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Research letters

Identification of a Kunjin/West Nile-like in brains of patients with New York Thomas Briese, Xi-Yu Jia, Cinnia Huang, Leo J Grady, W Ian Lipkin See page 1221

Molecular analysis of brains from patients of the recent New Patient Region York City encephalitis outbreak reveals the presence of a NS3-1 NS5-1 NS5-2 NS5-3 NS5-2/3 flavivirus not previously described in the Americas. 1 SEQ SEQ SEQ PCR PCR In late August, 1999, health officials reported an outbreak of 2 SEQ SEQ SWQ ·· ·· encephalitis accompanied by severe weakness and axonal 3 PCR PCR SNPCR ·· ·· neuropathy in Queens, New York, USA. Serological analysis 4 ·· ·· SNPCR ·· ·· of five of six patients for the presence of antibodies to North 5·········· American yielded results consistent with SEQ=sequence analysis; PCR=RT-PCR (40 cycles); SNPCR=semi--nested RT-PCR (2ϫ40 infection with St Louis encephalitis , which prompted cycles; primers NS5-2/3 followed by NS5-3). establishment of a eradication programme by the Table 1: Positions of primers and amplification products in State and City of New York. Concurrently, wildlife observers prototypic flavivirus noted increased mortality of avian species including free- Region Kunjin virus St Louis virus ranging crows and exotic birds housed in the Bronx Zoo.1 Histological analysis of birds revealed NS3-1 88 (98) 82 (94) NAS NS5-1 87 (99) 80 (95) 70 (76) and myocarditis. Avian mortality is not characteristic of St NS5-2 88 (100) 80 (96) 80 (89) Louis Encephalitis virus; therefore, efforts were made to Percentage identity of nucleotide sequence for three regions of the New York isolate identify other pathogenic arboviruses. genome with Kunjin and West Nile . Percentage identity of deduced aminoacid Samples of brain material from five patietns with sequences shown in parentheses. NAS=no available sequence. encephalitis were selected for molecular analysis. Based on Table 2: Sequence-analysis data for New York isolate genome findings in one brain consistent with the presence of with Kunjin and West Nile viruses flavivirus antigen (Centers for Disease Control and Prevention, Atlanta, GA, USA; unpublished data), ACM ACY TGN CC). Primer set NS5-3 (EDL/Fla- oligonucleotide primers were designed to hybridise to U10098, 5'-TGG ATG CAN ACD GAA GAY ATG CT conserved regions within the NS3 and NS5 of a wide and EDL/Fla-L10750, 5'-GGG GTC TCC TMT AAC variety of . CWC TAG) spanning the 3' end of NS5 and untranslated 3 Several degenerate primer sets were designed for use in sequence was based on the universal primers of Tanaka and reverse transcription (RT) PCR (figure 1, table 1). One Pierre and colleagues.4 Primer set NS5-2 used an inverted primer set (NS3-1), which encompassed conserved motifs in version of the universal forward primer (EDL/Fla-L10098 the serine protease and nucleotide triphosphatase or RNA 5'-TGG ATG CAN ACD GAA GAY ATG CT) in helicase domains of NS3 sequences (EDL/Fla-U5004, 5- conjunction with primer EDL/Fla-U9954 (5'-GSS AAA GGA ACD TCM GGH TCN CCH AT and Fla-L5457, 5'- KCH TAY GCN CAV ATG TGG). GTG AAR TGD GCY TCR TCC AT) was based on the Four of the five brain samples contained flavivirus primer sequences published by Chow and colleagues.2 sequences. Amplification products from two patients were Various nested primer sets towards the 5' end of NS5 submitted for dideoxy DNA sequencing (figure 1). The sequences, including conserved aminoacid motifs sequences obtained from these two patients were virtually A K G S R A I W X M W L G A R X L E F E A L G F L N X H W , identical and alignments of sequences from from both with DDTAGWDT, and QRGSGQVXTY were selected for use other flavivirus sequences showed the presence of a in domain-specific differential display. This method enables Kunjin/West Nile-like virus (figure 2, table 2). detection of differences in nucleic-acid populations. The Kunjin virus and West Nile virus are closely related cloned and sequenced amplification products were members of the virus serogroup; the generated with primer sets NS5-1.1 (EDL/Fla-U9093, 5'- former has been reported in , and the latter in AGY MGR GCH ATH TGG TWY ATG TGG and Africa, Asia, and Europe. Kunjin virus has not been EDL/Fla-L9279, 5'-TCC CAV CCD GCK GTR TCA associated with mortality in human beings. A West Nile virus TC), NS5-1.2 (EDL/Lp146, 5'-TGG AAA AGC YAA AGG outbreak in Romania in 1996 5 had a death rate of 4–8%; the NAG and EDL/Lp 154, 5'-GTG TCC CAN CCD GCD ratio of apparent to inapparent infections was estimated to GTR TC) and NS5-1.3 (EDL/Lp151, 5'-GAY ACH GCH be about 1:325. Neither Kunjin virus nor West Nile virus GGN TGG GAC AC and EDL/Lp153, 5'- GCA TAD GTS infections have been described in the Americas.

