Epidemiology and genetic diversity of bovine leukemia virus

The BLV genome consists of 8714 nucleotides (nt) [18] including essential structural protein and enzyme coding genes and a pX region, flanked by two identical long terminal repeats (LTRs) (Fig. 1a). The structural protein and enzyme coding genes, namely, gag, pro, pol, and env, have essential and indispensable roles in the viral lifecycle, viral infectivity, and the production of infectious virions [19‐24]. The gag gene of BLV is translated as the precursor, Pr45 Gag, and processed to generate three mature proteins [19, 23]: the matrix protein, p15, which binds viral genomic RNA and interacts with the lipid bilayer of the viral membrane [25]; the capsid protein, p24, which is the major target of the host immune response, with high antibody titers against this molecule found in the serum of infected animals [26, 27]; and the nucleocapsid protein, p12, which binds to packaged genomic RNA [28] (Fig. 1b). The env gene encodes the mature extracellular protein, gp51, and a transmembrane protein, gp30 [19]. The pX region, which is located between env and the 3′ LTR [2], encodes the regulatory proteins Tax and Rex, and the accessory proteins R3 and G4 (Fig. 1a). The regulatory proteins are important for regulation of viral transcription, transformation of BLV-induced leukemogenesis, and nuclear export of viral RNA into the cytoplasm [29‐36]. The R3 and G4 accessory proteins contribute to the maintenance of high viral loads [37, 38]. In addition to the genes described above, the BLV genome also contains RNA polymerase-III-encoded viral microRNAs (miRNAs) between the env and pX regions. Viral miRNAs are strongly expressed in preleukemic and malignant cells, and may have roles in tumor onset and progression [39, 40] through their effects on proviral load and consequently viral replication in the natural host [41]. Besides, Van Driessche et al. revealed the recruitment of positive epigenetic marks on BLV miRNA cluster, inducing strong antisense promoter activity [42]. They also identified cis-acting elements of an RNAPII-dependent promoter [42].

A variety of techniques have been developed for diagnosis of BLV and implemented worldwide. These diagnostic methods can be assigned into two main groups, consisting of antibody-based serological tests and detection of the proviral genome by nucleic acid-based polymerase chain reaction (PCR) assays (summarized in Table 1).

Studies of BLV genotypes for phylogenetic and epidemiological analyses have primarily focused on the env gene, the env gp51 gene in particular, because of its biological functions. The extracellular gp51 protein has key roles in the viral lifecycle and is indispensable for viral entry into host cells [20, 86]. In addition, because of the surface localization of the gp51 glycoprotein, it is also the target of neutralizing antibodies [87]. The conformational epitopes, F, G, and H, located in the N-terminal half of gp51, are important in syncytium formation and viral infectivity [87, 88]. Therefore, the env gp51 sequence region is frequently used for BLV phylogenetic analysis.

Over the years, a number of methods have been applied for BLV genotyping, as summarized in Table 2. In the early days of BLV genotyping, researchers clustered or genotyped BLV strains from different geographical regions based on restriction fragment length polymorphisms (RFLP) of PCR-products, generated using various restriction enzymes [86, 89‐96]. BLV clusters and genotypes were named after the geographical region of sample isolation, such as “Argentine type” or “Australian type”, or with reference to phylogenetic clustering (e.g., “cluster one”). A total of seven BLV clusters/genotypes were determined by PCR-RFLP [91]; however, PCR-RFLP genotyping studies were not consistent or comprehensive.

In 2007, Rodriguez et al. reported sequencing of the env gene (all of gp51 and part of gp30) of 28 BLV field strains, performed phylogenetic analysis of these sequences in comparison with published sequence data representative of established genetic groups by neighbor-joining, maximum likelihood, and Bayesian inference methods, and assigned BLV sequences into seven genotypes [97]. Subsequently, a new genotype, genotype-8, was identified in BLV samples from Croatia by Balic et al. [98], who concluded that BLV may be more divergent than previously thought, speculating that additional genotypes might be discovered in the future. Indeed, the presence of eight BLV genotypes was later confirmed in different geographical locations [74, 77, 99‐101]. Finally, in 2016, the novel BLV genotypes, genotype-9 and -10, were discovered in Bolivia [75], Thailand [102], and Myanmar [76], a totaling ten BLV genotype clusters (Fig. 2). Previously, almost all phylogenetic studies of BLV genotypes focused on the partial or entire env gene. However, for the first time in their study [75, 76], Polat et al. successfully concluded the existence of genotypes-1, −2, −4, −6, −9 and −10 among ten BLV genotypes (Fig. 3) by phylogenetic analysis using complete sequences of BLV strains newly determined by next generation sequencing and sequencing cloned, overlapping PCR products in their studies, and using complete BLV genome sequences available in the database (NCBI & DDBJ). These phylogenetic analysis of complete BLV genomes demonstrated that each BLV genotype encodes specific amino acid substitutions in both structural and non-structural gene regions.

