Diagnostic tests for caprine arthritis-encephalitis virus ( CAEV ) infection

Diagnostic tests for caprine arthritis-encephalitis virus (CAEV) infection Po-An Tu1, 2, Jen-Wen Shiau1, Fang-Yu Lai2, Shen-Shyuan Yang3 and Pei-Hwa Wang2* 1Hsinchu Branch, Livestock Research Institute, Council of Agriculture, Executive Yuan, No. 207-5, Bi-tou-mian, Wu-hoo village, Si-hoo Township, Miao-li County 36848, Taiwan 2Department of Animal Science and Technology, National Taiwan University, No. 50, Ln. 155, Sec. 3, Keelung Rd., Taipei City 10617, Taiwan. 3Hualein Animal Propagation Station, Livestock Research Institute, Council of Agriculture, No. 38, Chiang Road, Sec. 6, Chiang, Hualien 97362, Taiwan


Introduction: What is CAEV?
Caprine arthritis-encephalitis virus (CAEV) is one of the small ruminant lentiviruses (SRLV), viruses that belong to the genus Lentivirus of the family Retrovirdae 1 .The virus is able to infect goats and occasionally sheep and other related ruminants 2 .Epidemiological evidence indicates that the major transmission routes of the virus are through the ingestion of colostrum in milk from virus-infected adult goats by kids 3 , iatrogenic transmission, and lateral transmission through long-term close contact with infected goats 4 .All breeds and ages are susceptible to the virus, and once an infection occurs, it persists throughout the carrier's lifetime.Although most infected goats remain asymptomatic, the carriers continuously shed the virus into their environments, leading to more infections of naive goats.The infection of host cells involved in the immune system carries the virus throughout the body and into multiple organs, causing chronic inflammatory diseases.The disease manifestations in kid are normally CNS disease such as acute interstitial pneumonia, or leukoencephalomyelitis in kids 5 .Among adult animals, meanwhile, a CAEV infection may cause chronic polyarthritis and interstitial mastitis after prolonged virus incubation 6 .CAEV is mainly monocyte-tropic and macrophagetropic, while the expression of the viral genome depends on the maturation state of the cells.The viral transcripts are only produced when the cells mature into macrophages 7 .The international trade and movement of live goats and their germplasm play a major role in CAEV dissemination among large geographical regions 8 , while severe economic losses caused by CAEV infection are observed in particular in the context of intense goat husbandry, with decreases of about 10%-15% of milk yield in infected does being reported 8 .Relatedly, the milk contents produced by CAEV-infected does have been found to be significantly different from the milk contents of non-infected does 9 .In addition, there is a potential risk of viral dissemination through CAEV-infected semen 10 .Besides affecting production efficiency, the sanitary and economic impacts of CAEV infection are associated with interference in the international movement of goats and their germplasm due to sanitary restrictions imposed by countries that have regular control programs.

Intermittent seroconversion of CAEV antibodies
Goats produce anti-CAEV antibodies after CAEV infection.Seroconversion may then occur weeks to months after the time of infection.It is generally thought that following seroconversion, the level of anti-CAEV antibodies rises to a peak then declining to a lower but stable level 11 .However, some studies have reported some degree of intermittency in antibody levels, which could be the cause for false-negative results in serological tests 12,13 .In other words, some animals may present intermittent responses in serological tests, but the reasons for such fluctuations between positivity and negativity are not well understood 14 .Moreover, a loss of antibodies may also occur even after clinical signs appear 15 .The extent of this phenomenon is also variable, with some studies reporting that about 1/10 or 2/10 animals showed such fluctuations 13 .In any case, such fluctuations clearly constitute a confounding factor in the serological diagnosis of CAEV infection.

