Detection and Molecular Characterization of a Natural Coinfection of Marek’s Disease Virus and Reticuloendotheliosis Virus in Brazilian Backyard Chicken Flock

Marek’s disease virus (MDV) and the reticuloendotheliosis virus (REV) are two of the primary oncogenic viruses that significantly affect chickens. In Brazil, there have been no previous published reports on the presence of field REV alone or in coinfection. This retrospective study analyzes samples from a case of lymphoproliferative lesions from a backyard chicken flock. MDV and REV were detected by PCR and classified as MDV1 and REV3, respectively, through sequencing and phylogenetic analysis based on the glycoprotein B (gB) genes for MDV and the polymerase (pol) and envelope (env) genes for REV. Real-time PCR reactions were performed for MDV to rule out the presence of the Rispens vaccine strain. This is the first report of the presence of REV in coinfection with a MDV clinical case in Brazil and the first molecular characterization of REV in South America. This study highlights the importance of molecular diagnosis for REV and MDV in poultry. In addition, this study highlights the distribution of these two viruses worldwide and the latent risk of them solely or in coinfection to this part of the world.


Introduction
In chickens, neoplastic diseases typically have a viral etiology and cause a significant economic impact [1][2][3]. The three primary disease-causing agents are the Marek's disease virus (MDV), the avian leukosis virus (ALV), and the reticuloendotheliosis virus (REV). The prototype MDV is the Gallid alphaherpesvirus 2 (serotype 1, MDV1) and is grouped in the Mardivirus genus together with the following relative strains: The Gallid alphaherpesvirus 3 (serotype 2, MDV2) and Meleagrid alphaherpesvirus (serotype 3, MDV3), according to the latest information released on the Herpesviridae family at the International Committee on Taxonomy of Viruses (ICTV) [4].

Detection of MDV and REV Viruses through PCR Examination
Commercial vaccines (CVI988/Rispens, SB-1, and HVT) were used as positive controls for the MDV serotypes and synthetic DNA fragments (Invitrogen™ GeneArt™ Strings™) in the case of REV and ALV. Spleen from specific-pathogen-free (SPF) chicken resuspended in 1.5 mL of phosphatebuffered saline (PBS) was used as a negative control. DNA extraction of the controls and samples of liver, spleen, proventriculus, and small intestine were performed using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's specifications. The eluted suspensions were quantified with a NanoDrop One (ThermoFisher Scientific, Carlsbad, CA, USA) and stored at −80 °C for the subsequent PCR procedures. PCR methods for the detection of MDV and REV were performed as described previously (Table 1) [8]. An RT-Nested-PCR reaction for the detection of ALV was performed as previously described (Table 1) [18].

Detection of MDV and REV Viruses through PCR Examination
Commercial vaccines (CVI988/Rispens, SB-1, and HVT) were used as positive controls for the MDV serotypes and synthetic DNA fragments (Invitrogen™ GeneArt™ Strings™) in the case of REV and ALV. Spleen from specific-pathogen-free (SPF) chicken resuspended in 1.5 mL of phosphate-buffered saline (PBS) was used as a negative control. DNA extraction of the controls and samples of liver, spleen, proventriculus, and small intestine were performed using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's specifications. The eluted suspensions were quantified with a NanoDrop One (ThermoFisher Scientific, Carlsbad, CA, USA) and stored at −80 • C for the subsequent PCR procedures. PCR methods for the detection of MDV and REV were performed as described previously (Table 1) [8]. An RT-Nested-PCR reaction for the detection of ALV was performed as previously described (Table 1) [18]. previously described method, which targets a single nucleotide polymorphism (SNP) of nucleotide 320 in the pp38 gene [19]. The non-CVI988 reaction uses the Non-CVI988-F and the MDV pp38-R primers (Table 1) and is specific for amplification of MDV1 strains different of CVI988. The CVI988 specific reaction uses the CVI988-F and the MDV pp38-R primers (Table 1) and is specific for amplification of the Rispens vaccine.

PCR Amplification of the Glycoprotein B Gene of the MDV Serotypes
To confirm the MDV serotype by sequencing, a PCR method was developed using generic primers (Table 1) to amplify a fragment of glycoprotein B (gB) that is common to all three MDV serotypes (MDV-1: Gallid herpesvirus 2, MDV-2: Gallid herpesvirus 3, MDV-3: Meleagrid herpesvirus 1), using the reference sequences (NC_002229, NC_002577, and NC_002641, respectively). The volume of the reaction mix was 25 µL, including 50 ng of template DNA, 0.2 mM of each dNTP, 2 mM MgCl 2 , 0.5 µM of each primer, 1× PCR buffer, and 1 U of Platinum Taq DNA Polymerase (ThermoFisher Scientific, Carlsbad, CA, USA). The thermal conditions included an initial denaturation step at 94 • C for 3 min, followed by 35 cycles of 94 • C for 60 s, 54 • C for 60 s, and 72 • C for 90 s, followed by a final elongation step at 72 • C for 10 min.