BLV has spread to all continents via the trade in breeding animals, and is prevalent in cattle worldwide. BLV infection levels vary between and within countries, as shown in Table 3 (data obtained on March 17th, 2017; updated and detailed information is available at http://​www.​oie.​int/​wahis_​2/​public/​wahid.​php/​Diseaseinformati​on/​statuslist) [17, 103]. BLV eradication programs and control measures have been established in European Community member countries since the second half of the twentieth century, and eradication programs have been very successful in the majority of western Europe [104‐107]; indeed, some countries, including Denmark, Finland, Switzerland, Estonia, The Netherlands and Poland, are completely free of BLV [104, 108‐110]. Despite the majority of countries in Western Europe being free from disease, EBL still exists in eastern European nations, including Poland, Ukraine, and Croatia [98, 100, 111‐113]. In addition, in Italy, Portugal, Belarus, Latvia, Greece, Romania, and Bulgaria, BLV is present, although disease is either absent or limited to specific areas [103].

Nationwide BLV eradication and control programs were introduced in Australia and New Zealand in 1983 and 1996, respectively, and 99.7% of Australian dairy herds were declared free from EBL in December 2013, while those in New Zealand have been free from BLV-induced EBL since 2008 [113, 114].

In North America, an epidemiological study of BLV prevalence in US dairy cattle conducted by the Department of Agriculture’s National Animal Health Monitoring System demonstrated that 83.9% of dairy cattle were BLV-positive at herd level and 39% of beef herds had at least one BLV-infected animal [115]. In Canada, studies of BLV prevalence revealed that up to 37.2% of cows and 89% of herds were BLV-positive [116‐118]. BLV is also present in both beef and dairy cattle in Mexico [119]; however, disease is either absent or limited to specific areas [17] (accessed on 22 Dec 2016).

In South America, relatively high levels of BLV prevalence have been observed, and BLV-induced leukosis is present in the majority of countries. In Brazil, BLV prevalence varies among states, with infection rates ranging from 17.1% to 60.8% [120‐123]. Individual and herd level BLV prevalence in Argentina are as high as 77.4% and 90.9%, respectively [75, 95, 124]. Moreover, individual infection rates between 19.8% and 54.7% have been reported in Chile, Bolivia, Peru, Venezuela, Uruguay, Paraguay, and Columbia [75, 94, 125‐131].

BLV infection is widespread in Chinese dairy farms. Infection rates are up to 49.1% among individual dairy cattle, while 1.6% of beef cattle are BLV-positive [132]. Moreover, serological tests revealed that 20.1% of yaks in China were BLV-positive [133]. Epidemiological studies in Japan revealed varying levels of BLV prevalence throughout the country, based on different detection methods [83, 134‐136], and BLV infection rates of 40.9% of dairy and 28.7% of beef cattle, with infection rates in animals over 2-years-old reaching 78% in dairy herds and 69% in beef cattle herds [136]. Less than 6% of cattle were infected with BLV in Mongolia (3.9%) [77], Cambodia (5.3%) [137], and Taiwan (5.8%) [48], while a serological survey in Iran revealed that the prevalence of BLV was between 22.1% and 25.4% in that country [138, 139]. Lee et al. [102] demonstrated an average prevalence of BLV of 58.7% in Thailand, reaching maxima of 87.8% and 100% of cattle when assayed using PCR and ELISA, respectively. In Korea, 54.2% of dairy cattle and 86.8% of dairy herds were BLV-positive, whereas only 0.14% of beef cattle were infected with BLV [101]. BLV infection levels in The Philippines ranged from 4.8% to 9.7% [74] while it was 9.1% in Myanmar [76]. BLV infections in Middle Eastern countries are relatively low. The prevalence of BLV infection is approximately 5% in Israel [140], while in Saudi Arabia, 20.2% of dairy cattle tested as BLV-positive [141]. Compared to these countries, BLV infection rates in Turkey are higher, with 48.3% of dairy herds including sero-positive animals [142].

