during july  december 2012 and 2013 , sick raccoon dogs and arctic foxes , which were farmed for fur products on 6 farms ( farms a 
f ) in jilin and liaoning provinces , china , were received for quarantine inspection at the fur animal disease laboratory , institute of special animal and plant sciences , chinese academy of agricultural sciences .
several infant raccoon dogs from 1 litter became ill 40 days after birth , and the numbers of sick animals increased by the time they were 3 months of age .
clinical signs included anorexia , emaciation , growth retardation , thirst , chronic diarrhea , and unkempt fur ; necropsy often revealed cyanosed splenomegaly , enlargement of mesenteric lymph nodes , and renal cortex congestion and brittleness .
for the raccoon dogs showing similar clinical signs , rate of illness was 4%8% ; death rate was 60% before the age of 4 months ; and rate of illness increased by years on the farms that initially had sick animals . among arctic foxes , signs varied : emaciation and growth retardation in 3-month - old cubs with pale and swelling kidneys in dead foxes ; and severe diarrhea or intermittent tar - like feces in 37-month - old cubs .
because signs in the sick animals sent for quarantine inspection were similar to those in aleutian mink disease , we first used amdv - specific counter - immunoelectrophoresis ( ciep ) ( 5 ) to test serum samples of six 3-month - old sick raccoon dogs from farm a. all 6 were positive .
next , we designed conserved amdoparvovirus primers ( av7 ; table 1 ) for pcr detection .
viral nucleic acids were extracted by using a minibest viral rna / dna extraction kit ( takara , dalian , china ) .
dna extracted from spleen , kidney , mesenteric lymphonodus , and mucosal tissue and blood of the 4 sick raccoon dogs was all rfav dna positive .
after 10 days , the 4 raccoon dogs remained rfav dna pcr positive in blood and ciep positive in serum .
dna extracted from blood of two 37-month - old sick raccoon dogs from farm c was rfav dna pcr positive , and serum samples from these animals were ciep positive .
two 7-month - old raccoon dogs from farm d appeared healthy but on necropsy showed cyanosed splenomegaly .
the overall positive rates of rfav dna and ceip antibody in sick raccoon dogs were 90% and 100% , respectively .
serum iodine agglutination test ( iat ) ( 6 ) was positive or strongly positive in 20 sick raccoon dogs that were 7 and 19 months old but was negative in healthy animals .
of the 29 sick raccoon dogs , two 37-month - old animals were both canine parvovirus 2 and rfav dna positive by pcr in blood or spleen samples .
however , canine distemper virus was not detected in spleen samples from any sick raccoon dogs . in arctic foxes
raised together with sick raccoon dogs on farm b , intestinal mucosa samples from 3 of 7 foxes that died of diarrhea - associated dehydration were rfav dna pcr positive .
one kidney sample from a 3-month - old fox of 3 tested on farm f showed renal enlargement and was rfav dna pcr positive .
blood , urine , and feces of two 7-month - old arctic foxes on farm c , which had tar - like feces , were rfav dna pcr positive .
results of all samples tested by pcr , ciep , and iat are summarized in table 2 .
* ciep , counter - immunoelectrophoresis ; iat , serum iodine agglutination test ; na , samples not available ; nd , assays not done ; + , positive . 
farm : a , heishan county ; b , qianan county ; c , jilin county ; d , panjin county ; e , haicheng county ; f , nongan county .
qianan , jilin , and nongan are located in jilin province ; heishan , panjin and haicheng are located in liaoning province . 
for raccoon dogs , pcr+ for all the tested tissues ( blood , spleen , kidney and mesenteric lymphonodus ) . for arctic foxes , pcr+ results only for 1 of the tested tissues ( blood , kidney , or intestinal mucosae ) . 
 blood and spleen samples were tested separately by pcr from healthy animals except for the blood samples from the 3-month - old healthy raccoon dogs .
we next applied a semiquantitative pcr to quantify the level of rfav dna in the blood and spleens of four 7-month - old sick raccoon dogs ( 3 from farm d , 1 from farm c ) .
briefly , dna samples were diluted from 10 to 10 viral genomic copies ( vgc)/l by using a quantified rfav dna template and were amplified by using av3 primers with the detection threshold of 100 vgc in a volume of 15 l .
virus titer was determined on the basis of the maximum dilution at which viral dna was detected by agarose gel electrophoresis .
rfav dna ranged from 2  10 to 5  10 vgc / ml in blood and 7  10 vgc / g in spleen . in spleen and kidney tissues of 2 raccoon dogs euthanized at 3 months of age ( from farm a ) and four 7-month - old sick raccoon dogs ( from farm d ) , bacterial infections , performed by standard methods , were not found .
