A reassortant G3P[12] rotavirus A strain associated with severe enteritis in donkeys (Equus asinus).

BACKGROUND: In contrast to horses, the only evidence suggesting gastrointestinal disease in neonatal donkeys is associated with Group A rotaviruses (RVAs) is the detection of viral antigens by ELISA in just 1 of 82 symptomatic donkey foals. No additional, more comprehensive investigations have been conducted, and RVAs if circulating in donkey populations have not been molecularly characterised. OBJECTIVES: To investigate if RVAs are associated with an outbreak of severe enteritis in neonatal donkeys and if associated determine the genotype(s) along with the phylogenetic relationship to RVA strains circulating in horses. STUDY DESIGN: Cross-sectional. METHODS: RT-PCR-based techniques were used for RVA diagnosis and gene amplification. Statistical significance was determined by Chi-square and Fisher's exact two-sided tests. Genotyping was performed by RotaC and phylogenetic analysis by neighbour joining. RESULTS: In 2019, acute enteritis occurred in 119 of 206 donkey foals (≤4 months) at two intensive donkey farms in the Shandong province of China. The highest morbidity (68.1%), mortality (29.5%) and fatality levels (45.5%) occurred in foals in the 30-89 day, 30-59 day and 0-29 day age groups respectively. RVA gene sequences were detected in 107 (89.9%) of the symptomatic individuals while further analysis demonstrated the outbreak was associated with the same G3P[12] RVA strain designated RVA/Donkey-wt/CHN/Don01/2019/G3P[12]. Although the VP4 gene of Don01 exhibited close phylogenetic relationships with equivalent RVA sequences commonly circulating in horses, encoding VP7 was more closely associated with sequences isolated from bats suggesting this new donkey strain arose via an intergenogroup reassortment event. MAIN LIMITATIONS: Actual prevalence not determined because <7% of asymptomatic donkey foals were included in this study. The complete genomic sequence of RVA/Donkey-wt/CHN/Don01/2019/G3P[12] remains to be determined. CONCLUSIONS: Valuable new information about the molecular epidemiology of rotaviruses in different equid species is provided by isolation and molecular characterisation of a novel RVA strain from neonatal donkeys.

Epitope shifting of gp90-specific cellular immune responses in EIAV-infected ponies.

Unlike other lentiviruses, EIAV replication can be controlled in most infected horses leading to an inapparent carrier state free of overt clinical signs which lasts for many years. While the resolution of the initial infection is correlated with the appearance of virus specific cellular immune responses, the precise immune mechanisms responsible for control of the infection are not yet identified. Since the virus undergoes rapid mutation following infection, the immune response must also adapt to meet this challenge. We hypothesize that this adaptation involves peptide-specific recognition shifting from immunodominant variable determinants to conserved immunorecessive determinants following EIAV infection. Forty-four peptides, spanning the entire surface unit protein (gp90) of EIAV, were used to monitor peptide-specific T cell responses in vivo over a six-month period following infection. Peptides were injected intradermally and punch biopsies were collected for real-time PCR analysis to monitor the cellular peptide-specific immune responses in vivo. Similar to the CMI response to HIV infection, peptide-specific T cell recognition patterns changed over time. Early post infection (1 month), immune responses were directed to the peptides in the carboxyl-terminus variable region. By six months post infection, the peptide recognition spanned the entire gp90 sequence. These results indicate that peptide recognition broadens during EIAV infection.

Comparative analysis of LTR and structural genes in an equine infectious anemia virus strain isolated from a feral horse in Japan.

Although equine infectious anemia virus (EIAV) poses a major threat to the equine industry worldwide, the molecular epidemiology of this virus is poorly understood. Recently, an EIAV strain (EIAVMiyazaki2011-A) representing a new monophyletic group was discovered in feral horses in southern Japan. In the present study, the EIAVMiyazaki2011-A proviral genome is compared with evolutionarily divergent EIAV isolates to investigate conservation of functional elements or motifs within the long terminal repeats (LTRs) and structural genes. This analysis represents a significant step forward in increasing understanding of the molecular conservation and variation between geographically distinct strains of this equine lentivirus.

Is a diagnostic system based exclusively on agar gel immunodiffusion adequate for controlling the spread of equine infectious anaemia?