Sequence-based phylogeny of CAEV
Sequence-based phylogenetic analyses have demonstrated that CAEV is closely related to the maedivisna virus (MVV), a lentivirus often found in sheep.The CAEV and MVV share many features, and they are both considered to be SRLV.Based on their respective sequences of gag and pol genes, SRLV are divided into five phylogenetic groups (A to E), where group B includes the prototypes of goat CAEV subtypes 16 .The heterogeneity of viral strains is directly related to transcription errors in viral RNA caused by reverse transcriptase 17 .The genetic heterogeneity of regional virus strains should be taken into account when designing CAEV infection diagnosis and control strategies.

Diagnostic test
Various laboratory methods for the diagnosis of CAEV are available.These methods can be categorized as either serological or molecular techniques.The agar gel immunodiffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA) testing are the two types of serological monitoring, while molecular assays include polymerase chain reaction (PCR) and isothermal amplification methods for the detection of CAEV proviral DNA.The Western Blot (WB) is less used for screening but is seen as "gold standard" test for diagnosis.

AGID
One of the tests most commonly used to diagnose CAE is the AGID tests recommended by the World Animal Health Organization (the OIE recommends both AGID and ELISA) (OIE, 2008), which is based on CAEV serology.The precipitation line in an AGID test is due to multiple interactions between antibodies present in the serum and viral epitopes derived from the cell culture 18 .The antigenantibody precipitation line can be seen within 24 h, but the results should be read at 48-72 h in order to be sure of their stability 19 .The simplicity, specificity, and sensitivity of the test are considered suitable for initial use in control and screening programs.False-negative results may occur in an AGID test due to delayed seroconversion, slower antibody production, or as a result of the antibodies being at an undetectable level at an early stage of infection.Thus, the AGID test appears to have lower sensitivity compared to ELISA 20 .Furthermore, milk samples cannot be used for an AGID test for CAEV infection.

ELISA
The ELISA test is another serological technique that has been recommended for regulatory purposes by the OIE since 2008 21 .Broadly, these methods can be categorized into assays that use whole virus, recombinant proteins (or synthetic peptides) as antigens, or competitive ELISAs based on the use of anti-viral monoclonal antibodies.Whole-virus MVV ELISA exhibited a sensitivity of 98.6% and a specificity of 99.3% relative to 678 sheep reference sera determined by Western Blot (WB) and recombinant gag ELISA 22 .Indirect ELISAs based on recombinant or peptide antigens including recombinant p55 gag 23 , p25, p16, p14 core proteins 24 , gp46 transmembrane protein 25 , or purified gp135 envelope protein 14 have been described.Synthetic peptides derived from p25 or TM have also been used.The results suggest that whole virus ELISAs tend to be more sensitive than single recombinant ELISAs.Inclusion of both a core antigen and an envelope antigen inclusion in the assay are possible to reach equivalent sensitivities and specificities to that of whole virus ELISAs.For competitive ELISA, Herrmann et al. (2003) 26 described an assay based on CAEV-63 captured by one monoclonal antibody and measuring the displacement of another monoclonal antibody by test serum samples.The sensitivity and specificity of the ELISA relative to radio immunoprecipitation was determined to be 100% and 96.4%, respectively, relative to RIPA.
In most countries, the routine laboratory diagnosis of CAEV infection is based on ELISA testing 27 .Such testing uses recombinant envelope glycoprotein subunits as antigens, and ELISAs have proven to be more sensitive than AGID 25 .Furthermore, serological analysis by ELISA can use samples other than serum, such as milk or milk whey samples 28 .The use of milk samples is more advantageous since collection of milk material is considered non-invasive.This approach also reduces exam costs and facilitates sample collection.The serum generally requires a dilution from 1:10 to 1:100 when performing ELISA tests to minimize the background signal.However, such dilution increases the number of false-negative results, especially in the case of a weaklypositive sample serum 18 .Also, as with other serological tests for CAEV infection, delayed seroconversion or intermittency in antibody levels may cause false-negative and ambiguous results in ELISA tests 13,14 .The phylogenetic variability of SRLV affects the results of ELISA.It was shown that the use of antigens derived from phylogenetically different viruses generates false negative results 29 .Thus, in a diagnostic setting the combination of at least these two subtype-specific peptides is necessary to cover a wide range of infections 30 .

