Human Parvovirus B19 Infections Have Emerged but Given a Back Seat: A Pioneer

work From India indicates High Disease Burden

Prof Janak Kishore Division of Virology Department of Microbiolgy Sanjay Gandhi Post-Graduate

Institute of Medical Sciences

Lucknow, INDIA

Discovered: 1974 : Emeritus Professor Yvonne Cossart (born 1934 Sydney) Australian virologist working in London, Blood Donors Hepatitis B

B19: Occupying position 19 in plate B Nakatani” virus by Okochi et al in Japan {Lancet 1975,1985} 18-23 nm Small (Gk. Parvum=small) Non-enveloped, ssDNA virus, 96 capsomeres Genus Erythrovirus, family Parvoviridae Genome 5.5Kb, 3 ORF : NS1, VP1 and VP2 Genetically stable : 1.5% variations No association found between genotype & a particular clinical feature

Human Parvovirus B19 Genome and transcription map

EPIDEMIOLOGY

• Incubation period: 6-11 days

• Mode of Transmission:

Through upper Respiratory tract by droplets

Transfusion of Blood or its components Transplacental

• Host: All ages, School children, Females• Both immunocompromised & immunocompetent• Season: Early Spring late Winter, thro- Yr

5 European Countries: Mossong J et al 2008

• On 13,449 serum • Age-specific risk in all five countries : highest in 7-9 years

and lower in adults. • Susceptibilty in Women of child-bearing age & Risk of

acquiring B19 infection during pregnancy was:-• 26% and 0.61% in Belgium, • 38% and 0.69% in England & Wales,• 43.5% and 1.24% in Finland, • 39.9% and 0.92% in Italy {India: Kishore J, Kapoor A; 2010} • 36.8% and 1.58% in Poland, respectively.

Receptor & Cell tropism

Blood group “P antigen” Gb4Ceramide (globoside) : binding {Brown K E, Anderson SM, Young NS;1993} Co-receptors: Binding and Entry :

α5β1 integrins or 51 integrins (K562) {Kirsten A et al;2003}

Ku80 (autoantigen) {Munakata Y et al; 2005}

Erythroid Progenitor cells : both P antigen and α5β1

great tropism : highly efficient systemic dissemination

• Fetal Hepatocytes

• Vascular Endothelial cells: Myocardial cells, Liver, Kidneys, Brain

• Megakaryocytes ( Some)

• Synoviocytes

• Monocytic Cell-line U937 {Munakata Y et al; 2006}

Immunopathogenesis• B19 inflicts direct

virus injury through

NS1, VP1u proteins

virus Persistence

Immune mediated

injuries

Occasionally fatal

Macrophage activation

syndrome

Clinical Features of B19 Asymptomatic ~ 80% [Marks JL; 1884] Erythema Infectiosum [Pattison JR,1987]

1981 : Transient Aplastic Crisis [Thurns J; 1988} 1985 : Arthropathy [Anderson LJ ,1990] 1987 : Hydrops fetalis [ Kerr JR, 1996] Fetal loss Hematol: {Kishore J, 2000} Severe Anemia {Kishore J et al 2011}

PRCA , Aplastic anemia , Death {Kishore J et al 2004} 3 Novel: Non-occlusive Gangrene of stomach bowel {Kishore J et al 2009} Erythema Infectiosum with Myositis {Kishore J et al 2006} B19- Amegakaryocytic Thrombocytopenia {Kishore J et al 2005} Oncolytic Property …Leukemia {Kishore J et al 2014, 2015}

EM :Serum, tissue sections (~1012paticles/ml ) Inclusion body : Lantern cells B/M

Antigen : Antibody : IgM capture ELISA,RIA NAT: PCR, Nested PCR, Real- time PCR hybridization DNA Sequencing Epidemiological: Genotyping

Standardization of in-house ELISA for antibody detection against B19: Our Experience

Treatment

• Erythema infectiosum : No treatment • B-19 induced arthralgia : Symptomatic• TAC: Blood transfusion

( Hb % )

• Fetal Hydrops : Intrauterine BT• Persistent infection: I.V.I.G

(PRCA, HIV / AIDS ) 0.4 gm/kg x 5 days OR1.0 gm/ kg x 2-3 days

Recently: RITUXIMAB : B cell-depleting agent :4 weekly doses of 375 mg/m2

• Future proposal: Monoclonal Ab to B-19 proteins

Clinical Impact of B19 infections

 S. No.

  Clinical conditions  

YearTotal No. Sera Tested Infected

  % +ve

1. Juvenile Rheumatoid Arthritis 1998 69 19 27.52. PRCA - case report 2003 1 1

3.  Amegakaryocytic Thrombocytopenia (Novel report) 2005 1 1  

4. Myositis : Erythema infectiosum (Novel report)

2006 1 1  

5.Recurrent spontaneous abortions (≥ 3

abortions)

