Abortion storm of Yili Horses is Associated with Equus Caballus Papillomavirus 2 Variant Infection

Background: Nine different species of Equus caballus papillomavirus (EcPV) and three bovine papillomavirus (BPV) have been reported to infect horses, however, there are so far no describing such infections in China. In January 2021, an abortion storm occurred in Yili horses, as a result of which 50 out of 93 aborted fetus samples were found to be negative for equid herpesvirus (EHV) and equine arteritis virus (EAV).Results: In our pioneer study with Chinese horses, we first found EcPV-2 in the nasal swabs (4/230, 1.7%) of Yili horses, and semen (3/18, 16.7%) of the Thoroughbred horses. This indicated that EcPV can be indeed hosted by horses in China, and that EcPV-2 might be transmitted though breeding. Further detection of EcPVs in the lung tissues of aborted fetus in Yili horses, which were originally negative for equid herpesviruses, established that EcPV-2 was positive in 19 of 50 samples, thereby indicating that EcPV-2 might be a new pathogen causing of abortions. Thereafter, the sequence analyses for L1 genes sequences of 26 China’s EcPV-2 were performed which indicated that EcPV-2, that primarily infected the horses in China, shared 98.3%-99.9% nt identity with the already published sequences for EcPV-2. These observations indicated that EcPV-2 identified in the current study were highly similar variants of the previously identified strains of EcPV-2. Phylogenetic analysis based on L1 genes in GenBank showed that EcPV-2, found in the Chinese horses, was closely related to and clustered together with an already known EcPV-2a lineage. Conclusion: Our study provides the first evidence related to EcPV-2 infection in the Chinese horses, which can serve as a causative agent for Yili horse abortions, and thus can possibly lay the foundation for a systematic and detailed epidemiological study of this infection in the Chinese horses.

Equine Arteritis Virus (EAV) Outbreak in a Show Stallion Population

(1) Background: Equine arteritis virus (EAV) infection causes reproductive losses and systemic vasculitis in susceptible equidae. The intact male becomes the virus’ reservoir upon EAV infection, as it causes a chronic-persistent infection of the accessory sex glands. Infected semen is the main source of virus transmission. (2) Here, we describe acute EAV infection and spread in a stallion population after introduction of new members to the group. (3) Conclusions: acute clinical signs, acute phase detection of antigen via (PCR) nasal swabs or (EDTA) blood, and seroconversion support the idea of transmission via seminal fluids into the respiratory tract(s) of others. This outbreak highlights EAV’s horizontal transmission via the respiratory tract. This route should be considered in a chronic-persistently infected herd, when seronegative animals are added to the group.

Methodology of Selecting the Optimal Receptor to Create an Electrochemical Immunosensor for Equine Arteritis Virus Protein Detection

The study reports a methodology of selecting the optimal receptor to create an electrochemical immunosensor for equine arteritis virus (EAV) protein detection. The detection was based on antigen recognition by antibodies immobilized on gold electrodes. Modification steps were controlled by electrochemical impedance spectroscopy and cyclic voltammetry measurements. In order to obtain the impedance immunosensor with the best parameters, seven different receptors complementary to equine arteritis virus protein were used. In order to make the selection, a rapid screening test was carried out to check the sensor’s response to blank, extremely low and high concentrations of target EAV protein, and negative sample: M protein from Streptococcus equi and glycoprotein G from Equid alphaherpesvirus 1. F6 10G receptor showed the best performance.

