The clinical signs, morbidity and mortality associated with current cases of BEF are different from those of BEF cases reported before 2000. The current disease cases showed more severe symptoms, and the morbidity and mortality have increased significantly. Luoyang city, in Henan Province, central China, is an epidemic area for BEF, and there have been eight BEF epidemics in the area from 1983 to 2011. The three BEF epizootics in 2011, 2005 and 2004 caused considerable economic loss to dairy cattle farming. During the latest an outbreak, which occurred in 2011, the infected dairy cattle showed a sudden onset of fever, stiffness, and nasal and ocular discharges. Moreover, difficulty in breathing and shortness of breath were the most obvious clinical symptoms shown by the infected dairy cattle. Some of the severe cases died between 6 and 12 hours after infection. The morbidity was about thirty percent, and the mortality rate was about five percent. However, the morbidity was from ten to twenty percent and the mortality was lower than one percent before 2000. During the 2004 BEF epizootic, about 12,200 dairy cattle were affected, of which, 2,198 cases died. Similar data were obtained in the 2005 epizootic. In the 2011 outbreak, the infected and dead dairy cattle rose to about 32,051 and 5,360, respectively. Certainly, the numbers of dairy cattle were increased from 44,000 in 2004 and 2005 to 107,300 in 2011 (http://henan.people.com.cn/news/2011/08/02/558227.html). The high feeding density of dairy cattle and the suffocation caused by the BEF may be the leading reasons for the high mortalities.
The phylogenetic relationships of the G gene sequence of BEFV isolated in Japan, Taiwan, Turkey, Israel and Australia had been analyzed previously [25, 26]. To date, the genetic relationships of BEFV derived from mainland China and those from other countries or areas have not been studied. In order to clarify the variation in the BEFV G gene with time and location, the G genes of four BEFV strains (LS11, LYC11, JT02L and JB76H) isolated from mainland China were amplified and sequenced. The G gene sequences of the three field isolates was repeatedly amplified and sequenced from infected blood, suckling brain and BHK-21 cells. The results showed that no change was found in the nucleotide sequences, indicating that the adaptation to suckling mice and BHK-21 cells through low passages had no significant effect on the nucleotide sequences of the BEFV G gene. However, it is worth noting that only one G gene sequence of the JB76H strain was used, because the original samples could not be obtained. It was unclear whether the extensive passages in BHK-21 cells affected variation in the G gene sequence of JB76H isolate.
The nucleotide and deduced aa sequences of the region encoding the ectodomain of BEFV G protein were well conserved among the BEFV isolates. In particular, the strains that originated from mainland China, Taiwan and Japan had higher identities. The corresponding sequences of the isolates derived from Turkey and Israel were highly conserved. However, the identities of the sequences were slightly lower among Australian isolates and other strains.
The phylogenetic relationships of the sequences of the ectodomain region of the BEFV G gene were analyzed in this work. The analysis revealed that the clusters of the BEFV isolates were closely related with geographical location. The strains derived from oriental areas (mainland China, Taiwan and Japan) had a close relationship. Turkish and Israeli isolates were grouped into one cluster, which had a close relationship. The Australian isolates were grouped into an independent cluster, and had a distant relationship with the Asian strains. The results revealed that the phylogenetic relationships among the BEFV isolates were closely interrelated with geographical location. Close genetic relationships among BEFV strains can be deduced if the isolates originate from adjacent areas. Similarly, the BEFV isolates derived from widely separated regions have distant genetic relationships. This may indicate that BEFV circulates in neighboring region for a long time.
The clusters of the isolates were also chronologically related. In cluster I, the JT02L strain clustered with other East Asian isolates from 2001–2004, suggesting that the same BEF outbreak spread through mainland China, Taiwan and Japan across the borders. The LS11 and LYC11 strains slightly diverged from the isolates from previous epizootics in East Asia, which indicated that the new BEFV possibly invaded mainland China from a neighboring area via infected vectors carried on the seasonal wind over a long distance or the import of live cattle. In fact, some evidence has shown that both winds and animal transport have an important role in trans-boundary transmission of BEFV [25, 26]. The oldest Chinese mainland vaccine strain, JB76H, and the oldest Japanese strain, YHL, sat separately. The Japanese and Taiwanese isolates from 1984–1989 clustered together. Similar results were obtained in the clusters II and III.
The variation in the aa sequences of the antigenic sites G1, G2 and G3 of the BEFV isolates was analyzed. The mentioned aa sequences of the three field strains obtained from mainland China corresponded identically with those of the Japanese isolates from 2001–2004 and the Taiwanese strains except for the 1984/TW/TN1 and 2001/TW/TN10. The other Japanese isolates from 1988–1989 had the same aa sequences mentioned above except for a substitution in ON-BEF-89-3 strain. No residues were changed among the isolates derived from Turkey and Israel. Three to five substitutions were found in the antigenic sites of G1 and G3 of Australian isolates compared with the residues of the Chinese mainland strains. These results indicated that the antigenic sites G1, G2 and G3 of BEFV isolates that related closely in place or time were highly conserved.