surprisingly , during summer and autumn 2014 , sbv reappeared in germany to a greater extent .
several samples from various federal states were submitted to the friedrich - loeffler - institut , federal research institute for animal health ( insel riems , germany ) , to confirm an sbv infection ( table ) and to further characterize these reemerging viruses . to evaluate sequence variations among sbv variants circulating in germany since 2011 , the original sbv isolate ( bh80/11 ) and viruses isolated in 2012 from the blood of viremic sheep ( bh619/12 ) or cattle ( d495/12 - 1 and bh652/12 )
were compared with 3 genomes obtained from the viruses in acutely infected adult cattle in 2014 ( bh119/14 - 1/2 , bh119/14 - 3/4 , bh132/14 ) .
rna was extracted by using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) according to the manufacturer s recommendations , and the open reading frames of all 3 genome segments ( large [ l ] , medium [ m ] , and small [ s ] segment ; primer sequences available on request ) were sequenced as described ( 7 ) .
kp731865kp731882 ) . sequence alignments and translation in amino acids were supported by geneious version 7.1 ( biomatters , auckland , new zealand ) .
we generated a maximum - likelihood tree ( hasegawa - kishino - yano model , 1,000 bootstrap replicates ) using mega5 ( 8) . for the phylogenetic analysis of the m segment , sequences previously obtained from organ samples from newborns malformed because of sbv infection also were integrated ( 7 ) . for the s segment ( 830-nt long ) , which encodes the nucleocapsid protein ( n ) and a nonstructural protein ( nss ) ,
the samples obtained from acutely infected animals in 2014 , bh619/12 and bh652/12 , were 100% identical to the original sbv strain bh80/11 from 2011 . in the sample d495/12 - 1 from 2012 , a single nucleotide was substituted ( technical appendix figure ) . the viral rna - dependent rna polymerase encoding l segment ( 6864 nt ) also showed high stability , and
only a few nucleotide substitutions compared with bh80/11 were found in the latest samples ( d495/12 - 1 and bh652/12 : 6 nt ; bh619/12 : 10 nt ; bh119/14 - 1/2 , bh119/14 - 3/4 . and bh132/14 : 18 nt ) . overall , sequence identity was 99.7%99.9% ( technical appendix figure ) .
the m segment ( 4415-nt long ) encodes 2 glycoproteins ( gn and gc ) and a nonstructural protein ( nsm ) .
it is the most variable genome segment of sbv and related viruses ( 7,9,10 ) .
despite the identification of a highly variable region within the gc - coding sequence in viruses in malformed newborns ( 7,9 ) , there was a high sequence stability of the viruses detected in the blood of acutely infected adult animals ; all sequences clustered closely ( figure 1 ) .
in contrast to the maximum of 77 nt and 43 aa substitutions or 12 aa deletions or 2 aa insertions found in organ samples of lambs or calves ( 7 ) , we detected only 612 nt and 2 aa ( bh619/12 ) , 3 aa ( d495/12 - 1 , bh119/14 - 3/4 , bh132/14 ) or 4 aa ( bh652/12 , bh119/14 - 1/2 ) substitutions ( technical appendix figure ) .
because gn and gc are major immunogens of orthobunyaviruses ( 10 ) , the mutation hot spot was supposed to be involved in immune evasion mechanisms and/or adaption of the cell tropism within the individual host ( 7,9 ) . however , insect - transmitted viruses such as sbv have to adapt to 2 hosts and undergo replication cycles in both the arthropod vector and the mammalian host . thus , the high sequence stability of virus strains detectable in viremic animals might be necessary for transmission to the vector .
notably , in comparison with the original sbv isolate , ke substitutions at aa 746 and 1340 of the m segment were found in all other samples .
because these mutations have now been consistently present for at least 2 years , they might have occurred during the adaptation to european ruminants or insects and could provide a growth advantage within the individual host or might be beneficial for transmission between host and vector .
phylogenetic analysis based on nucleotide sequences of the medium segment of schmallenberg virus samples isolated from blood of acutely infected animals in 2011 or 2012 ( blue ) or sequenced directly from the blood of viremic cattle in 2014 ( red ) and from organ samples of malformed newborns ( black ) ( 7 ) .
scale bar indicates nucleotide substitutions per site . to investigate whether the detected sequence variations correlated with any change in pathogenicity , 5 female sheep and 1 female calf were subcutaneously inoculated with pools of up to 5 serum or whole blood samples from 1 of the holdings with new cases confirmed in 2014 ( sheep 1 and 3 and the calf with samples from lower saxony ; sheep 2 , 4 , and 5 with samples from saxony [ permit no .
blood samples were taken daily for the first 2 weeks after inoculation and analyzed by real - time reverse transcription pcr ( 11 ) .
thereafter , serum samples were taken in weekly intervals and tested in a microneutralization assay ( 12 ) and a commercially available sbv antibody elisa ( i d screen schmallenberg virus competition ; idvet , grabels , france ) .
the calf and sheep 25 became infected ; viral genome was detectable in their blood for 46 days ( figure 2 ) , which agrees with the short - lived viremia previously observed after experimental infection of cattle and sheep with the original sbv sample or the first cell culture isolate ( 1,13 ) .
antibodies were first detected on day 7 after infection ( sheep 2 , sheep 5 ) or day 14 after infection ( calf , sheep 3 , sheep 4 ) in both tests .
detection of schmallenberg virus genome in the blood of experimentally infected cattle and sheep , germany , 2014 .
virus genome with a high sequence identity to the first sbv sample was repeatedly detected in the blood of acutely infected adult cattle , and in experimentally infected animals , viremia developed that was identical to the original sbv isolate .
the renewed virus circulation during the 2014 vector season was observed primarily in an area less affected in the 2 previous years .
the missing or markedly reduced virus circulation led to a decline in herd seroprevalence caused by a missing infection of the young stock ( 14,15 ) ; further reasons for the unexpected recurrence of sbv could be persistence within the insect vectors . as a consequence ,
the infection of naive animals in autumn 2014 resulted in an increasing frequency of the birth of malformed offspring in the following winter .
comparison of the small- , medium- , and large - segment sequences of schmallenberg viruses isolated in 2012 from the blood of viremic ruminants or detected in 2014 in acutely infected adult cattle with the first sbv isolate from 2011 , germany .
 schmallenberg virus ( sbv ) emerged in germany in 2011 , spread rapidly across europe , and almost disappeared in 2013 . 
 however , since late summer 2014 , new cases have occurred in adult cattle . 
 full - genome analysis revealed some amino acid substitution differences from the first sbv sample . 
 viremia developed in experimentally infected sheep and cattle for 46 days . 