2006 116 

  19.8

6. Fulminant hepatitis and Thrombocytopenia 2009 1 17. Seroprevalence in Blood Donors (IgG) 2010 1000

  399

39.98. Gangrene of stomach or intestines owing to

non-occlusive bowel infarction : Novel finding

2010  08 08 100

9. Haematological Pediatric Malignancies 2011 35 07  28

10 Multitransfused Thalassemia major, IgM ELISA 2011 90 37   41.1

11. High-risk pregnant women with BOH 

2011  60   36 6012. Hemophagocytic Lymphohistiocytosis EBV B19

2014 

1 1

B19 induced MYOSITIS : Erythema Infectiosum

Blocked gastric blood vessel with fibrinous

deposit in its lumen

Problems with B19• All Clinical Features of B19 are not known to all clinicians• Diagnostic facilities still limited• Cryptic sites of virus replications• DNAemia : is brief• B19 virus Titers : be low : –ve in Wks : except in persistent • IgM antibodies to B19 : low titers in immunocompromised• ELISA Kits : prone to False –ve/+ve• EM: very limited, detects very high virus titers • Clinicians Ignore B19 infections, self–limiting• Treatment I.V.I.G. is : Costly• Relapses : seen in 20-25% cases, in HIV or transplant cases• Prophylactic : Vaccine : None licensed

RECENT ALARMS

Heart:

• Myocarditis • Dilated Cardiomyopathy,• Arrhythmogenic : Familial long QT Brugada syndrome

Brain: Stroke

Liver: Hepatitis, FHF

Kidney :

Collapsing Glomerulopathy Thrombotic Microangiopathy

Lungs

New Cardiotropic agents : Inflammatory Cardiomyopathy {Bock CT et al 2005}

B19 infects of intracardiac endothelial cells of small arterioles and veins causing• Endothelial dysfunction, impaired

myocardial microcirculation, penetration of inflammatory cells and secondarily myocyte { Kandolf R 2004}

SUDDEN Death: Myocarditis 5 yr child { Murrey CE et al 2001} 8 yr child: {Dettmeyer R etal 2003} Two children { Koehl B et al 2012} Mimicking Acute Myocardial Infarction

{Kuhl U et al 2003}

Brugada syndrome : Genetic abnormality of sodium channels in the myocardium with characteristic ECG Sudden Death {Juhasz Z et al 2014}

Agent of Inflammatory Cardiomyopathy

• Dilated Cardiomyopathy• Pediatric and adult inflammatory cardiac diseases• Clues to etiopathogenetic role by ISH and PCR

Infection of Endothelial cells of small intracardiac arterioles and venules, which may be associated with endothelial dysfunction

impairment of Myocardial microcirculation Penetration of inflammatory cells in the myocardium

(Bock CT 2006)

B19 +ve in Aplastic anemia.

• B19 IgM and viral DNA were detected in higher proportions in patients than controls

40.7% vs 5%, P < 0.01 37% vs 0%, P < 0.001 {Mishra B et al; Am J Hematol 2005} • 16 (26.7%) of 60 Aplastic anemia cases were B19

DNA positive { Qian X et al 2001}

B19 in Human Tissues• in 13 of 50 (26%) bone marrow samples from

rheumatic patients indicating persistent infection {Lundqvist A et al 2005} • B19V persists in a wide range of erythroid and non-

erythroid tissues• Low-level viral gene expression occurs in some

persistently infected cells:-- Bone marrow, Heart, Kidney, Liver, Lymphoid, Synovial, testicular and thyroid tissues {Adamson-small LA et al; Virus Res 2014 }

Am J Kidney Dis 2000 Jun;35:1166-74

• Focal Segmental Glomerulosclerosis

• Small and medium renal vessel Vasculitis

• Thrombotic Microangiopathy

FATAL CLINICAL CASE -1

Fatal Missed case of Hemophagocytic

Lymphohistiocytosis in an Infant

co-infected with

Parvovirus B19 and Epstein-Barr virus

{Kishore J, Kishore D; 2014}

Hemophagocytic Lypmphohistiocytosis (HLH) Its a life-threatening clinical condition, Mortality ~ 40% despite treatment

Dysregulation of the Immune system -- runs amok--- { Janka G; 2009} Uncontrolled Activation and proliferation of Lymphocytes , macrophages and

