these studies were approved by institutional review boards at the national institutes of health ( nih ) and washington university . a 28-year - old woman with job syndrome pathologic examination of a bronchoalveolar lavage ( bal ) sample showed scattered cells , primarily columnar bronchial cells , with cytomorphologic changes reminiscent of bk polyomavirus ( bkpyv)infected decoy cells . the cells stained positive with pab416 , a monoclonal antibody against the sv40 large t antigen . the patient had bkpyv viremia ( 8.1  10 copies / ml ) and viruria ( 6.9  10 copies / ml ) . the bal sample was weakly positive for bkpyv by pcr ( < 250 copies / ml ) and negative for jc polyomavirus . nonenveloped virions were purified from the bal sample by using ultracentrifugation with optiprep ( # d1556 ; sigma - aldrich , st . dna was extracted from the virion preparation and subjected to random - primed rolling circle amplification ( rca ) and restriction enzyme digestion , which yielded 2 strong bands . the bands were cloned and identified as wupyv by using sanger sequencing . the complete genomic sequence of the isolate , designated j1 ( genbank accession no . kj643309 ) , was confirmed by using miseq analysis ( illumnina , san diego , ca , usa ) of the rca product . a second wupyv variant with 2-nt polymorphisms and a single base insertion was also detected in the rca product . we developed an immunohistochemical ( ihc ) assay to detect the wupyv viral protein 1 ( wu - vp1 ) by using an igg2b designated nn - ab06 . recombinant histidine - tagged wu - vp1 protein was generated by expressing wu - vp1 ( genbank accession no . abq09289 ) in escherichia coli from a gateway pdest17 plasmid ( life technologies , carlsbad , ca , usa ) and purifying the protein by using an affinity ni - nta column ( pierce biotechnology , rockford , il , usa ) . after generation of hybridomas , we identified clones producing antibody against wu - vp1 by elisa and immunoblot . clones that cross - reacted with ki polyomavirus vp1 ( ki - vp1 ) were identified by elisa with glutathione s - transferase  tagged ki - vp1 ( 2 ) and eliminated . to generate positive control cells for ihc assay optimization , we transfected 293 t cells with plasmid pdest26-wu - vp1 ( life technologies ) . a subset of cells was fixed in 10% neutral - buffered formalin and embedded in paraffin . ihc testing was performed by deparaffinizing slides in xylene and rehydrating them in a series of ethanol solutions . after treating slides with 3% hydrogen peroxide , antigen was retrieved in citrate buffer , ph 6.0 ( 10 mmol / l citric acid , 0.05% tween 20 ) in a pressure cooker ( pc625 ; nesco , two rivers , wi , usa ) for 3 min on the high setting . slides were blocked in 1.5% normal horse serum ( # s-200 ; vector laboratories , burlingame , ca , usa ) and incubated with nn - ab06 , then with biotinylated anti - mouse igg ( ba-2000 ; vector laboratories ) . biotin complex kit ( # pk-6100 ; vector laboratories ) and ( 3,3-diaminobenzidine ) ( # sk-4100 ; vector laboratories ) , we counterstained tissues with hematoxylin . a serial section of the same cell block stained with an isotype - matched antibody ( # 557351 , mouse igg2b : bd biosciences , san jose , ca , usa ) ( figure 1 , panel b ) and mock transfected cells stained with nn - ab06 ( figure 1 , panel c ) showed negative results . western blotting was performed as an independent means of evaluating specificity of nn - ab06 ( 11 ) . nn - ab06 reacted with wu - vp1 protein lysate but not with ki - vp1 lysate , which is the most closely related virus to wupyv . wu polyomavirus antigen in bronchoalveolar lavage specimens from lungs transplanted into a recipient ( 28-year - old woman ) with job syndrome . immunohistochemical analysis of 293 t cells transfected with pdest26-wu  virus protein 1 and stained as follows . d ) bronchoalveolar lavage specimen stained with nn - ab06 showing prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes ( arrows ) . original magnifications 400 in panels a  c and 600 in panels d and e. we applied the wu - vp1 ihc assay to formalin - fixed , paraffin - embedded sections of the bal sample . prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes were observed ( figure 1 , panel d ) . staining was not