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the nonstructural protein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

Virus transmission by ultrasonic scaler and its prevention by antiviral agent

ABSTRACTDuring an ultrasonic scaler (USS) operation, droplets and aerosol are generated that may contribute to the transmission of viruses contained in saliva and gingival crevicular fluid. The purpose of this research was to develop an experimental model for testing the spread of viruses during USS instrumentation and to examining the prevention of spreading by replacing the coolant with an antiviral agent. In a virus transmission tunnel, USS operation with saline coolant and delivery of a viral suspension to the vicinity of USS tip generated droplets and aerosol containing Equine Arteritis Virus (EAV). Evaluation of droplet transmission was evaluated with adherent 48h cell culture monolayer RK13 cell lines in standard 48-well-plates positioned at a distance from 30 to 55 cm. The aerosol was collected by a cyclone aero-sampler flow of 100l/min. Antiviral activity of 0.25% sodium hypochlorite or electrolyzed water (EOW) was tested by suspension test. The two tested antiviral agents’ transmission prevention ability was evaluated by repeating the same experiment as with saline coolant. All experiments were repeated twice. With saline coolant, the cytopathic effect on cells was found in cells up to the distance of 45 cm, with the number of infected wells decreasing with distance. Viral particles were detected in only one AS in a very low concentration (≤4.2 TCID50/ml). In suspension test of 0.25% NaOCl and EOW, the TCID50/ml was below detection limit after 5s. With both antiviral agents, no cytopathic effect was found. However, the cytotoxic effect of 0.25% NaOCl was evident up to the distance of 35 cm. By USS activity, EAV could be transmitted by droplets up to a distance of 45 cm. Both antiviral agents could prevent virus droplet transmission. The transmission of EAV by aerosol yielded inconclusive results.

Anti-GnRH vaccination of stallions shedding equine arteritis virus in their semen: a field study

Stallions are natural reservoirs of equine arteritis virus (EAV) in their semen, representing a potential source of outbreaks. The carrier-state is testosterone-dependent, and clears spontaneously in 4 to 40% stallions. Reduction of testosterone secretion may be obtained with the anti-GnRH vaccine Equity. In this report, 16 naturally infected stallions excreting EAV in their semen were vaccinated twice with the vaccine EquityTM and monitored irregularly under field conditions for EAV viral load in their semen and plasmatic testosterone concentration. The results are indicated in months (M) after the first vaccine injection. Testosterone concentrations decreased from 1.7 to 0.2 ng/mL (P<0.002) after 3M. The EAV viral load decreased from 3.2×109 to 1.1×106 RNA copy/mL of semen (P<0.001) after 5M. One stallion died at 7M for other reason. At M3-10, 12/15 stallions ceased to shed the virus in their semen. At M5-10, 9/15 stallions had plasmatic testosterone concentrations of ≥ 0.5 ng/mL but the 6 others showed a persistently low testosterone concentration (≤0.3 ng/mL). Of the 14 stallions that were expected to recover their reproductive activity at the time of the next breeding season (<M12), 8 were EAV negative and produced foals, and 6 were not usable (4 for reproductive deficiency and 2 for EAV positivity). All the stallions were EAV negative at M22, with one stallion being vaccinated a third time at M15. These results suggest that the anti-GnRH vaccination could help to clear EAV shedding in stallions, without a significant effect on reproductive capacity for most of them, but some present a long lasting reduced testosterone secretion.

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

SummaryCoronaviruses, like SARS-CoV-2, encode a nucleotidyl transferase in the N-terminal NiRAN domain of the non-structural protein (nsp) 12 protein within the RNA dependent RNA polymerase (RdRP) 1-3. Though the substrate targets of the viral nucleotidyl transferase are unknown, NiRAN active sites are highly conserved and essential for viral replication 3. We show, for the first time, the detection and sequence location of GMP-modified amino acids in nidovirus RdRP-associated proteins using heavy isotope-assisted MS and MS/MS peptide sequencing. We identified lys-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of nucleotidylation in vitro that uses a phosphoramide bond to covalently attach with GMP. In SARS-CoV-2 replicase proteins, we demonstrate a unique O-linked GMP attachment on nsp7 ser-1, whose formation required the presence of nsp12. It is clear that additional nucleotidylation sites remain undiscovered, which includes the possibility that nsp12 itself may form a transient GMP adduct in the NiRAN active site that has eluted detection in these initial studies due to instability of the covalent attachment. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.