Rapid Identification of ASFV, CSFV and FMDV from Mongolian Outbreaks with MinION Short Amplicon Sequencing.

African swine fever virus (ASFV), classical swine fever virus (CSFV), and foot-and-mouth disease virus (FMDV) cause important transboundary animal diseases (TADs) that have a significant economic impact. The rapid and unequivocal identification of these pathogens and distinction from other animal diseases based on clinical symptoms in the field is difficult. Nevertheless, early pathogen detection is critical in limiting their spread and impact as is the availability of a reliable, rapid, and cost-effective diagnostic test. The purpose of this study was to evaluate the feasibility to identify ASFV, CSFV, and FMDV in field samples using next generation sequencing of short PCR products as a point-of-care diagnostic. We isolated nucleic acids from tissue samples of animals in Mongolia that were infected with ASFV (2019), CSFV (2015), or FMDV (2018), and performed conventional (RT-) PCR using primers recommended by the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH). The (RT-) PCR products were then sequenced in Mongolia using the MinION nanopore portable sequencer. The resulting sequencing reads successfully identified the respective pathogens that exhibited 91-100% nucleic acid similarity to the reference strains. Phylogenetic analyses suggest that the Mongolian virus isolates are closely related to other isolates circulating in the same geographic region. Based on our results, sequencing short fragments derived by conventional (RT-) PCR is a reliable approach for rapid point-of-care diagnostics for ASFV, CSFV, and FMDV even in low-resource countries.

Experimental Infection of Domestic Pigs with African Swine Fever Virus Isolated in 2019 in Mongolia.

African swine fever (ASF) is an infectious viral disease caused by African swine fever virus (ASFV), that causes high mortality in domestic swine and wild boar (Sus scrofa). Currently, outbreaks are mitigated through strict quarantine measures and the culling of affected herds, resulting in massive economic losses to the global pork industry. In 2019, an ASFV outbreak was reported in Mongolia, describing a rapidly progressing clinical disease and gross lesions consistent with the acute form of ASF; the virus was identified as a genotype II virus. Due to the limited information on clinical disease and viral dynamics within hosts available from field observations of the Mongolian isolates, we conducted the present study to further evaluate the progression of clinical disease, virulence, and pathology of an ASFV Mongolia/2019 field isolate (ASFV-MNG19), by experimental infection of domestic pigs. Intramuscular inoculation of domestic pigs with ASFV-MNG19 resulted in clinical signs and viremia at 3 days post challenge (DPC). Clinical disease rapidly progressed, resulting in the humane euthanasia of all pigs by 7 DPC. ASFV-MNG19 infected pigs had viremic titers of 108 TCID50/mL by 5 DPC and shed virus in oral secretions late in disease, as determined from oropharyngeal swabs. Whole-genome sequencing confirmed that the ASFV-MNG19 strain used in this study was a genotype II strain highly similar to other regional strains. In conclusion, we demonstrate that ASFV-MNG19 is a virulent genotype II ASFV strain that causes acute ASF in domestic swine.

Development of an Indirect ELISA for the Detection of SARS-CoV-2 Antibodies in Cats.

Companion animals are susceptible to a variety of coronaviruses, and recent studies show that felines are highly susceptible to SARS-CoV-2 infection. RT-PCR diagnostic is currently the method of choice to detect the presence of SARS-CoV-2-specific viral nucleic acids in animal samples during an active infection; however, serological assays are critical to determine whether animals were exposed to the virus and to determine the seroprevalence of SARS-CoV-2-specific antibodies in a defined population. In this study, we utilized recombinant nucleocapsid (N) protein and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 expressed in E. coli (N) and mammalian cells (N, RBD) to develop indirect ELISA (iELISA) tests using well-characterized SARS-CoV-2-positive and -negative cat serum panels from previous experimental cat challenge studies. The optimal conditions for the iELISA tests were established based on checkerboard dilutions of antigens and antibodies. The diagnostic sensitivity for the detection of feline antibodies specific for the N or RBD proteins of the iELISA tests was between 93.3 and 97.8%, respectively, and the diagnostic specificity 95.5%. The iELISAs developed here can be used for high-throughput screening of cat sera for both antigens. The presence of SARS-CoV-2-specific antibodies in a BSL-2 biocontainment environment, unlike virus neutralization tests with live virus which have to be performed in BSL-3 laboratories.

Middle East Respiratory Syndrome-Coronavirus Seropositive Bactrian Camels, Mongolia.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic disease that was first identified in humans in 2012 in Saudi Arabia. MERS-CoV causes acute and severe respiratory disease in humans. The mortality rate of MERS in humans is ∼35% and >800 deaths have been reported globally as of August 2020. Dromedary camels are a natural host of the virus and the source of zoonotic human infection. In experimental studies, Bactrian camels are susceptible to MERS-CoV infection similar to dromedary camels; however, neither the virus, viral RNA, nor virus-specific antibodies were detected in Bactrian camel field samples so far. The aim of our study was to survey Mongolian camels for MERS-CoV-specific antibodies. A total of 180 camel sera, collected in 2016 and 2017, were involved in this survey: 17 of 180 sera were seropositive with an initial enzyme-linked immunosorbent assay (ELISA) test performed at the State Central Veterinary Laboratory in Mongolia. These 17 positive sera plus 53 additional negative sera were sent to the Rocky Mountain Laboratories, NIAID/NIH, and tested for the presence of antibodies with a similar ELISA, an indirect immunofluorescence assay (IFA), and a virus neutralization test (VNT). In these additional tests, a total of 21 of 70 sera were positive with ELISA and 10 sera were positive with IFA; however, none was positive in the VNT. Based on these results, we hypothesize that the ELISA/IFA-positive antibodies are (1) non-neutralizing antibodies or (2) directed against a MERS-CoV-like virus circulating in Bactrian camels in Mongolia.

Equine influenza A(H3N8) virus isolated from Bactrian camel, Mongolia.

Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission.

Isolation and characterization of H3N8 equine influenza A virus associated with the 2011 epizootic in Mongolia.

BACKGROUND: Equine influenza virus (EIV) epizootics affect 2.1 million Mongolian horses approximately every 10 years and critically impact economy and nomadic livelihood of Mongolia. OBJECTIVES: An active surveillance program was established in 2011 to monitor influenza viruses circulating among Mongolian horses. METHODS: Nasal swabs were collected from horses in free-ranging horse herds in Töv, Khentii, and Dundgovi aimags (provinces) from January to September 2011. Real-time reversetranscriptase-polymerase chain reaction (rRT-PCR) was used to determine the presence of influenza A virus. Influenza A-positive specimens were cultured to amplify virus; viral RNA was extracted, and gene segments were amplified and sequenced by Sanger sequencing. RESULTS: A total of 745 horses were swabbed; most horses were without clinical signs of illness. In July 2011, reports of influenza-like illnesses emerged among horses in Mongolia's capital, and subsequently, surveillance efforts were adjusted to swab horses associated with the epizootic. Thirty-four specimens of rRT-PCR influenza-positive virus were collected in May, June, August, and September. Three specimens yielded detectable virus. Gene sequence studies suggested that all three isolates were identical H3N8 viruses. Phylogenetic analyses indicated the strain was very similar to other H3N8 EIVs circulating in central Asia between 2007 and 2008. CONCLUSIONS: As large Mongolian equine herds often seem to suffer from EIV epizootics, it seems prudent to continue such routine equine influenza surveillance. Doing so will provide an early warning system, should novel viruses emerge, help in assessing if EIV is crossing over to infect humans and provide data to assess the likely effectiveness of current EIV vaccines.