Histiocytes with excessive cytokine release Impaired Function of cytotoxic T cells and NK cells is seen

Results : Phagocytosis : Erythrocytes, Platelets by Macrophages Types: Primary (Genetic) or Secondary : triggered by Viral infection Rheumatologic,

malignant or metabolic conditions Epstein Barr virus (EBV) and Cytomegalovirus (CMV) are usual Parvovirus B19 (B19) infection or EBV co-infections are much rare Diagnostic difficulty : Low Specificity of symptoms, requirement of

multiple criteria and many triggering agents with varied clinical presentation

Posing a diagnostic Dilemma {Balweirz et al 2010} Hence Clinical, Virological and Hematological features of a

Missed Fatal case of HLH in an infant is presented

HLH: Case History

• A 2 mo Male infant (5.4 Kg) delivered by Caesarean section • Fever high grade, Rash, Anemia, Thrombocytopenia --2 wks • abnormal movements -1d, was admitted in I.C.U. of a private hospital• Despite supportive and multiple antimicrobial therapy the child

continued with moderate fever for the next-- 2 wks • Referred to our tertiary care institute, after 1 mo• Pallor, few petechial rash on abdomen with hepato-splenomegaly

(liver 5 Cm and spleen 4 Cm below the costal margin) • Severe anemic (Hemoglobin 6g/dL; 5.7 g/dL ; PCV 19.4%)• Leucocytosis (17x109/L; 26x109/L) and Lymphocytosis (89%) with 6%

polymorphs, 4% monocytes and 1% eosinophils • Thrombocytopenia ( 42x109/L , 12x109/L ; 6 x109/L) • Liver functions : deranged with raised Alkaline Phosphatase and LDH • Renal functions : Normal

HLH: Hospital Course and Virological Investigations

• Maculopapular rash on face resembling “Slapped cheek”, became generalised and persisted for 3 wks

• I.V.I.G. was given: Infant did not respond • Referred to our Tertiary Care InstituteClinical and Lab profiles were reviewed, investigatedSerum : Parvovirus B19 specific IgM, IgG were positive

by ELISA • B19 DNA was not detected by PCR and Real-time PCR

(Shanghai, China)• CMV IgM antibodies and CMV real-time PCR: -veEBV (VCA) IgM antibodies and EBV DNA (329.81x103

copies/ml) were positive

“Slapped Cheek”

Maculo-Papular Lacy Rash on face

later spread to entireBody

Persisted-2 wks

Lesson Learnt : HLH

All cases of prolonged Fever, Hepatosplenomegaly and severe Bicytopenia (Hemphagocytosis)

Be screened by simple Biochemical tests : - Hypertriglyceridemia and Hyperferritinaemia (reliable, 93%) cost-effective {Switala JR et al; 2012} Early clinical Suspicion, diagnosis of HLH Robust treatmentment Cytotoxic (cyclophosphamide, azithiprine), immunotherapy, Stem cell transplantation only can reduce mortality Further genetic studies are needed for future guidance and

etiological classification of HLH.

2nd : FATAL CASE

of

Fulminant Hepatic Failure Co-infected with

Parvovirus B19

INTRODUCTION: FHF

• Viral Hepatitis in children is mainly caused by hepatotropic viruses (Hepatitis A-E), occasionally by TORCH agents and Epstein-Barr virus (EBV)

• FHF is a rare and devastating event during childhood and mortality varies from 18-80% {Ayogdu et al, 2003]

• In a third to half of all children with fulminant hepatitis (FH): No cause is found.

• Parvovirus B19 infection has been recently reported to be associated with acute, chronic or fulminant hepatitis either alone or together with other hepatitis viruses.

EVIDENCE : B19 can be HEPATOTROPIC

B19 DNA has been found in with acute, unexplained, hepatitis in:-

Liver tissues of patients with FHF may be with bone-marrow aplasia (Langnas et al, 1995)

Serum of patients (with or without B19 specific IgM)

(Yoto et al, 1996, Hillingso et al,1998) Complications observed in patients with acute hepatitis and B19

infection are:

Aplastic anemia (Langnas et al, 1995 ) Pure red cell aplasia (Chehal et al,2002 ) Thrombocytopenia (Yoto et al,1996)

Case History : FHF with Thrombocytopenia and Anemia

• A 5 yr old male child admitted in Critical Care Unit• Fever high to moderate, poor appetite- 3 wks• Yellowish discolouration of eyes and urine—5 d• Not vaccinated against HAV or HBV• O/E : Drowsy, febrile wt 15 Kg, pallor and icterus• Heart rate 110/min, Systolic BP 80mmHg• Crepitations at base of lungs, Hepato-splenomegaly • (liver 6cm, spleen 5cm below costal marin)• Consciousness : M4V3E3 on Glassgow Coma scale

