The Nucleocapsid Protein Gene as Excellent target for Detection of Canine Distemper Virus by Reverse Transcriptase-Polymerase Chain Reaction

The genome consists of about 15.7 kilobases (kb), which codes for six structural proteins: the nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the polymerase (L) and the envelope glycoproteins, the fusion protein (F) and the haemagglutinin (H). These last two, located in the lipid envelope surrounding the virion, are responsible for the recognition and entry of the virus into the host cell [1-4]. The helical nucleocapsid contains the N, P and L proteins, which initiate intracellular replication. The M protein connects the glycoproteins of the surface and nucleocapsid during viral maturation [1,5].


Introduction
The Canine Distemper Virus (CDV) belongs to the genus Morbillivirus of the Paramixoviridae family, Mononegavirales order and is a pleomorphic virus with a lipid envelope, a diameter of 150 to 300 nm, whose genome is a single-stranded RNA, with a negative sense and with a helical nucleocapsid.
The genome consists of about 15.7 kilobases (kb), which codes for six structural proteins: the nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the polymerase (L) and the envelope glycoproteins, the fusion protein (F) and the haemagglutinin (H). These last two, located in the lipid envelope surrounding the virion, are responsible for the recognition and entry of the virus into the host cell [1][2][3][4]. The helical nucleocapsid contains the N, P and L proteins, which initiate intracellular replication. The M protein connects the glycoproteins of the surface and nucleocapsid during viral maturation [1,5].
The N gene, of around 1.5 kb, is one of the most conserved genes of the CDV genome and it has highly conserved regions in the first 2/3 of the gene between members of the genus Morbillivirus and encodes the most abundant of the structural viral proteins, the nucleocapsid protein, which has regulatory functions of transcription and replication, as well as the encapsidation of the RNA genome in a resistant RNAsa nucleocapsid. However, despite being a highly conserved region of the CDV genome, some variations of the N gene have been shown among field isolates [6][7][8][9].
Diagnosis of CDV. Of the diagnostic methods, the clinical signology is the main tool used by veterinarians, however, the non-specificity of the signs in some cases makes it impossible to reach a final diagnosis, so it is necessary to use laboratory tests.
Among them, one of the most used is the ELISA (Enzyme-Linked Immune Sorbent Assay), which can detect serum antibodies (Abs) IgM (against the nucleocapsid proteins, N and P of the CDV) or IgG (against the antigens of the envelope, H and F of the CDV). However, this test does not differentiate if the Abs produced correspond to maternal Abs, vaccine or infection. Otherwise, the production of Abs depends on the stage of the disease and may not be in the initial and final phases of it. In dogs not vaccinated with acute infection, they can die without presenting Abs against the virus. In animals with neurological signs, the determination of specific Abs against CDV in cerebrospinal fluid not contaminated with blood, is a definitive diagnosis of DC encephalitis, however this is a very invasive method and its results are variable [7,10].
Another diagnostic method used is the detection of eosinophilic intracytoplasmic inclusion bodies by cytology or by immunofluorescence, however, these can only be visualized in certain periods of the disease, generally being absent when clinical signs are generated [10,11]. Immunohistochemistry is used to detect viral antigens and/or inclusion bodies in tissues, with reliable results only when there is a marked viremia, and this technique can be performed exclusively on samples taken postmortem [7,9]. The isolation of the virus has a very high sensitivity and specificity, however it is a laborious and very slow procedure, so it is not routinely used for diagnosis [11]. The RT-PCR consists of an exponential amplification of DNA fragments of the virus, after reverse transcription from RNA to complementary DNA, allowing the presence of the virus RNA to be detected early, being a Unlike the other techniques mentioned, a sensitive, specific and rapid test for the diagnosis of CDV can be positive even when other tests fail to detect the virus [7,10,11]. In the last decades, several methods based on RT-PCR for the diagnosis of CDV have been developed, being the main targets for amplification, genomic regions that present a high degree of conservation among the virus isolates. This is how, as in 1999, Frisk et al. developed a method, based on the amplification by RT-PCR of a conserved 287 bp fragment of the N gene, which was later used by several authors, proving to be a very effective technique for the detection of the virus genome [7,8,12]. The main objective of this title memory is to diagnose CDV molecularly, through the implementation of the RT-PCR assay of the nucleocapsid protein gene.

Material and Methods
This work was carried out in the Virology and Microbiology

Controls
Positive controls: For positive vaccinal controls, blood was used from the negative control without vaccination, contaminated with live attenuated CDV virus obtained from the vaccines "Nobivac® Puppy DP" (Onderstepoort strain), "Canigen MHA2PPi/L" (Lederle strain) and "Vanguard" Plus 5/CV-L "(Snyder Hill strain). For this, each vaccine was reconstituted with 0.5 mL of 0.9% sodium chloride, and then this mixture was added to 0.5 mL of blood, to later extract the RNA. The ten positive controls from national isolates of the virus corresponded to viral RNA. These isolates came from domestic dogs from Santiago de Chile and were confirmed as positive by a previous RT-PCR based on the hemagglutinin gene [13,14].
Negative controls: Blood was used with anticoagulant of two uninfected animals, of an adult dog without clinical signs of the disease, without a risk and without vaccination history and of an adult dog without clinical signs of the disease and without a risk history, but with a calendar of daily vaccination (vaccinated for the last time ten months ago).