PCR
Infections caused by CAEV persist due to the integration of proviral DNA into the cellular genome followed by replication in cells of the immune system.Thus, molecular biology techniques such as PCR can be used to detect the presence of the CAEV provirus.This capability has facilitated the diagnostic procedure.The different PCRbased diagnostic techniques vary according to their targets and include, for example, reverse transcription PCR for the detection of viral RNA 31 , as well as double-nested PCR 32 , semi-nested PCR 33 , nested PCR 15 , real-time PCR 4 , and TaqMan-based qPCR 34 for the detection of viral DNA.The real-time PCR diagnostic methods have been found to provide earlier positive detection results (∼15 days postinfection) than the traditional serological AGID and ELISA methods (∼40-60 days post-infection) 4 , indicating the higher sensitivity of the former methods.The two main difficulties in developing suitable PCR tests for CAEV are strain sequence variation 16 and low virus loads in vivo.Since only around 1×10 6 leukocytes are virus-infected 35 , PCR assays may produce false negative results simply because a virus load is too low to detect.Also, sophisticated thermal cyclers are required for all these PCR-based methods, with some methods requiring identification by agarose electrophoresis or by the capillary sequencing method 36 , further restricting the application of the methods for onsite diagnosis on farms.

Isothermal amplification methods
Several isothermal amplification methods have been developed into useful technology in the past decade.Huang et al. (2012) 37 and Balbin et al. (2014) 38 used LAMP for the rapid detection of CAEV proviral DNA.This method utilized a Bst DNA polymerase with strand displacement activity along with two outer primers (F3 and B3) and two inner primers (FIP and BIP) that recognize six specific regions within the target CAEV sequence; additional Loop-F and Loop-R primers were employed in the reaction to accelerate the LAMP amplification.However, the high variability of retroviruses (and RNA viruses) has its basis in the lack of elaborate proofreading and repair mechanisms during some (or all) of the steps in the replication of the given viral genome 11 .Therefore, the highly mutagenic property of proviral sequences in different CAEV strains makes designing multiple primers for use with the LAMP detection method difficult without specific software.Tu et al. (2017) 39 developed the recombinase polymerase amplification lateral flow dipstick (RPA-LFD) method for the field diagnosis of CAEV infection.Under the optimal incubation conditions, specifically, 30 min at 37 • C for RPA followed by 5 min at room temperature for LFD, this assay was found to be sensitive to a lower limit of 80 pg of total DNA and 10 copies of plasmid DNA.The RPA method applied similar primer design principles as traditional PCRbased methods, and the RPA-LFD method used the LF probe to allow for specific amplification only.The single-strand binding protein GP32 contained in the RPA mixture can enhance the nucleic acid detection limit 40 .The isothermal feature also makes these methods more applicable for onsite utilization at farms for both eradication programs and epidemiological studies.

Western blot (WB)
Western blot is often used as ''gold standard'' tests for SRLV diagnosis.However, the complex and time-consuming procedures makes WB less suitable for regular screening.The specificity is represented by visual band confirmation of the correct molecular weight.For the sensitivity of WB, researchers have shown the WB is either equally sensitive or more sensitive than ELISA [41][42][43] .

Conclusions
CAE is a chronic disease which causes significant losses in goat breeding.Due to delayed seroconversion or intermittent antibodies and the genetic heterogeneity of regional virus strains, the actual state of animal infection with CAEV is difficult for both serological or molecular diagnostic tests to determine with certainty.There are also other factors which affect the effectiveness of the selected technique, including the availability of commercial reagents, personnel training, the antigen/primer used, and the cost of the technique.It is clear that ELISA is generally more sensitive than the AGID test for the detection of CAEV antibodies, while molecular techniques like PCR and isothermal amplification methods appear to be more suitable to detect infected animals prior to seroconversion.Currently, there is still no gold standard method for CAEV diagnosis.However, by combining the advantages of both serological and molecular techniques, it might be possible to create the optimal detection method for CAEV infection.