Armando Damiani

A single-nucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease.

Epidemiological studies have shown that a single-nucleotide polymorphism in the equid herpesvirus type 1 DNA polymerase gene is associated with outbreaks of highly lethal neurological disease in horses. Reverse genetics experiments further demonstrated that a G(2254) A(2254) nucleotide mutation introduced in neurovirulent strain Ab4, which resulted in an asparagine for aspartic acid substitution (D(752) N(752)), rendered the virus nonneurovirulent in the equine. Here, we report that the nonneurovirulent strain equid herpesvirus type 1 strain NY03 caused lethal neurological disease in horses after mutation of A(2254) G(2254) (N(752) D(752)), thereby providing final proof that the D(752) allele in the viral DNA polymerase is necessary and sufficient for expression of the lethal neurovirulent phenotype in the natural host. Although virus shedding was comparable between the N(752) and D(752) variants, infection with the latter was accompanied by efficient establishment of prolonged cell-associated viremia in peripheral blood mononuclear cells and neurological disease in 2 of 6 animals.

A Potentially Fatal Mix of Herpes in Zoos

Pathogens often have a limited host range, but some can opportunistically jump to new species. Anthropogenic activities that mix reservoir species with novel, hence susceptible, species can provide opportunities for pathogens to spread beyond their normal host range. Furthermore, rapid evolution can produce new pathogens by mechanisms such as genetic recombination. Zoos unintentionally provide pathogens with a high diversity of species from different continents and habitats assembled within a confined space. Institutions alert to the problem of pathogen spread to unexpected hosts can monitor the emergence of pathogens and take preventative measures. However, asymptomatic infections can result in the causative pathogens remaining undetected in their reservoir host. Furthermore, pathogen spread to unexpected hosts may remain undiagnosed if the outcome of infection is limited, as in the case of compromised fertility, or if more severe outcomes are restricted to less charismatic species that prompt only limited investigation. We illustrate this problem here with a recombinant zebra herpesvirus infecting charismatic species including zoo polar bears over at least four years. The virus may cause fatal encephalitis and infects at least five mammalian orders, apparently without requiring direct contact with infected animals.

Development of a surveillance scheme for equine influenza in the UK and characterisation of viruses isolated in Europe, Dubai and the USA from 2010-2012

Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010-2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic inter-relationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida clade 2, all those from the USA belonged to Florida clade 1. Two subpopulations of clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida clade 1 and clade 2.

Effective Treatment of Respiratory Alphaherpesvirus Infection Using RNA Interference

Background Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNAs), delivered intranasally, could prove a valuable alternative for infection control. siRNAs against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrease EHV-1 infection in a mouse model. Methodology/Principal Fndings siRNA therapy in vitro significantly reduced virus production and plaque size. Viral titers were reduced 80-fold with 37.5 pmol of a single siRNA or with as little as 6.25 pmol of each siRNA when used in combination. siRNA therapy in vivo significantly reduced viral replication and clinical signs. Intranasal treatment did not require a transport vehicle and proved effective when given up to 12 h before or after infection. Conclusions/Significance siRNA treatment has potential for both prevention and early treatment of EHV-1 infections.

Equine Herpesvirus Type 4 UL56 and UL49.5 Proteins Downregulate Cell Surface Major Histocompatibility Complex Class I Expression Independently of Each Other

ABSTRACT Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.

Elimination half-life of intravenously administered equine cardiac troponin I in healthy ponies

REASONS FOR PERFORMING STUDY To date, no information is available on the true biological elimination half-life (T(1/2) ) of cardiac troponin I (cTnI) in the equine species. Such data are required to better evaluate the optimal time to acquire the cTnI sample following acute myocardial injury. OBJECTIVE To determine the T(1/2) of equine cTnI. METHODS Four healthy ponies received i.v. injections of recombinant equine cTnI. Plasma cTnI concentrations were measured with a point-of-care cTnI analyser at multiple time points after injection. Standard pharmacokinetic analysis was performed to establish the T(1/2) of cTnI. RESULTS The average T(1/2) of cTnI was determined to be 0.47 h using a single rate elimination model. CONCLUSION The elimination of recombinant equine cTnI following i.v. administration is very rapid. Establishing the T(1/2 ) of troponin provides critical information in understanding the clinical application of this cardiac biomarker in equine practice.