PCR for the Gag, Polymerase, and Envelope Genes of REV
To confirm the integrity of the REV provirus in the positive samples, two PCRs were developed to amplify fragments of the gag + polymerase (pol) and envelope (env) genes (Table 1), according to the REV sequences of the complete genomes that are available at the GenBank (Table 2). In both cases, a 25 µL PCR was performed, including 50 ng of template DNA, 0.2 mM of each dNTP, 2 mM MgCl 2 , 0.6 µM of each primer, 1× PCR buffer, and 0.75 U of Platinum Taq DNA Polymerase (Thermo Fisher Scientific, Carlsbad, CA, USA). The thermal conditions included an initial denaturation step at 94 • C for 3 min, followed by 35 cycles of 94 • C for 45 s, 60 • C for 45 s, and 72 • C for 90 s, followed by a final elongation step at 72 • C for 10 min.

Sequencing and Phylogenetic Analysis
The PCR product of gB, gag + pol, and env from the respective positive control (MDV vaccines) and the sample with the highest concentration were purified with the IllustraTM GFX PCR and Gel Band Purification Kit (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) according to the manufacturer's instructions. Sequencing was performed on an ABI 3730 DNA Analyzer with the BigDyeTM Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA). The sequencing products were assembled using Geneious Prime ® 2019.0.4. (www.geneious.com).
The sequences of glycoprotein B from the MDV samples and vaccines were analyzed together with other previously published MDV sequences (Table 3). Partial REV sequences corresponding to the pol and env genes were analyzed and compared with the reference sequences ( Table 2). Multiple sequence alignments were performed using Clustal W [20], and an identity matrix of nucleotides and inferred amino acids was generated using Geneious Prime ® 2019.0.4. The selection of the best-fit substitution models and the construction of phylogenetic trees were performed using MEGA v7.0 [21].

Histopathologic Examination
Marked uniform small to medium size neoplastic lymphocytes were infiltrating and replacing multifocal to diffuse the liver and spleen parenchyma (Figure 2a,b). There were multifocal neoplastic lymphoid cells in the kidney and lung. Trachea, proventriculus, gizzard, and small intestine lamina propria showed increased thickness by uniform neoplastic lymphocytes infiltration. Peripheral nerves and ganglion were moderate to marked infiltrated by uniform neoplastic lymphocytes, as showed in Figure 2c,d, respectively. There were few heterophils and plasma cells among neoplastic lymphocytes in all organs examined. Thymic atrophy also was observed in part and there were lymphocytes arranged in focal nodules. showed in Figure 2c,d, respectively. There were few heterophils and plasma cells among neoplastic lymphocytes in all organs examined. Thymic atrophy also was observed in part and there were lymphocytes arranged in focal nodules.  In the case of REV, a PCR product of 201 bp corresponding to a fragment of LTR was detected in all four DNA samples analyzed as well as the positive synthetic control. PCRs designed to amplify the junction between the gag + pol genes and the last part of the env gene tested positive and amplified fragments of 767 and 703 bp, respectively, in all organ samples. All MDV positive controls tested negative in all REV-specific PCRs. The RT-Nested-PCR reactions for ALV were negative for all the organ samples and positive in the synthetic control.

