of urine/ day; return to normal renal activity Anorexia, fatigue due to dehydration 2-3 months Progressive improvement in: glomerular filtration, renal blood flow, and urine concentrating ability Clinical stages: 1 - Incubation (4-40 d)‏ 2 - Febrile Phase ‏ 3 - Hypotensive Phase 4 - Oliguric Phase 5 – Recovery: - Diuretic Phase - Convalescent Phase

65. 65 Hantavirus Clinical characteristics - NE Puumala hantavirus strain Common mild form of HFRS in Europe Similar sequence of symptoms as HFRS, but much milder Only 6% of serologically confirmed cases require hospitalization

66. 66 Hantavirus Diagnosis Early symptoms resemble influenza, though common signs of upper respiratory disease such as sore throat, sinusitis, and ear pain not usually present More serious symptoms of hypotensive phase of HFRS or cardiopulmonary phase of HPS have acute onset Hantavirus is difficult to culture, so morphological identification is difficult. RT-PCR using primers for conserved genome regions allows confirmation of infection. PCR product can be sequenced and compared to known viral sequence database for species identification

67. 67 Hantavirus Diagnosis - HPS Abdominal pain often misinterpreted as appendicitis Many doctors outside endemic regions fail to recognize or have sufficient testing Many lab tests and radiographs appear normal Suggestive labs

68. 68 Hantavirus Diagnosis - HPS Serological tests more effective ELISA IgM capture assay, using either SNV, Laguna Negra, or Andes antigens are used in all countries that have previously detected cases Immunofluorescent test for the presence of antibodies Blood analysis also may find thrombocytopenia with platelet count less than 150,000 mm in 98% of cases

69. 69 Hantavirus Treatment No FDA approved antiviral drugs available for HFRS. Prompt diagnosis and good management of illness improves patient’s survival. No vaccine HPS is more serious and rapidly progressive illness. IV ribavirin effective for HFRS, not for HPS. Aggressive supportive care. Extra Corporeal Membrane Oxygenation (ECMO)‏ = $

70. 70 Hantavirus Control and prevention Avoid contact with rodents and their habitats. Do not keep wild rodents as pets. Keep all food in sealed containers. Virions may be stable for 2 days on a dry surface so disinfect areas contaminated by rodents using a 10% solution of household bleach or detergent. Eradication unlikely due to ubiquitous rodent populations Prevent aerosolization of virus from rodent excrement General hygiene

71. 71 Hantavirus HPS in Mexico