Development and evaluation of a blocking ELISA for serological diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) is an important viral disease characterized by persistent infection in equids worldwide. Most EIA cases are life-long virus carriers with low antibody reactions and without the appearance of clinical symptoms. A serological test with high sensitivity and specificity is required to detect inapparent infection. In this study, a B-cell common epitope-based blocking ELISA (bELISA) was developed using a monoclonal antibody together with the EIAV p26 protein labelled with HRP. The test has been evaluated against the standard and with field serum samples globally. This bELISA test can be completed within 75 min, and the sensitivity is higher than those of either the AGID or one commercial cELISA kit. This bELISA assay was 8-16 times more analytically sensitive than AGID, and 2 to 4 times more analytically sensitive than one cELISA kit by testing three sera from the USA, Argentina, and China, respectively. The 353 serum samples from Argentina were tested, in comparison with AGID, the diagnostic sensitivity and specificity of our bELISA assay were 100% (154/154) and 97.0% (193/199), respectively, and the accuracy of the bELISA test was 98.3%. The bELISA test developed in this study is a rapid, sensitive, specific method for the detection of EIAV infection, and could be a promising candidate for use in the monitoring of the EIA epidemic worldwide. KEY POINTS: • A universal epitope-based blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies to EIAV. • The bELISA assay can be used to test EIAV serum samples from different regions of the world including North America, South America, Europe, and Asia. • The bELISA assay was evaluated in three different international labs and showed a better performance than other commercial kits.

First molecular characterization of poxviruses in cattle, sheep, and goats in Botswana.

BACKGROUND: Poxviruses within the Capripoxvirus, Orthopoxvirus, and Parapoxvirus genera can infect livestock, with the two former having zoonotic importance. In addition, they induce similar clinical symptoms in common host species, creating a challenge for diagnosis. Although endemic in the country, poxvirus infections of small ruminants and cattle have received little attention in Botswana, with no prior use of molecular tools to diagnose and characterize the pathogens. METHODS: A high-resolution melting (HRM) assay was used to detect and differentiate poxviruses in skin biopsy and skin scab samples from four cattle, one sheep, and one goat. Molecular characterization of capripoxviruses and parapoxviruses was undertaken by sequence analysis of RPO30 and GPCR genes. RESULTS: The HRM assay revealed lumpy skin disease virus (LSDV) in three cattle samples, pseudocowpox virus (PCPV) in one cattle sample, and orf virus (ORFV) in one goat and one sheep sample. The phylogenetic analyses, based on the RPO30 and GPCR multiple sequence alignments showed that the LSDV sequences of Botswana were similar to common LSDV field isolates encountered in Africa, Asia, and Europe. The Botswana PCPV presented unique features and clustered between camel and cattle PCPV isolates. The Botswana ORFV sequence isolated from goat differed from the ORFV sequence isolated from sheep. CONCLUSIONS: This study is the first report on the genetic characterization of poxvirus diseases circulating in cattle, goats, and sheep in Botswana. It shows the importance of molecular methods to differentially diagnose poxvirus diseases of ruminants.

Comparison of eleven in vitro diagnostic assays for the detection of SARS-CoV-2 RNA.

Transmission mitigation of SARS-CoV-2 requires the availability of accurate and sensitive detection methods. There are several commercial ad hoc molecular diagnostic kits currently on the market, many of which have been evaluated by different groups. However, in low resource settings the availability and cost of these commercial kits can be a limiting factor for many diagnostic laboratories. In such cases alternatives need to be identified. With this in mind, eight commercial reverse transcription quantitative real-time PCR (RT-qPCR) master mixes from Applied Biosystems (Thermo Fisher Scientific), Bio-Rad, Biotech Rabbit, Promega, Qiagen, QuantaBio, Invitrogen (Thermo Fisher Scientific) and Takara using the same commercial primer and probe mix [LightMix® Modular SARS and Wuhan CoV E-gene mix (TIB MolBiol, Germany)] were evaluated. Three ad hoc molecular diagnostic kits [GeneFinder™ COVID-19 Plus RealAmp kit (Osang Healthcare); genesig® Real-Time PCR Coronavirus COVID-19 (Primerdesign); and ViroReal® Kit SARS-CoV-2 & SARS-CoV (Ingenetix)] were also included in the study. The limit of detection was calculated for each assay using serial dilutions of a defined clinical sample. The performances of the assays were compared using a panel of 178 clinical samples and their analytical specificity assessed against a panel of human betacoronaviruses. Inter assay agreement was assessed using statistical tests (Bland-Altman, Fleiss-Kappa and Cohen's Kappa) and was shown to be excellent to good in all cases. We conclude that all of the assays evaluated in this study can be used for the routine detection of SARS-CoV-2 and that the RT-qPCR master mixes are a valid alternative to ad hoc molecular diagnostic kits.

African swine fever in North Sumatra and West Java provinces in 2019 and 2020, Indonesia.

African swine fever (ASF) is a highly lethal and contagious viral haemorrhagic disease of domestic and wild pigs, caused by the ASF virus (ASFV). After entering China in 2018, the disease has continued to spread through Asia. In September 2019, a team from the Indonesian Research Center for Veterinary Science, Bogor, investigated outbreaks in backyard pigs in the Dairi and Humbang Hasundutan districts of North Sumatra province. In January 2020, three pigs purchased from a pig seller in Bogor District, West Java province were also tested. Real-time PCR results confirmed ASFV DNA in sixteen out of twenty-nine samples, with nine positive samples from North Sumatra and seven from West Java. Four partial or full-length genes (i.e. p72, p54, pB602L and CD2v) and a 356-bp fragment between the I73R and I329L genes were sequenced from representative samples. Phylogenetic analysis established that the ASFV in the samples from both North Sumatra and West Java were identical, indicating a common source of infection, and that they belonged to the p72 genotype II and serogroup 8. The sequences from the Indonesian ASFVs were also identical to other genotype II ASFV from domestic pigs in Vietnam, China and Russia.

Molecular characterization of rabies viruses from two western provinces of the Democratic Republic of the Congo (2008-2017).

Although rabies is enzootic in the Democratic Republic of the Congo, there is very little molecular epidemiological information about the viruses circulating in animals. In this study, a fragment of the rabies virus (RABV) nucleoprotein gene was amplified and sequenced from 21 animal brain samples collected in two western provinces of the country between 2008 and 2017. The samples tested were from cat (n = 1), dog (n = 17), goat (n = 2), and sheep (n = 1). Phylogenetic analysis revealed that the sequences generated were highly similar to each other and belonged to lineage Africa 1b clustering with a single sample identified in a canine in the Republic of Congo in 2014. This is the first molecular epidemiological study of RABV in the DRC and the data generated will assist authorities in the development of effective control strategies for rabies in the country.