Sequence and Phylogenetic Analysis
The similarity analysis between the USP386 MDV sequence with respect to the reference sequences grouped by serotype showed the highest similarity with the MDV1 serotype at the nucleotide level (99.8-100%) and at the amino acids level (100%) ( Table 4). The phylogenetic analysis showed that the sequence corresponding to USP386 MDV (MH825642) was found in the cluster corresponding to the MDV1 serotype and the positive control of the same serotype (vaccine Rispens, MH825643). Similarly, the positive controls of serotype 2 (vaccine SB-1, MH825644) and serotype 3 (vaccine HVT, MH825645) were grouped in the corresponding clusters with 100% bootstrap support in all cases ( Figure 3). The analysis of similarity in the case of the pol gene of REV for the sample USP386 showed greater similarity with the subtype REV3 at the nucleotide level (99.5-100%) as well as at the amino acid level (98.9-100%). Similarly, the env gene analysis revealed greater similarity with the REV3 subtype at the nucleotide level (99.2-100%) as well as at the amino acid level (98.5-100%) ( Table 5). The phylogenetic analysis for the REV pol gene (Figure 4) showed that the grouping of the sequence corresponded to the USP386 sample within the REV3 subtype. Likewise, in the case of the env gene ( Figure 5), the sample USP386 was grouped within the subtype REV3. The similarity analysis between the USP386 MDV sequence with respect to the reference sequences grouped by serotype showed the highest similarity with the MDV1 serotype at the nucleotide level (99.8-100%) and at the amino acids level (100%) ( Table 4). The phylogenetic analysis showed that the sequence corresponding to USP386 MDV (MH825642) was found in the cluster corresponding to the MDV1 serotype and the positive control of the same serotype (vaccine Rispens, MH825643). Similarly, the positive controls of serotype 2 (vaccine SB-1, MH825644) and serotype 3 (vaccine HVT, MH825645) were grouped in the corresponding clusters with 100% bootstrap support in all cases (Figure 3). The analysis of similarity in the case of the pol gene of REV for the sample USP386 showed greater similarity with the subtype REV3 at the nucleotide level (99.5-100%) as well as at the amino acid level (98.9-100%). Similarly, the env gene analysis revealed greater similarity with the REV3 subtype at the nucleotide level (99.2-100%) as well as at the amino acid level (98.5-100%) ( Table 5). The phylogenetic analysis for the REV pol gene (Figure 4) showed that the grouping of the sequence corresponded to the USP386 sample within the REV3 subtype. Likewise, in the case of the env gene ( Figure 5), the sample USP386 was grouped within the subtype REV3.

Discussion
The coinfection of MDV and REV in the field case of lymphoma was described for the first time in Brazil. The histopathological findings correspond primarily to Marek's disease characterized by neoplastic small to medium size lymphocytes infiltrations in several visceral organs as described previously [1,5]. In addition to these lesions, enlargement of the spleen and liver is a frequent finding in cases of MDV infection with increased mortality and have already been reported in cases of coinfection of MDV with REV [9,13,15] as well as with another avian viruses [22][23][24]. All organ samples tested positive in the PCRs for the pp38 and gB MDV genes and the LTR, gag, pol, and env REV genes, indicating the coinfection in all samples examined. Real-time PCR assays based on the SNP of nucleotide 320 in the pp38 gene [19] was used to differentiate the field strains from the Rispens vaccine (CVI988). In this process, the possibility of infection by the MDV1 serotype vaccine was excluded. To further exclude the presence of the MDV2 and MDV3 serotypes, a generic PCR based on the gB gene was designed, as this gene is one of the most conserved genes among the three serotypes, including the field and vaccine strains [25][26][27][28][29][30][31]. Sequencing demonstrated high levels of identity in the amino acid comparison of the gB gene among the three serotypes, and a lower identity in the comparison of nucleotides. A higher level of discrimination at the nucleotide level among the three serotypes was demonstrated in the phylogenetic tree, with bootstrap supports of 100% for each serotype. Thus, this gB fragment can be used for differential diagnosis and molecular typing. The