As mentioned above, phylogenetic analyses of whole genome (Fig. 3) and env gp51 sequences (Fig. 2) of BLV strain showed that BLV can be classified into ten genotypes. Three genotypes of BLV, namely genotype-1, genotype-4 and genotype-6, were mainly detected from across the world, as shown in Table 4. Genotype-1 is the most dominant genotype of BLV and is distributed across almost all continents, including Europe, America, Asia, and Australia. In particularly, genotype-1 spread to South and North America, and these continents still have a high prevalence of BLV infection. In addition, genotype-1 continues to spread worldwide, including Asian countries. The second most widely distributed genotype is genotype-4, which is primarily detected in Europe and some American countries. However, it is only found in Mongolia among Asian nations. Interestingly, although genotype-4 used to exist in Europe, it decreased because of BLV eradication in European countries. Genotype 6 may have come from South America and spread to South Asia by animal trading. Of the other genotypes, genotype-2 is restricted to South American countries and is only found in Japan among Asian nations, while genotype-8 is restricted to Europe. Genotypes-5 (in Brazil and Costa Rica) and −10 (in Thailand and Myanmar) are only observed in geographically proximal areas, where there may be an exchange of animals across national boundaries [76, 102]. By contrast, genotypes-7 is distributed across geographically dispersed regions [74, 77].

In detail, in Europe, a total of five different BLV genotypes have been detected (genotypes −1, −3, −4, −7, and −8): genotype-4 in Belarus [100] and Belgium [86, 143]; genotypes-4, −7, and −8 in Russia and Ukraine [100]; genotype-8 in Croatia [98]; genotypes −4 and −7 in Poland [100]; genotypes −3 and −4 in France [86]; genotypes −1 and −4 in Germany [91]; and genotype-7 in Italy [144]. In Australia, only genotype-1 was detected [90]. In North America, genotypes −1, −3, and −4 have been detected in the USA [86, 143, 145], and genotype-1 was reported in the Caribbean [146]. In Central America, genotypes −1 and −5 were detected in Costa Rica [143]. A variety of BLV genotypes (−1, −2, −4, −5, −7, and −9) were detected in South America: genotypes −1, −2, −4, and −6 in Argentina [93, 95, 97, 147, 148]; genotypes −1, −2, −5, −6, and −7 in Brazil [122, 123, 127]; genotypes −4 and −7 in Chile [94]; genotypes −1, −2, −6, and −9 in Bolivia [75]; genotypes −1, −2, and −6 in Peru and Paraguay [75]; and genotype-1 in Uruguay [126]. In Asia, a total of seven BLV genotypes have been confirmed (−1, −2, −3, −4, −6, −7, and −10): genotypes −1 and −3 in Korea [101, 149]; genotypes −1, −2, and −3 in Japan [93, 99, 143, 150]; genotypes −1 and −6 in The Philippines [74]; genotypes −1, −6, and −10 in Thailand [102]; genotypes −1, −4, and −7 in Mongolia [77]; genotype-10 in Myanmar [76]; and genotypes −1 and −6 in Jordan [151].

Based on the European Food Safety Authority panel on animal health and welfare, BLV-induced EBL may have originated and spread widely from an area of Memel in East Prussia (now Klaipeda in Lithuania) [113, 152]. The worldwide distribution of the disease occurred due to the introduction of cattle from European countries into herds in other countries free of the disease, and also through the international trade of bred animals [113]. Interestingly, genotype-4 existed primarily in East Prussia as shown in Table 4. Then, infected cattle were reintroduced into some European countries; for example, BLV was introduced into the UK via bred animals from Canada in 1968 and 1973 [113]. As detailed in some previous publications, the widespread distribution of BLV genotypes within and between distant geographical locations may be driven by the spread of virus through the movement of live animal populations, associated with human migration and animal domestication, and also with viral transmission during close contact between individual animals [97].

It appears that at least ten different BLV genotypes of BLV strains are circulating in various geographical locations worldwide. The completion of whole genome sequencing of these BLV strains has revealed that BLV genomes contain a number of unique genotype specific substitutions not only in the env region, but also in the LTR, Gag, Pro, Pol, Tax, Rex, R3, G4, and miRNA encoding regions, distinguishing each genotype [75]. However, the BLV genome sequences of strains from different geographic origins, especially the important sites on the regulation of viral replication of BLV, are relatively stable and highly conserved among BLV strains, assigned to different genotypes. By contrast, several groups recently reported that the expression or pathogenesis of BLV does not depend on strains, but rather, is related with the specific site of mutation in their BLV genome [153, 154]. These results clearly demonstrate that BLV strain should be determined by full genome sequencing. However, although BLV is present worldwide, BLV genotyping studies are limited to certain areas, as shown in Table 4. Therefore, the accumulation of the full genome sequencing of BLV strains, assigned to different genotypes worldwide may define the genotype-dependent pathogenesis and association between genetic variability in each genotype and its infectivity, and differences in its functions in the future.