Lab Investigations• Anemia: Hemoglobin : 7.7g/dl-- 2d later--5.7g/dl; PCV=36.7%• Hypochromia, anisocytosis• Leucocytosis: 22x10 (9)/L--55x10 (9)• Thrombocytopenia: Platelets: 44x10 (9) • Prothrombin time raised: 22.4 sec(control 11.8sec)• Hypoalbuminemia : 2.8 later 2.0 gm/dl• Total Bilirubin: 5.2 mg%• Raised ALT , AST and alkaline phosphatase• Malarial parasite, Widal titers were negative• Anti-HAV and HEV IgM antibodies : Positive • B19 DNA was positive by PCR while anti-B19 IgM antibodies was

positive by ELISA (IBL Hamburg, Germany) • Blood Culture (BACTEC 9120,USA): Coagulase –ve Staphylococci

Clinical Course

• Clinical Diagnosis: Acute viral hepatitis with Encephalopathy Grade-1 with moderate anemia, sepsis and severe pneumonia associated with bilateral pleural effusion

• Treatment: Amoxycillin Clavulanate, I.V. fluids, Oxygen inhalation• Day-2: Air-entry was poor, chest X-ray: Pleural effusion• And bronchopneumonia• Ultrasound Abdomen : showed hepatosplenomegaly,• Collection of fluids in pelvis• Subsequently, Respiratory symptoms and X-ray worsened and • Child could not maintain Oxygen saturation• Developed Seizures : I.V. diazepam and phenytoin was given• Sent to I.C.U. : put on Ventilator further fall in PO2 to 50-60%• Cardiac arrest : Revived by I.V. dopamine and Ventilator support• Condition worsened further and child died on 4 day

Interpretations of the Case• Since B19 receptors are on fetal hepatocytes • B19 DNA found in the Liver tissues in 67% cases of non-A-B-C FHF,

50% of Cryptogenic FHF, 15% of chronic liver • Pathophysiology : B19 induced FHF is unclear • B19 direct virus cytotoxicity and apoptosis by NS1 protein• Immunenological mediation

• Multiple viral infection: B19 HAV HEV : may have caused massive liver necrosis

• B19 Mycarditis may also have caused death • B19 thrombocytopenia is rare {Frotscher B et al, 2009; Kishore J 2005}

• B19 circulating in the child’s serum : 2.4x103 genome• equivalent/ml besides anti-B19 IgM antibodies• Further presence of anemia and thrombocytopenia too indicate B19

infection

3rd Study: Our unpublished data on B19 infection

• 50 children Viral Hepatitis; Controls= 37 (1 B19 IgM +ve)• 34 Acute Viral Hepatitis (mean age 6.70±3.32yrs) : 12 (35.3%) had anti-B19 IgM and 5 had B19 DNA also • 16 FHF children (Mean age 6.25±2.88yrs) 6 (37.5%) had B19 IgM and 3 had B19 DNA also • Mortality in FHF: 4 of 16 (25%) died of 4 children 3 (75%) had B19 infection• B19 possibly aggravated FHF • B19 should be considered as a possible hepatotropic virus as a co-pathogen with other such viruses e.g. HBV and HVD B19 co-infection should be considered and tested in all

severe cases of acute viral hepatitis

Fatal Case-4

Persistence B19 Infection in Pure Red Cell Aplasia Resulting in Myelodysplasia

Case- Report

• 46 yr old female, immunocompetent was referred with • Fever high grade, Cough, expectoration—1 wk• H/O: 5 units blood transfusion • Normotensive, non-diabetic• No history of rash, arthropathy• O/E: Pallor, mild hepatomegaly• Hemoglobin: 5.4 g/dl, 6.6 g/dl), TLC =8,500/cmm• ESR 64mm/1hr, platelets 2,58000/cmm• Reticulocytes 0.3%• No Iron overload• Blood and urine cultures : sterile

Investigations : PRCA case

Renal and Liver function tests : Normal Bone- Marrow histology: normocellular with multiple

myeloid cells and megakaryocytes Reduced erythropoiesis, Early erythroid precursors seen but with maturation arrest Great paucity of erythroblast myeloid: erythroid ratio= 15:1, plasma cells 5% blast cells < 5% with occasional dysplastic features Diagnosis of PRCA was made Etiology unexpained, Refered to us for B19 infection