Discussion
The coinfection of MDV and REV in the field case of lymphoma was described for the first time in Brazil. The histopathological findings correspond primarily to Marek's disease characterized by neoplastic small to medium size lymphocytes infiltrations in several visceral organs as described previously [1,5]. In addition to these lesions, enlargement of the spleen and liver is a frequent finding in cases of MDV infection with increased mortality and have already been reported in cases of coinfection of MDV with REV [9,13,15] as well as with another avian viruses [22][23][24]. All organ samples tested positive in the PCRs for the pp38 and gB MDV genes and the LTR, gag, pol, and env REV genes, indicating the coinfection in all samples examined. Real-time PCR assays based on the SNP of nucleotide 320 in the pp38 gene [19] were used to differentiate the field strains from the Rispens vaccine (CVI988). In this process, the possibility of infection by the MDV1 serotype vaccine was excluded. To further exclude the presence of the MDV2 and MDV3 serotypes, a generic PCR based on the gB gene was designed, as this gene is one of the most conserved genes among the three serotypes, including the field and vaccine strains [25][26][27][28][29][30][31]. Sequencing demonstrated high levels of identity in the amino acid comparison of the gB gene among the three serotypes, and a lower identity in the comparison of nucleotides. A higher level of discrimination at the nucleotide level among the three serotypes was demonstrated in the phylogenetic tree, with bootstrap supports of 100% for each serotype. Thus, this gB fragment can be used for differential diagnosis and molecular typing. The detection of the REV LTR, gag + pol, and env genes suggests that REV was present with the total proviral genome. REV presents a highly conserved genome [9,[32][33][34][35][36]. The USP386 REV strain reflects this fact and shows a high percentage of identity in nucleotides and amino acids for the two genes evaluated. Based on the analysis of both genes, there was greater identity with the strains of the REV3 subtype, intermediate identity with REV1, and lower identity with REV2.
In the phylogenetic analysis of the pol gene, the Brazilian REV (USP386) was grouped in subtype 3 (REV3) and was shown to be more closely related to strains 104865 (KJ56349), CY1111 (KJ909531), 1105 (JQ804915), and APC-566 (DQ387450). In the phylogenetic analysis of the env gene, USP386 was also grouped in the REV3 subtype and was shown to be more closely related to strains 104865 (KJ56349) and APC-566 (DQ387450). The phylogenetic distribution for both genes is consistent with previous reports [9,37,38]. It has been shown that there is no correlation between time, place, or specific host in the distribution of REV; however, most of the strains reported, including the present study, belong to the REV3 subtype [39][40][41]. The occurrence of cases caused only by REV is infrequent. However, the origin and distribution of these viruses are complex and can be influenced by several factors, including the contamination of vaccines with MDV [42], FPV [43,44], IBDV [34], biological products, or free-living birds [39,45,46]. REV can remain undetected for a long time while it is transmitted horizontally through the direct contact of birds or through certain insects [47], integrated into other field viruses [9][10][11][12]48], or transmitted vertically by eggs [49]. Additionally, REV has no specific host or specific geographic location, having been detected in chickens, ducks, turkeys, geese, pheasants, Japanese quails, peafowl, guinea fowls, and Attwater's prairie chickens in several countries and continents [3]. Particularly, in the case of South America, two serological reports in Peru [16] and Argentina [17] indicated the presumable presence of REV in this region several years ago. However, it will be difficult to determine the source of REV in Brazil until further studies of regional strains are available. The immunosuppressive capacity of REV can significantly influence poultry farming, leading to economic losses associated with reduced efficiency, morbidity, and increased mortality from coinfections with other pathological agents, which can be associated exclusively with the damages. The genome status of REV (partial or integral) in coinfection with other field viruses can lead to alterations in the severity of lesions [10,13,14] and the inefficiency of vaccines [9,15,50].
As reports of the MDV strain in Brazil are scarce, even more in backyard chicken flocks, this study remembers the importance of the vigilance. On the other hand, the apparition of MDV in backyard flocks is a common event [51] with the presence of low and high-virulence pathotypes [52,53]. The pathogenicity of the MDV strain detected in this study could be comparable with the highest virulent ones, based on the percentage of mortality (>10%) and the lymphoproliferative lesions observed in necropsy (Figure 1a,b) and histopathologic examination (Figure 2a-d). Some possibilities arise here, a virulent enough MDV strain could be responsible for the typical clinic manifestations without the participation of REV; or the last could be helping to a less virulent MDV strain to cause the pathogenic effects. However, conclusive evidences about this should be supported by different approaches as experimental infections and additional genomic studies.

Conclusions
The knowledge of the infective process and pathologies associated with REV is not fully clarified despite having spent many years since its first reports and studies in some countries with developed poultry industry. In fact, it is possible that this virus is circulating in several other countries, but its monitoring can be neglected. In this context, the South American region includes several countries with notable development of the poultry industry, as is the case of Brazil. Despite the "rumors" about some isolated cases of REV in this continent, this study represents the first description of REV with molecular characterization and in coinfection with MDV causing oncogenic manifestation. However, the main limitation of this study was the number of samples. Further studies involving more cases and samples could reveal or discard the presence of these viruses on field nowadays. We believe that the presence of REV in this region represents a serious threat to many avian species due to its wide spectrum of target hosts and may include not only commercial birds but also ornamental birds, pets, and free-living birds.
Due to the absence of a vaccine or treatment against REV, it is very important to ascertain its origin through monitoring so that measures for eradication and prevention must be taken. In addition, Marek's disease is associated with large economic losses in poultry and can be supplied with pathogenic viruses from the backyard chicken flocks as the presented strain.
Finally, the importance of this study highlights the REV and MDV detection into this region and provide molecular clues for future research about these viruses.