Suspicious samples of DC
Eleven samples of peripheral blood were analyzed with anticoagulant (2 mL), coming from dogs that showed nervous signs compatible with DC and that in some cases presented positive to the ELISA test specific for CDV, with IgM antibody titers ≥1: 80.
These samples were collected from veterinary clinics of Santiago de Chile, maintained in refrigeration at 4 °C and processed in less than two weeks. In addition, they were classified according to race, sex, age, vaccination status against CDV and anti-CDV IgM antibody titers (Table 1).

RT-PCR reaction
This was done using the "SuperScript ™ one step RT-PCR with platinum Taq" kit (Invitrogen®) according to the manufacturer's instructions. The reaction began with the synthesis of complementary DNA and a pre-denaturation performed in one cycle (45°C for thirty minutes and then two minutes at 94°C). The PCR amplification was carried out in forty cycles, which consisted of a denaturation (94°C for one minute), alignment of the starters (60°C for two minutes), elongation (72°C for two minutes), and a final extension in one cycle (72°C for ten minutes).

Detection of the DNA fragment synthesized in the RT-PCR
The RT-PCR products were analyzed by means of 2% agarose gel electrophoresis in Tris-HC1 buffer (100 mM Tris-HC1, 10 mM EDTA) and comparison against a molecular size standard: Hyperladder IV (Bioline®). The PCR product was mixed with a commercial loading product (Fermentas®) and then the electrophoresis was

Sequencing of Amplified Fragments
The fragments of DNA obtained from two positive samples were sent to be sequenced, to the Sequencing Center of Genytec Ltda.

Bioinformatic analysis of the nucleotide sequences of DNA fragments
The sequences obtained were aligned using the free online program Clustal Ω for obtaining a consensus sequence for each sample used. In addition, the consensus sequences were entered the BLAST program to identify the origin of the DNA fragments obtained in the RT-PCR.

Analysis of results
Those samples that after RT-PCR synthesized a DNA fragment of approximately 290 bp were considered positive and after analysis with the BLAST program their nucleotide identity corresponded to CDV.

Implementation of the RT-PCR assay of the CDV Nucleocapsid Protein Gene
Once the RT-PCR protocol of the nucleocapsid protein gene of the CDV was established, the operation of the method was tested with positive and negative controls, to later analyze the field samples suspected of DC. Three positive controls from commercial vaccines, ten positive controls corresponding to CDV RNA (national isolates) and two negative controls from uninfected dogs were processed. All these

Am J Biomed Sci & Res
Copy@ Navarro C controls were subjected to the RT-PCR of the nucleocapsid gene of the CDV and after the visualization of the products, all the positive controls generated intense bands of around 290 bp and in the case of the negative controls, they did not present visible bands ( Figure   1).

Suspicious samples of DC
Eleven samples of peripheral blood of dogs with nervous signs concordant with DC were collected and, in some cases, they were positive to the specific ELISA test for CDV (IgM) ( Table 1). All these samples were submitted to the RT-PCR of the nucleocapsid gene of the CDV and after visualization of the products, ten samples (canine 1 to canine 10, Table 1) presented bands of around 290 bp, the expected DNA fragment. On the contrary, the sample from canine 11 did not generate visible bands (Figure 2).  Table 1).

Sequencing of amplified fragments and bioinformatic analysis of the nucleotide sequences of the DNA fragments
The DNA fragments from two samples considered positive (canine 1 and canine 3, Table 1), were sent to be sequenced in triplicate to the Sequencing Center of Genytec Ltd., obtaining five sequences for each sample. The selection criteria for these samples was based on the ages of the animals, where the six-year-old canine 1 was chosen, which is the age where chronic encephalitis would most frequently occur due to CDV infection, which could be related to greater nucleotide variations according to [7]. Likewise, dog 3, eleven months old, was chosen, which is the age in which more animals are usually infected by CDV (less than one year old). The sequences were then aligned using the Clustal Ω program, obtaining a consensus sequence for each sample used: from canine 1 the sequence CDV/CMC1 was obtained and from canine 3 the sequence CDV/CMC2 (Annex 1) was obtained. Next, the consensus sequences were entered into the BLAST program, to identify the origin of the DNA fragments obtained in the RT-PCR, which revealed that the sequence CDV/CMC1 presented a percentage of nucleotide identity of 95% and the CDV/CMC2 sequence of 94%, with respect to the first one hundred results corresponding to CDV, and that in the case of both sequences, these corresponded to CDV (Annex 2).

