Introduction

Bluetongue virus (BTV) belongs to the genus Orbivirus and the family Reoviridae, and causes bluetongue disease (BTD) affecting both domestic and wild ruminants¹. The virus has a negative-sense double-stranded RNA (dsRNA) genome consisting of 10 segments². It is a non-enveloped virus with an icosahedral capsid, with a diameter of approximately 90 nm. The genome codes for the structural proteins that make up the external and internal capsid or core (VP1 - VP7), and the four non-structural proteins, called non-structural (NS) that are involved in the replication, maturation, or exit of the virion from infected cells). Nonstructural genes are highly conserved across the genus^3,4. The NS1 gene encodes for a protein of the same name, which is expressed in the largest quantity during BTV replication and is the most abundant cytoplasmic protein. On the other hand, the NS3 gene encodes for the NS3 protein that acts as a viroporin, which is related to cell lysis^3,4. Because of the above, the two genes have been used as targets in screening assays for the identification of BTV⁵.

The virus is transmitted by the bite of mosquitoes of the genus Culicoides spp; therefore, the occurrence of the disease is associated with the spread of this vector, although other vectors such as ticks have been reported⁶; it is known that the virus can remain viable throughout the life of the vector.

Currently, 28 different BTV serotypes have been described worldwide⁷, and the virus is distributed in practically all countries where cattle and sheep are raised. Bluetongue disease (BTD) can occur both subclinically and clinically, especially in sheep, since in cattle it is mostly asymptomatic. In countries where the disease is endemic, it causes severe economic losses to producers⁸. The name "blue tongue" was given to this disease by Africans who observed cyanosis on the tongue of some animals; however, this sign is not observed in all infected animals, as the signs vary between species and depend on the strain. Lesions such as hyperemia and edema of the lips and face, oral erosions and ulcers, and the typical cyanosis of the tongue are due to infection of the endothelial cells that allow increased cell permeability⁹.

The World Animal Health Organization (OIE)¹⁰ classifies bluetongue disease as a notifiable disease; therefore, timely diagnosis is important. The degree of severity of the disease depends on the serotype, the virus strain, and the species, age and immune status of the animal, with sheep and white-tailed deer being the most affected¹¹; in sheep, the incubation period of BTV is six to eight days; on the other hand, cattle rarely show clinical signs, but maintain a prolonged viraemia¹². Deer can also be infected by a closely related orbivirus responsible for epizootic hemorrhagic disease¹³. BTD is not contagious and is only transmitted by Culicoides insects; its distribution is therefore associated with the prevalence of the vector. Up to five serotypes have been identified in North America; however, seven serotypes have been reported only in the United States¹⁴. The occurrence of the virus in the Americas is mainly associated with the presence of two vector species, C. sonorensis and C. insignis¹⁵. In Mexico, although the disease is considered exotic, in the 1980s, positive serology to the virus was reported in both sheep and cattle in different regions of the country^16,17. On the other hand, in 2015, the detection of a viral genome fragment in three Culicoides species (C. variipennis, C. sonorensis, and C. occidentalis) was published¹⁸. The VP2 gene is used to define the 28 BTV serotypes described so far⁷.

Finally, in February 2021, the Secretary of Agricultural Development, Fisheries and Aquaculture (SEDPA) of Oaxaca in the Municipality of San Pedro Mixtepec notified the CPA of oral lesions in sheep. The CPA detected two bluetongue virus-positive samples using the RT-PCR technique and the notification was made in the CPA's AVISE Newsletter¹⁹.

BTD is notifiable to the OIE, mainly because new outbreaks lead to movement and trade restrictions, resulting in severe economic losses. However, active surveillance is implemented worldwide to detect BTV infection through different tests such as virus isolation or other screening or serological tests¹¹. The detection method par excellence is the isolation of the virus in permissive cell cultures, for subsequent genetic analysis of the virus to determine the serotype present in the sample of the affected animal. In virus endemic areas, vector control is recommended to prevent the spread of the virus, in addition to vaccination programs. Live attenuated vaccines have been used in the United States and Europe. The objective of this work was to establish molecular techniques using a synthetic gene as a positive control that included the NS1 and NS3 genes, in order to subsequently evaluate sheep samples from different regions of the country.

Material and methods

Results

As an assay to evaluate the quality of the genetic material, the amplification of a fragment of approximately 400 bp of the ovine GAPDH gene was carried out as previously above. All samples used for detection of a viral genomic fragment were positive for GAPDH amplification by RT-PCR, which indicates that the genetic material was intact and in good condition for use in RT-PCR assays (Figure 1).

Lane 1, 50bp fragment size marker (Low Mass ladder); Lanes 2-7, sheep samples. Amplification products were run on a 1.5% agarose gel.

Figure 1 RT-PCR for amplification of the sheep’s constitutive GAPDH gene 

As for the assay for the detection of a fragment of the NS1 gene by RT-PCR endpoint, the identification of this gene was obtained in 12 samples of sheep blood from the states of Chiapas, Coahuila, and Tamaulipas. In order to harmonize the methods, it was decided to use primers suggested by the OIE, as described above. The analysis of the sequences obtained showed in the alignment an identity with sequences reported in GenBank with the NS1 fragment from 89 % (p= 1e-12) to 98 % (p= 4e-13), corresponding to serotypes 10, 11 and 12.