seen in serial sections stained with the isotype antibody ( figure 1 , panel e ) or with no antibodies . many wupyv - positive cells were cuboidal to columnar and showed other morphologic features consistent with respiratory epithelial cells . to determine their etiology , we developed a double immunofluorescence ( dif ) assay with a polyclonal antibody against wu - vp1 ( 2 ) , designated nn - ab01 , and a monoclonal antibody against cytokeratins ( # m3515 ; dako , carpinteria , ca , usa ) . deparaffinization and antigen retrieval were accomplished as noted above , and sections were blocked in superblock t20 ( # 37516 ; thermo scientific , waltham , ma , usa ) . to validate the assay , we performed immunofluorescence analysis with nn - ab01 on positive control 293 t cells expressing wu - vp1 . several wu - vp1-positive cells were observed ( figure 2 , panel a ) ; a serial section stained with preimmune serum at the same dilution showed a negative result ( figure 2 , panel b ) . wu polyomavirus antigen in respiratory epithelial cells from lungs transplanted into a recipient ( 28-year - old woman ) with job syndrome . immunofluorescence of 293 t cells transfected with pdest26-wu  virus protein 1 and stained with a ) wu virus protein 1 polyclonal antibody ( nn - ab01 ) or b ) preimmune serum . c ) double immunofluorescence with nn - ab01 ( red ) and a monoclonal antibody against cytokeratin ( green ) showing a double - positive cell from the bronchoalveolar lavage specimen . d ) bronchoalveolar lavage specimen with multiple wu virus protein 1/cytokeratin double - positive cells . original magnifications 100 in panels a and b , 600 in panel c , and 400 in panel d. for dif , bal sections were incubated first with the primary antibodies and then with fluorescently labeled secondary antibodies ( # a10042 anti - rabbit-568 and # a10042 anti - mouse-488 ; life technologies ) . we observed cells positive for wu - vp1 and cytokeratin ( figure 2 , panels c , d ) , which identified these cells as epithelial cells . of the 136 wu - vp1-positive cells , 77 ( 57% ) were also cytokeratin positive . a serial section of the bal sample stained with an isotype - matched antibody to the cytokeratin antibody ( # 555746 mouse igg1 ; bd biosciences ) and preimmune rabbit serum was negative . we hypothesized that the remaining 43% of wu - vp1-positive , cytokeratin - negative cells might be macrophages . however , a dif assay using nn - ab01 and an antibody against cd68 ( # m0814 ; dako ) , a macrophage marker , showed wu - vp1 and cd68 single - positive cells but no double - positive cells . in addition , a stain with nn - ab01 and an antibody against cd45 ( # m3515292 ; dako ) , a marker for hematopoietic cells , also showed negative results . before this study , to our knowledge , no specific cell type had been identified as susceptible to wupyv infection . the presence of nuclease - resistant viral dna from the optiprep gradient and detection of wu - vp1 , which is believed to be expressed concomitantly with dna replication ( 12 ) , suggests that the cells were infected by wupyv and that the wupyv life cycle reached at least the stage of late gene expression . the clinical role of infection by wupyv is uncertain , given that the patient was not experiencing any recognizable symptoms . although we attempted to identify a second population of wu - vp1-positive , cytokeratin - negative cells , the etiologic features of these cells remains uncertain . our patient had job syndrome , a primary immunodeficiency not previously associated with polyomavirus susceptibility . it is possible that immunosuppressant medications , which include prednisone and tacrolimus , altered susceptibility to virus infection . other human polyomaviruses are believed to exclusively cause disease in immunocompromised hosts this case suggests that immunosuppression might also play a role in wupyv infection and expands our understanding of wupyv biology .
we detected wu polyomavirus ( wupyv ) in a bronchoalveolar lavage sample from lungs transplanted into a recipient with job syndrome by using immunoassays specific for the wupyv viral protein 1 . 
 co - staining for an epithelial cell marker identified most wupyv viral protein 1positive cells as respiratory epithelial cells .