Discussion
The CD is a disease of worldwide distribution, viral and highly contagious, affecting a wide range of terrestrial carnivores and some marine mammals, which have had a progressive increase in recent years, infecting even animals with their vaccination plans at day. It is due to this increase in hosts, that a true diagnosis of the disease caused by the CDV is crucial, to limit the spread of it to other animals and to give an opportune treatment to the sick.
Although the diagnosis of CD is based mainly on the clinical signs of the patient, in certain occasions, it is difficult to reach a final diagnosis in this way, since the main respiratory and digestive signs are common to other diseases that affect the canines and in the case of the more specific clinical signs of DC, such as nervous signs, these most commonly occur in the later stages of the infection, after it has become more widespread. Also, the different clinical presentations of DC vary from one animal to another, making the final diagnosis more complex [1,10]. Although there are several techniques to help the diagnosis of CD, such as virus isolation, immunofluorescence and ELISA, most of these methods are laborious, time-consuming and may give false results, which is why they are convenient for the definitive ante-mortem diagnosis of the disease. For this reason, a sensitive, specific and rapid method is necessary to detect a small amount of the virus early in the infection, which, thanks to the development of molecular biological techniques such as RT-PCR, may be possible, where a good white sequence is required.
conserved between different strains of the CDV, to avoid mismatches of the splitters and the subsequent failure to amplify it.
In this work a diagnostic method for CDV was implemented through RT-PCR, based on the detection of the nucleocapsid protein gene, a highly conserved region of the CDV genome, which makes this an excellent target gene for the molecular detection of this virus.
In fact, several authors have revealed the suitability of molecular biology methods based on the nucleocapsid protein gene for the detection of CDV [1,[6][7][8]12,15]. Additionally, the primers used in this study are in the most conserved region of the nucleocapsid protein gene, the central zone, which would increase the sensitivity of the method [7,8].
Although two Faculty reports based on the CDV RT-PCR have been carried out in our Faculty [13,14], these were based on the hemagglutinin gene, which has the highest antigenic and genetic variation among the CDV genes, presenting about 10% variability between different strains of CDV, which is not the most suitable gene for the diagnosis of the virus [8,11].
In the present study, the previous observations of the RT-PCR utility of the CDV nucleocapsid protein gene were confirmed, with the virus RNA found in about 91% (10/11) of the field samples suspected of DC (Figure 2). This result suggests that the protocol used in this title memory, from the obtaining of the phlogistic layer to the visualization of the amplified products, could be highly sensitive. At the same time, this method would be more sensitive than the protocols implemented in previous studies, since [13] only found viral RNA in 7% (3/42) of the samples analyzed that came from dogs suspected of DC and in the case of [13], he found the RNA of the virus in 83% (5/6) of the samples analyzed, which had previously been confirmed as positive by another RT-PCR, however in twenty field samples of dogs suspected of DC, did not find viral RNA [13]. However, to corroborate the sensitivity of the method implemented, additional studies are required. The specificity of the method was corroborated with the amplification of the expected fragment in 100% (10/10) of the positive controls and in the nonamplification of the fragment expected in the negative controls ( Figure 1). As for the negative controls, it is important to note that one of the animals used for these controls, had its vaccination schedule up to date and had been vaccinated for the last time ten months ago, which supports the preliminary observations that a previous vaccination does not causes false-positive results [7].
On the other hand, CDV can remain active indefinitely at -70°C, -192°C (liquid nitrogen) or lyophilized and only remains active for about a month at -10°C [16], without However, in the positive controls corresponding to CDV RNA (coming from national isolates), it is important to note that this RNA had been stored for more than a year at -20 °C, which is not the ideal temperature to maintain integrity and not degradation, but despite this, the stability of the virus RNA was not affected.
Regarding the type of sample chosen for the field samples, blood was chosen with anticoagulant (ethylenediaminetetraacetic acid "EDTA"), because it is one of the most suitable as a substrate for RT-PCR, since it is an easy sample of obtain, maintain cell morphology for a longer time preventing the viral RNA from being degraded by endogenous RNAses when leaving the cell and also the amount of EDTA present in the tubes to obtain blood, would not inhibit the result of the RT-PCR [17]. However, similar results have also been seen with serum and urine samples, further increasing the sensitivity of detection if more than one is used for the diagnosis of the disease [1,7]. The field sample suspected of DC that was negative after being analyzed by the proposed method (canine 11, Table 1), could have been due to the absence of viral RNA, since either because the virus was not in blood, it could be in other epithelia of the body or because the sample was poorly preserved or poorly processed.
Although DC disease is less frequent in developed countries due to immunization, in recent years, the virus that produces it has emerged as a significant pathogen of vaccinated animal populations, and in the case of this study, the presence of some animals suffering from DC, which were at least once vaccinated against the virus, which raises the hypothesis of failure in the immunization of the vaccine, which could be due to incorrect vaccination plans or genetic variations of the virus [6], however, in order to respond to these presumptions it is necessary to carry out other studies on the subject.

Annexx 1:
After obtaining the sequences of two of the samples that amplified a fragment of approximately 290 bp and defining the consensus sequence for each of them (Annex 1), the BLAST program was used, where it was obtained that both sequences presented a percentage of nucleotide identity greater than 94% with respect to the first one hundred results corresponding to CDV and that in both cases the amplified fragments belonged to this virus, corroborating the specificity of the method implemented (Annex 2).