To improve the efficiency of the National equine infectious anaemia (EIA) surveillance program in Italy, a three-tiered diagnostic system has been adopted. This procedure involves initial screening by ELISA (Tier 1) with test-positive samples confirmed by the agar gel immunodiffusion test (AGIDT) (Tier 2) and, in the case of ELISA positive/AGIDT negative results, final determination by immunoblot (IB) (Tier 3). During this evaluation, 74,880 samples, principally collected from two Regions of Central Italy (Latium and Abruzzo) were examined, with 44 identified as negative in AGIDT but positive in both ELISA and IB. As the majority of these reactions occurred in mules, an observational study was conducted in this hybrid equid species to investigate if there is a correlation between plasma-associated viral loads and serological reactivity, to test the hypothesis that false-negative or very weak positive AGIDT results are associated with elite control of EIA virus (EIAV) replication accompanied by reduced transmission risks. The study animals consisted of 5 mules with positive AGIDT readings, along with another 5 giving negative or very weak positive results in this test. All mules were seropositive in Elisa and IB. Samples were collected routinely during an initial 56-day observation period, prior to dexamethasone treatment lasting 10 days, to determine the effect of immune suppression (IS) on clinical, humoral and virological responses. All mules were monitored for a further 28 days from day 0 of IS. None of the animals experienced relevant clinical responses before IS and there were no significant changes in antibody levels in ELISA, IB or AGIDT. However, plasma-associated viral-RNA (vRNA) loads, as determined using TaqMan(®) based RT-PCR, showed unexpectedly high sample to sample variation in all mules, demonstrating host-mediated control of viral replication is not constant over time. Furthermore, there was no apparent correlation between vRNA loads and antibody reactivity in serological tests. Analysis of PCR products established all mules were infected with viruses possessing nucleotide sequence similarity, varying from 77 to 96%, to previously identified European EIAV strains. Following IS, all mules showed increases in plasma-associated vRNA loads, suggesting control of EIAV replication is mediated by immune responses in this hybrid species. However, only three mules showed anamnestic humoral responses to rises in viral loads, as defined by at least a four-fold increase in ELISA titre, while two remained AGIDT-negative. This study demonstrates that viral loads in equids with consistent ELISA/IB positive-AGIDT negative to very weak positive test results (Group N) can be equivalent to those that produce clearly positive results in all three serologic tests (Group P). Therefore, such animals do not pose inherently lower risks for the transmission of EIAV. Consequently, the exclusive use of the AGIDT, as prescribed by the World Organization of Animal Health (OIE) for diagnosis of EIA prior to the international movement of horses, can report as negative some EIAV-infected equids. These results dramatically underscore the necessity of combining the specificity of AGIDT with tests with higher sensitivity, such as the ELISA and the power of the IB to enhance the accuracy of EIA diagnosis.

Identification of a novel equine infectious anemia virus field strain isolated from feral horses in southern Japan.

Although equine infectious anemia (EIA) was described more than 150 years ago, complete genomic sequences have only been obtained from two field strains of EIA virus (EIAV), EIAV(Wyoming) and EIAV(Liaoning). In 2011, EIA was detected within the distinctive feral Misaki horse population that inhabits the Toi-Cape area of southern Japan. Complete proviral sequences comprising a novel field strain were amplified directly from peripheral blood of one of these EIAV-infected horses and characterized by nucleotide sequencing. The complete provirus of Miyazaki2011-A strain is 8208 bp in length with an overall genomic organization typical of EIAV. However, this field isolate possesses just 77.2 and 78.7 % nucleotide sequence identity with the EIAV(Wyoming) and EIAV(Liaoning) strains, respectively, while similarity plot analysis suggested all three strains arose independently. Furthermore, phylogenetic studies using sequences obtained from all EIAV-infected Misaki horses against known viral strains strongly suggests these Japanese isolates comprise a separate monophyletic group.

Development of a nested PCR assay to detect equine infectious anemia proviral DNA from peripheral blood of naturally infected horses.

Equine infectious anemia (EIA) has posed a major challenge and caused significant losses to the equine industry worldwide. PCR detection methods have considerable potential as an adjunct to conventional serological diagnostic techniques. However, most published PCR methods, including that recommended by the OIE, were designed using laboratory-adapted virus strains and do not function with field isolates of EIA virus (EIAV). In the present study, a nested PCR assay for detection of EIAV proviral DNA in peripheral blood cells of naturally infected horses was developed. Primer sets were designed based on conserved 5' regions of the viral genome extending from the LTR to the tat gene. Preliminary studies demonstrated that the method has a detection limit of 10 genomic copies and, when applied to a naturally EIAV-infected feral horse population, shows 100 % correlation with conventional serological diagnostic techniques. This assay provides a powerful new tool in the control of EIAV.

The determination of in vivo envelope-specific cell-mediated immune responses in equine infectious anemia virus-infected ponies.

Distinct from human lentivirus infection, equine infectious anemia virus (EIAV)-infected horses will eventually enter an inapparent carrier state in which virus replication is apparently controlled by adaptive immune responses. Although recrudescence of disease can occur after immune suppression, the actual immune correlate associated with protection has yet to be determined. Therefore, EIAV provides a model for investigating immune-mediated protective mechanisms against lentivirus infection. Here, we have developed a method to monitor EIAV-envelope specific cellular immunity in vivo. An EIA carrier horse with no clinical signs infected 7 years ago and 4 related experimental ponies infected 6 months previously were used in this study. Forty-four 20-mer peptides, representing the entire surface unit protein (gp90) of EIAV, were combined into 14 peptide pools and intradermally injected into the neck of EIAV-infected horses. An identical volume of saline alone was injected into a fifteenth site as a negative control. After 48 h, those sites with palpable infiltrations were measured prior to the collection of 2mm and 4mm punch biopsies. Total RNA was extracted from each 2mm biopsy for determination of CD3 and interferon-γ (IFN-γ) mRNA expression by real-time PCR. The 4mm skin biopsies were formalin-fixed and paraffin-embedded for immunohistochemistry (IHC) staining for CD3, CD20, CD25 and MAC387 (macrophage marker). Peripheral blood mononuclear cells (PBMC) were obtained prior to the injection and tested for in vitro reactivity against the same peptides. Histological examination showed that some of the envelope peptides elicited a lymphocytic cellular infiltration at the injection site, as evidenced by positive staining for CD3. Gp90 peptide-specific increases in CD3 and IFN-γ gene expression were also detected in the injection sites. Furthermore, differences were found between in vivo and in vitro responses to gp90 specific peptides. These results demonstrate a novel method for detecting in vivo cell-mediated immune responses to EIAV-specific peptides that is readily applicable to other host/pathogen systems.

Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus.

The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the "clades" described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.