in january 2012 , a previously unknown severe disease was observed in pigs on several farms in northern and eastern china . in shandong province , > 80,000 pigs were infected .
the affected pigs had high fever ( > 40.5c ) , anorexia , coughing , respiratory distress , conjunctival serous and mucinous secretion , and posterior paralysis .
the disease was first observed in older pigs and spread within 23 days to younger pigs .
viscera ( e.g. , lung , kidney , heart , liver , and spleen ) and serum samples were collected from dead pigs from different provinces .
pathologic examination showed the most striking gross lesions were consolidated in the lungs ( figure 1 , panel a ) , with edema and hemorrhage ( figure 1 , panel b ) .
in addition , foci of yellow - white necrosis were observed in the kidneys of some dead pigs ( figure 1 , panel c ) .
c ) kidney with many yellow - white necrotic spots ( arrows ) .   to gain insight into the etiologic agent of the disease
, we conducted extensive and systematic diagnostic testing , including pcr , elisa , viral isolation , immunohistochemical staining , and standard bacteriologic culture , to evaluate the specimens .
a specific prv monoclonal antibody was used , and immunopositive cells were observed in infected tissue ( technical appendix 
 figure 1 , panels a , b ) .
the pcr for inocula samples showed that many glycoprotein ( g ) genes of prv could be amplified by using the primers specific to the unique gene fragments ( technical appendix 
 figure 2 ) .
the prv ge - elisa assays ( idexx laboratories , westbrook , me , usa ) indicated that serum samples from the sick pigs contained antibodies against wild - type , virulent prv glycoprotein e but not against the vaccine strain ( table ) .
our results also ruled out other suspected agents , such as classical swine fever , african swine fever , porcine reproductive and respiratory syndrome virus , and some bacterial infections .
the 3 isolates found here are referred to as nvdc - prv - bj , nvdc - prv - heb , and nvdc - prv - sd , according to the provinces from which they were isolated .
alignment the partial sequences of glycoprotein ( g ) c ( a ) , gd ( b ) , and ge ( c ) genes of pseudorabies virus at the nucleotide level .
* s / n ratio was calculated as a ratio of the absorbance of a well with serum to the absorbance of a control well without serum .
the pigs vaccinated with attenuated live prv vaccines still showed clinical signs of prv during the outbreak . to confirm the presence of prv in these herds , 15 pigs were vaccinated with the current vaccine strain and then challenged with the nvdc - prv - sd strain 21 days after vaccination .
the results indicated that the vaccinated pigs had not been given completely effective protection against infection and exhibited obvious clinical signs of disease similar to the typical symptoms observed in the field , suggesting that the virulence of the newly isolated prv strains had changed .
to better understand the genetic relationship of the 3 prv isolates found here to other prv isolates , we amplified and sequenced the 15 major genes of the 3 isolates ( technical appendix table 1 ) . compared with other prv isolates ( technical appendix table 2 ) , there was a 21-nt insertion from nucleotide positions 185205 in the gc gene , similar to sa215 , bj , dg , ea , fa , and p - prv strains ( figure 2 , panel a ) , which shared 100% nt identity with each other . the gd gene , like isolates ea , fa , sa215 , and yangsan , had 6 nt at positions 801806 and shared 99.4%99.8% identity with each other ( figure 2 , panel b ) .
there were 2 insertions of 6 discontinuous nucleotides each at positions 138140 and 14721474 in the ge gene ( figure 2 , panel c ) .
the rest of the genes from the 3 isolates had no nucleotide insertions or deletion in common with the other prv isolates .
we further analyzed the relationship of these 3 isolates with other prv isolates using a phylogenetic tree based on the ge gene ; the 3 isolates formed a tightly clustered branch and were very closely related to other isolates from asia ( technical appendix figure 3 ) .
we describe and analyzed a major outbreak of prv in pigs in china . in these herds ,
all pigs had been vaccinated against prv 3 times a year , at approximately the same time as each other . the disease spread to > 6 provinces ( including autonomous cities and regions ) and caused considerable economic losses among local pig farms .
prv has been recognized as a source of infection for pigs and continues to circulate globally among herds ( 9 ) .
in particular , pigs receiving attenuated live prv vaccines showed typical clinical symptoms , which suggest that these isolates might have evolved new types of pathogenicity .
even though the prv isolates showed nucleotide insertions in the gc , gd , and ge genes , the molecular mechanisms underlying their high pathogenesis have yet to be elucidated .
the origin of these lethal isolates within china is still obscure , although phylogenetic trees based on the ge gene here indicated that the 3 isolates are more closely related to the asia prv isolates , especially the china isolates , than to isolates from other countries . because the virulence and origin of prv is thought to be associated with multiple factors , whether such insertions are related to the virulence of prv remains an issue and requires further investigation . in summary
, our study indicates that an outbreak of disease in pigs in china , which was of unprecedented scale , was caused by prv infection .
pseudorabies virus target genes and primers ; sequences obtained from genbank used in this study ; immunohistochemical staining of lung and brain specimens of clinically sick pigs ; pcr of infected tissues with specific primers for pseudorabies virus glycoprotein d  specific primers ; and phylogenetic trees of glycoprotein e sequence .
 in 2012 , an unprecedented large - scale outbreak of disease in pigs in china caused great economic losses to the swine industry . isolates from pseudorabies virus epidemics in swine herds 
 were characterized . 
 evidence confirmed that the pathogenic pseudorabies virus was the etiologic agent of this epidemic . 
The Study
Conclusions
None