Figure 1: Positions of primers and amplification products in prototypic flavivirus genome

THE LANCET • Vol 354 • October 9, 1999 1261 Retinoic acid for treatment of multicentric Castleman’s disease Philippe Rieu, Dominique Droz, Antoine Gessain, Jean-Pierre Grünfeld, Olivier Hermine Multicentric Castleman’s syndrome has an aggressive course with poor prognosis, and its treatment remains uncertain. We report a woman with multicentric Castleman’s disease that was successfully treated with prednisone and retinoic acid. A 35-year-old woman had been in excellent health until 1 month before admission, when she developed progressive weakness, in the evening, and oedema with weight gain. Physical examination on admission showed normal blood pressure, multiple enlarged small peripheral lymph nodes, hepatosplenomegaly, pleural effusion, and marked peripheral oedema. No neuropathy or skin changes were noted. Investigation showed mild renal insufficiency (creatinine=140 ␮mol/L) with proteinuria (0·7 g/d) and microscopic haematuria. Blood count showed a normocytic anaemia (Hb=10 g/dL; MCV=87 fL) without laboratory signs of haemolysis. Leucocyte and platelet counts were normal. C- reactive protein was 53 mg/L. Plasma albumin and globulin concentrations, serum immunoelectrophoresis, fibrinogen, antinuclear antigen, serum complement, antiphospholipid antibodies, blood culture, and viral serological tests (HBV, HCV, HIV-1) were normal or negative. Biopsy of an axillary lymph node showed angiofollicular lymphoid hyperplasia, consistent with Castleman’s disease of the hyaline vascular type. PCR analysis for HHV-8 sequences on lymph-node biopsy material was negative. Renal biopsy showed lesions Figure 2: Sequence of New York flavivirus isolate in patient one similar to those of thrombotic microangiography. No immune Accession numbers for bank shown in parentheses. deposits were identified by immunofluorescence. In hospital, Jap enceph=Japanese encephalitis. hypertension developed and was gradually controlled with the Since Oct 4, 1999, 37 patients with neurological disease use of three antihypertensive agents. Once blood pressure was have been identified by clinical and serological criteria as controlled, oral prednisone (0·5 mg/kg day) was started. being infected with a Kunjin/West Nile-like virus, including After 2 weeks, fever, weakness, oedema, peripheral four with fatal encephalitis. Efforts are underway to clone lymphadenopathy, and splenomegaly persisted. All-trans- and sequence the entire viral genome and to establish retinoic acid treatment (45 mg/m2 daily orally) was then added sensitive real-time thermal cycling methods for the detection to prenisone. This resulted in marked improvements in her of flavivirus sequences similar to those used for diagnosis of symptoms within a week. After 1 month of treatment, physical infections of the central nervous system with herpesviruses examination and laboratory tests were normal. Prednisone was and enteroviruses. These methods are anticipated to gradually reduced and completely discontinued after 12 facilitate surveillance in human beings, wildlife, and months. Retinoic acid was initially given for 6 weeks, and domestic animals, as well as mosquito populations, and may thereafter for 14 days every 2 weeks for 9 months. After 1 year provide insight into the origins of this virus and its off treatment, she remains without symptoms and with normal mechanisms for neuropathogenesis. laboratory tests We thank the UCI Biotechnology Resource Facility for sequencing Retinoic acid have been recognised as an important expertise, and thank Yuri Chun and Linda Falsafi-Haghighi for technical immunoregulating agents. It has potential therapeutic effects assistance. This work was supported by EIP funding through the Centers in diseases associated with overproduction of interleukin-6 for Disease Control and Prevention (Wadsworth Center). (IL-6) and endothelial injury such as Kaposi’s sarcoma and 1 Asnis D, Conetta R, Waldmon G, et al. Outbreak of West Nile-like POEMS syndrome,1, 2 two diseases that have been reported in : New York, 1999. MMWR Morb Mortal Wkly Rep the course of multicentric Castleman’s disease. Recently, Raife 1999; 48: 845–49. et al reported a patient with thrombotic thrombocytopenia 2 Chow VTK, Seah CLK, Chan YC. Use of NS3 consensus primers for the polymerase chain reaction amplification and sequencing of dengue purpura (TTP), refractory to conventional therapy, who viruses and other flaviviruses. Arch Virol 1993; 133: 157–70. entered into remission after 13-cis retinoic acid treatment.3 It is 3 Tanaka M. Rapid identification of flavivirus using the polymerase noteworthy that IL6 is raised in serum of children with TTP chain reaction. J Virol Methods 1993; 41: 311–22. and that IL6 levels vary with disease activity.4 IL6 plays a 4 Pierre V, Drouet M-T, Deubel V. Identification of mosquito-borne central part in the pathophysiology of Castleman’s disease5 an d flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction. Res Virol 1994; 145: 93–104. vascular lesions are important features. The response to 5 Tsai TF, Popovici F, Cernescu C, Campbell GL, Nedelcu NI. West retinoic acid and prednisone seen in this patient may Nile encephalitis epidemic in southeastern Romania. Lancet 1998; therefore result from modulation of IL-6/IL-6R 352: 767–71. autocrine/paracrine loop or a beneficial effect on endothelial Emerging Diseases Laboratory, Departments of Neurology, in j u r y . Microbiology & Molecular Genetics (T Briese PhD, X-Y Jia MD, 1 Nagpal S, Cai J, Zheng T, et al. Retinoid antagonisms of NF-IL6: Prof W I Lipkin MD) and Anatomy and Neurobiology (W I Lipkin), insight into the mechanism of antiproliferative effects of retinoids in University of California, Irvine, CA 92697, USA, and Wadsworth Kaposi’s sarcoma. Mol Cell Biol 1997; 17 : 15 9 – 6 8 . Center, New York State Department of Health, Albany, NY 2 Authier FJ, Belec L, Levy Y, et al. All-trans-retinoic acid in POEMS syndrome. Therapeutic effect associated with decreased circulating levels (C Huang PhD, L J Grady PhD) of proinflammatory cytokines. Arth Rheum 1996; 39 : 14 2 3 – 2 6 . Correspondence to: Prof W I Lipkin 3 Raife TJ, McArthur J, Peters C, Kisker CT, Lentz SR. Remission after

1262 THE LANCET • Vol 354 • October 9, 1999