collectively , these results demonstrate that blood or tissues from the 2 sick animals contained rfav dna , and the dna levels were high in some samples .
we used indirect immunofluorescence assay ( ifa ) to probe rfav antigens in spleen and kidney of the 2 rfav pcr 
positive 3-month - old sick raccoon dogs from farm a. tissues of the sick raccoon dogs were amdv - g antigen ifa positive , but the healthy animal tissues were not ( figure 1 , panel a ) .
this result indicates that the spleen and kidney of the sick raccoon dogs contained viral antigens that share immunogenicity with amdv - g .
more importantly , we isolated an rfav strain , named xq - jlr , by infecting crfk cells .
ifa using anti - amdv serum showed positive green cells in rfav - infected crfk cells but not in mock - infected cells ( figure 1 , panel b ) . under a transmission electron microscope , virus particles at 23 nm in diameter
were visualized in a concentrated supernatant of infected crfk cells ( figure 1 , panel c ) .
tissue smears , as described below , were prepared from spleen and kidney samples of sick raccoon dogs and detected with a control mouse serum or anti - amdv - g serum by using an ifa ( 8) .
( a ) mock smear of a spleen tissue from a sick raccoon dog , detected with normal mouse serum as a primary antibody ; ( b ) control smear of a spleen tissue from a healthy raccoon dog ; ( c , d ) amdoparvovirus antigen - positive smears of spleen tissue ( c ) and kidney tissue ( d ) from a sick raccoon dog , detected with anti - amdv - g serum . original magnification 200 .
b ) one milliliter filtered ( 0.22 m ) pathological spleen samples collected from a sick raccoon dog that had a virus titer of 7 10 vgc / ml , was inoculated to confluent crfk cells in a t25 flask .
infected cells of the fourth passage were fixed and analyzed with ifa by using anti - amdv serum ( mock- or rfav - infected crfk cells ) .
c ) twenty - five milliliter supernatant ( 2 10 vgc / ml ) of rfav(xq - jlr)infected crfk cell cultures were concentrated .
virions were agglutinated with an anti - rfav raccoon dog serum and were visualized under a transmission electron microscope ( tem ) .
amdv , aleutian mink disease virus ; rfav , raccoon dog and fox amdoparvovirus ; ifa , indirect immunofluorescence assay ; vgc , viral genome copies .
we further proved that rfav is the predominant virus in the lesion tissues of sick animals .
a modified sispa ( 7 ) was performed for high - throughput sequencing with mixed lesion tissues of 3 spleens and 3 kidneys from 3 sick raccoon dogs . by using illumina miseq sequencing ( illumina , san diego , ca
, usa ) , we obtained 478,813 high - quality reads , and 668 contigs were assembled , including 17 contigs for new amdoparvovirus .
cytosine nucleotide content of 62-bp gap , were recovered , which are consistent with the sequences acquired by sanger sequencing of viral dna amplified from tissues .
there were 865 reads , by blastn ( http://www.blast.ncbi.nlm.nih.gov/blast.cgi ) , in alignment with amdoparvovirus sequences but only 16 reads with sequences of other non - mammal viruses , such as phage and baculovirus .
the identities were < 91% of the reads aligning with amdv sequences in genbank by blastn ( e value < 10 ) .
we sequenced av7 primer - amplified pcr products of rfav dna from the tissues of 32 animals ( genbank accession nos .
kj396347kj396350 ) only share a similarity of 82% and 76.7% in the nonstructural protein ( ns ) 1encoding sequence and ns1 aa sequence , respectively , with amdv .
phylogenetic analyses of 2 neighbor - joining trees of either ns1 or major structural protein ( vp2 ) strongly suggest that rfav strains cluster into a unique clade between amdv and gray fox amdovirus species ( figure 2 ) .
rfav and other published amdoparvovirus sequences were aligned by using the muscle program in mega5.2 ( 9 ) , which used a p - distance model with 1,000 bootstrap replicates to generate phylogenetic trees of ns1 and vp2 aa sequences .
amdv , aleutian mink disease virus ; gfav , gray fox amdoparvovirus ; rfav , raccoon dog and fox amdoparvovirus ; ns , nonstructural protein ; vp , viral structural protein .
we identified a new virus species , rfav , from farmed raccoon dogs and arctic foxes in jilin and liaoning provinces , china .
raccoon dogs are naturally susceptible to rfav infection , and rfav is most likely the etiologic agent responsible for the disease manifestations of the sick raccoon dogs .
 a new amdoparvovirus , named raccoon dog and fox amdoparvovirus ( rfav ) , was identified in farmed sick raccoon dogs and arctic foxes . 
 phylogenetic analyses showed that rfav belongs to a new species within the genus amdoparvovirus of the family parvoviridae . 
 an rfav strain was isolated in crandell feline kidney cell culture . 