Figure 2 shows the results of the phylogenetic inference, depicting the clustering of the samples primarily with serotypes 10 and 11. The positive results were corroborated by real-time PCR in two of the samples, one from Chiapas and the other from Tamaulipas; as mentioned above, RT-PCR uses the gene NS3.

The dendogram was obtained using the alignment of 100 bp of the NS1 gene from the sequences of the positive samples in this study and 30 sequences obtained from GenBank belonging to serogroups 10, 11, and 12. Sheep samples positive by end-point RT-PCR are identified as follows: OT= Tamaulipas sheep: OT1, OT2, OT3, OT4, OT5. OC= Chiapas sheep: OC1, OC2, OC3, OCF10, OCF39, OC5, OC4. The breeds are indicated by color: Creole , Pelibuey , Suffolk , and Dorper . The samples were taken between 2016 and 2017. The sequence of the epizootic hemorrhagic fever virus (AM745001) was used as outgroup, indicated by .

Figure 2 Phylogenetic inference of bluetongue virus NS1-positive sheep samples 

The positive result of RT-PCR detection was corroborated by the SENASICA - CPA laboratory and notified to SIVE. In this laboratory, the detection of a fragment of gene NS3 also serves as a positive control, using viral RNA obtained from the supernatant of cell cultures infected with the virus that are subsequently inactivated (CPA personal communication). With respect to the tests performed in collaboration with the official laboratory to corroborate the results of the animals that tested positive, the synthetic control proposed in this work showed that it could be used without problem in any BSL2 laboratory in order to perform virus detection in official BSL2 laboratories.

Discussion

As mentioned above, BTD in different hosts can be subclinical, and detection of the causative agent in sheep populations can be complex²¹. Therefore, for this study, clinically healthy animals were considered for sampling, where the status of viraemia in animals and signs may or may not have been observed, depending on the viral load or subtypes involved. In addition, sampling was carried out in those Federal Entities of the country located in areas where the vector transmitting the virus is present in a climate suitable for its development²², from sheep that were close to cattle farms, as bovines can be a healthy carrier of the virus.

Regarding the diagnosis of the causative agent of BTD, the recommended method is by virus isolation in a cell culture or in embryonated eggs²³. However, different versions of RT-PCR have been developed that can be used to detect BTV, specifically the Orbivirus serogroup, and to determine the BTV serotype. These molecular approaches are much faster than traditional virological and immunological methods, which can take up to four weeks to provide information on serogroups and serotypes. Currently, there are targeted assays mainly for VP1, NS1, NS2, VP6, and NS3 proteins. None of these proteins is related to virus serotyping, and they are strongly conserved among BTV serotypes, while some, such as NS3, have a higher degree of conservation among orbiviruses. Therefore, these assays lack the potential to classify isolates²⁴.

In addition, each technique offers a range of virus or genome detection; for instance, Bonneau et al²⁵ report that the RT-PCR assay is capable of detecting the genome within a period ranging from 3 to 122 days. Therefore, it is important to make the recommendation to carry out sampling and surveillance campaigns not only on ruminants but also on the potential vectors that are reported as transmitters of the virus.

BTD is considered exotic in Mexico; however, this status should be reconsidered taking into account the various notifications made since the 1980s to the present date in different regions of the country; this would allow to assess the presence of the virus in different hosts and vectors using a variety of methods. In 1981, Moorhead et al²⁶ determined the presence of antibodies by immunoprecipitation in sheep slaughtered at the slaughterhouse, finding 8.5 % positivity in serum. Subsequently, Vilchis et al (1986)²⁷, using immunodiffusion, demonstrated 27.4 % seropositivity in the animals sampled. Stott et al²⁸ reported seropositivity of 6 %, 35 % and 60 % in three independent studies on cattle from different states of the country. The most recent scientific publication by Lozano-Rendón JA and his work group¹⁸ proved a 14.4 % molecular detection of the NS1 gene of BTV in Culicoides vectors in the state of Nuevo León.

As for the results presented in this research, a 3.75 % positivity rate was detected in the samples of clinically healthy sheep using the same NS1 gene as Lozano-Rendón et al¹⁵. However, this study was conducted on the vector, where the probability of demonstrating the presence of the virus is greater than in sheep, where the viraemia time is shorter. This NS1 gene, as already mentioned, is one of the most conserved among the different BTV serotypes²⁹. The results of the detection of a fragment of the viral genome in sheep samples in this study are consistent with those reported this year by the CPA¹⁹.

On the other hand, the detection rate is similar to that described in older reports using ruminant samples. The results reported in the present work, as well as those presented by other authors, show the need to change the status of the disease, as well as to implement virus surveillance systems in both the vectors and the main hosts of the virus, whether these be domestic or wild animals.

Conclusions and implications

A synthetic positive control is presented herein as an alternative to viral RNA, which can only be utilized in the BSL3 laboratory of the country's official agencies. The use of such synthetic positive control would enlarge the network of laboratories capable of implementing the viral detection technique to determine the real status of the disease in the country.