Treatment : PRCA case• Pallor continued, Hb was 5.5 g/dl• Reticulocytes were 0.1%• Transfused : 2 Units of packed red cells blood : Hb raised to 8.3g/dl• Prednisolone : 45 mg/d• Cyclosporin : 5 mg/d/kg• Virological Investigations: • B19 DNA by PCR and anti-B19 IgM antibodies by an in-house ELISA : + ve {Kishore J and Muckopadhyay C 2004}• Advised I.V.I.G. : Poor economic condition and Clinician’s reluctance, it was not given

Follow -ups PRCA case• 6 mo later: Pedal edema and few months later • Fever low grade• Hb : 5.3 g/dl, TLC 7,900 /cmm• Visited other hospitals also• Bone Marrow : Hypocellular, with hyperplasia in myeloid

and megakaryocytes with monocytic/histiocytic infiltrations

early erythroid precursors with muration arrest, blasts < 5% blast cells < 5% with occasional dysplastic features• B19 DNA by PCR and anti-B19 IgM antibodies by an in-house ELISA : + ve• Persistent B19 infection was dignosed

PRCA: Follow up

• 3 yrs later : Hb 4.7 g/dl, TLC 5.08 • TLC 7,700/cmm; despite regular blood transfusions• Platelets was 3,08,000/cmm • Repeat Bone Marrow : picture changed to Mylodysplasia with over

all pancytopenia• Blood and urine cultures continued to be sterile• B19 DNA and IgM antibodies : - ve

• 5th Yr : Gross Pallor, severe anemia despite 2 wkly packed red cell transfusion

• Anasarca• Bone Marrow : Aspirate failed since no cells were seen• Died : over 500 units packed RBC transfusions over 5 yrs time • B19 DNA and IgM antibodies : - ve• I.V.I.G. therapy was not given?

Review from literature : PRCA given IVIG

• Review : of 123 patients 63 Solid-organ transplant, 39 HIV positive• Hemoglobin level was corrected • First IVIG course in 124 patients (93%)• Relapsed in 42 (33.9%) at a mean of 4.3 months. {Craboly et al; 2013} I.V.I.G. : 2-3 courses are required Relapses are common

Novel Clinical associations : Added

• Real Time PCR Reconfirmed Three Novel Clinical Associations of Parvovirus B19 : Non-occlusive Bowel Gangrene, Amegakaryocytic thrombocyto --penia and Myositis. Indian J Med Research (2015)

• Can Parvovirus B19 Infection Be Naturally

Oncolytic : Clinical Evidence Raises Such A Possibility in Leukemic Children. Indian J Med Research (2014)

• Prolonged remission in a child with chronic myeloid leukemia following Parvovirus B19 (B19V) infection. Indian J Med Microbiol (2015)

SUMMARY Human parvovirus B19 (B19) once listed as a newly emerging virus now has myriads of clinical manifestation Infections: Significant but Cryptic cause of morbidity and mortality Know : B19 spectrum of clinical features, pathogenesis Suspect B19 and Screening test: B19 IgM ELISA Our limited work and other studies denotes high/alarming

situation of B19 infections Major organ involvements like kidneys, heart, brain, gut and other

organs Hence B19 be looked/recognised and prevented B19 VACCINE : VLP: immunogenic but reactogenic: under phase-1

trials

My Hypothesis: Glaring Data• B19 Co-infections : found to be fatal as was the case oh HLH , FHF and

untreated PRCA

• B19: 80% get asymptomatic infections (20 left)• Vast majority (80-90%) of symptomatic: Self-limiting infection (16-18)• 2-4 (2% -4%) left behind perhaps gets the brunt and suffers sinister

complications • 2% to 3 % x 1/3 to 1/2 of World population of 6 billion• infected by B19 equals to 2 million• 2-3 million suffers sinister complications• Quarter or half of complicated B19 die it equals • in 0.5 to 1 or 2 million may be dying of severe B19 infection alone or

as co-infections• Limitations : no exact fact and figures exists • How many B19 infected get proper, timely Diagnosis ?• Treatment : UNKNOWN ?

Collaborators

Co-Investigators:--

Prof RN Misra, Head, Clinical Immunology

Prof Rajan Saxena, Head, G .I . Surgery

Prof S K Yachha, Head, Gastroenterology

Prof Archana Kumar, King Geoge University

Residents:- Dr Itisha Gupta Dr Jagdeep Singh

Dr A Maurya, KGMU Dr Abhiruch Paisal, KGMU

Technical Help:- Hemant V K Misra

janaksgpgi@ yahoo.co.in