Zebra-borne equine herpesvirus type 1 (EHV-1) infection in non-African captive mammals.

Equine herpesvirus type 1 (EHV-1) was detected in an Indian rhinoceros (Rhinoceros unicornis), which was euthanized because of severe neurological disease. Encephalitis was suspected and EHV-1 DNA was detected in brain, lung, and spleen tissues. The viral IR6 protein was detected in lung tissues by Western blot analysis. Phylogenetic analyses of EHV-1 sequences amplified from various tissues was nearly identical to one recently described that resulted in both non-fatal and fatal encephalitis in polar bears. This represents transmission of EHV-1 to a species that is not naturally sympatric with the natural host of the virus and broadens the host range to Asian non-equid perissodactyls.

Equine herpesvirus type-1 modulates CCL2, CCL3, CCL5, CXCL9, and CXCL10 chemokine expression.

Equine herpesvirus type 1 (EHV-1) is highly prevalent in horses and causes rhinopneumonitis, abortion, and encephalopathy. Studies on the related human herpes simplex virus and of murine models of EHV-1 suggest that chemokines play important roles in coordinating of innate and adaptive immune responses, and thus effective control of herpesvirus infection and prevention of severe clinical disease. Here, equine peripheral blood mononuclear cells (PBMC) were infected with one of three EHV-1 strains, which differ in pathogenicity (RacL11, NY03=abortogenic, Ab4=neurogenic). Changes in CCL2, CCL3, CCL5, CXCL9 and CXCL10 chemokine gene expression relative to non-infected PBMC were measured by real-time PCR. CXCL9 and CXCL10 gene expression was up-regulated 10h post infection and decreased to the level of non-infected cells after 24h. CCL2 and CCL3 were significantly down-regulated 24h post infection with NY03 and Ab4. CCL5 was up-regulated 24h after infection with RacL11. Ab4 infected PBMC had significantly lower expression of all chemokines except CCL2 24h post infection then RacL11 infected cells. While there was not a significant difference between NY03 and the other strains, there was a trend with each chemokine toward NY03 inducing less expression then RacL11 but more then Ab4. The data suggested that EHV-1 infection of PBMC induced up-regulation of inflammatory chemokines CCL5, CXCL9 and CXCL10, and down-regulation of chemotactic CCL2 and CCL3. The data also implies that different EHV-1 strains have varying effects on all five chemokines, with the nuropathogenic strain, Ab4, having the greatest suppressive potential.

The effect of siRNA treatment on experimental equine herpesvirus type 1 (EHV-1) infection in horses

Abstract Available vaccines fail to induce lasting and protective immunity to equine herpesvirus 1 (EHV-1) associated diseases. RNA interference is a novel approach showing promise for therapeutic use in outbreak situations. This study examined the effect of small interfering RNA (siRNA) on clinical signs as well as the presence of live virus and viral DNA in nasal secretions and peripheral blood mononuclear cells (PBMCs) in horses experimentally infected with EHV-1. siRNA targeting two EHV-1 genes (glycoprotein B and the origin binding protein) was administered 12h before and 12h after intranasal infection with EHV-1. Control horses received siRNA targeting firefly luciferase. A significantly smaller proportion (0/10) of horses receiving siRNA targeting viral genes required euthanasia due to intractable neurologic disease as compared to horses in the control group (3/4; p =0.01). There was no significant difference in the presence of live virus or viral DNA in the nasal secretions or PBMCs between the two groups. Future studies are necessary to define the relative contributions of host and virus factors in the development of the neurological form of the infection and to determine an optimal dosing regimen for metaphylactic or therapeutic use of siRNA for treating EHV-1 infection.

Serological Survey in Dogs and Cats for Influenza A(H1N1)pdm09 in Germany

A serological survey for the detection of antibodies to influenza A(H1N1)pdm09 was carried out in a population of dogs and cats in Germany. A total of 1150 sera collected in 2010 and 2011 were screened using an ELISA targeting anti‐nucleoprotein NP antibodies. Those initially screened positive samples were subsequently tested for antibodies to N1 neuraminidase followed by a virus neutralization test using A/Bayern/74/2009 strain. A prevalence of A(H1N1)pdm09‐specific antibodies of 0.13% and 1.93% was estimated among dogs and cats, respectively. Evidence of exposure to other influenza A virus subtypes was also observed.