Mathias Martins

Virological and clinico-pathological features of orf virus infection in experimentally infected rabbits and mice.

Many aspects of the biology of orf virus (ORFV) infection remain poorly understood and attempts to establish animal models have yielded conflicting and non-reproducible results. We herein describe the characterization of ORFV infection and disease in rabbits and mice. A protocol of intradermal inoculation was employed to inoculate 10(8.5)TCID₅₀/mL of ORFV strain IA-82 in the skin of ears, of the back and labial commissures. All inoculated rabbits presented a clinical course characterized by erythema, macules, papules/vesicles or pustules that eventually dried originating scabs. Local signs started around days 3 and 4 post-inoculation (pi) and lasted 3-10 days. Virus was recovered from lesions between days 2 and 14pi. Histological examination of lesions revealed focal proliferative dermatitis with ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies in keratinocytes, histological hallmarks of contagious ecthyma in sheep. A similar, albeit milder clinical course occurred in 5/10 inoculated mice; virus was recovered from lesions from three animals. Inoculated lambs - used as controls - developed severe lesions of contagious ecthyma. VN tests performed at day 28pi failed to detect neutralizing antibodies in all inoculated animals. In contrast, convalescent rabbit sera were positive by ELISA at dilutions from 100 to 400. These results show that rabbits are susceptible to ORFV infection and thus may be used to study selected aspects of ORFV biology.

Isolation and identification of feline calicivirus and feline herpesvirus in Southern Brazil

Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55) of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55) of the animals that tested positively, and co-infection were detected in 9.1% (5/55) of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary.

Immunogenicity of a recombinant parapoxvirus expressing the spike protein of Porcine epidemic diarrhea virus.

The parapoxvirus Orf virus (ORFV), has long been recognized for its immunomodulatory properties in permissive and non-permissive animal species. Here, a new recombinant ORFV expressing the full-length spike (S) protein of Porcine epidemic diarrhea virus (PEDV) was generated and its immunogenicity and protective efficacy were evaluated in pigs. The PEDV S was inserted into the ORFV121 gene locus, an immunomodulatory gene that inhibits activation of the NF-κB signalling pathway and contributes to ORFV virulence in the natural host. The recombinant ORFV-PEDV-S virus efficiently and stably expressed the PEDV S protein in cell culture in vitro. Three intramuscular (IM) immunizations with the recombinant ORFV-PEDV-S in 3-week-old pigs elicited robust serum IgG, IgA and neutralizing antibody responses against PEDV. Additionally, IM immunization with the recombinant ORFV-PEDV-S virus protected pigs from clinical signs of porcine epidemic diarrhoea (PED) and reduced virus shedding in faeces upon challenge infection. These results demonstrate the suitability of ORFV121 gene locus as an insertion site for heterologous gene expression and delivery by ORFV-based viral vectors. Additionally, the results provide evidence of the potential of ORFV as a vaccine delivery vector for enteric viral diseases of swine. This study may have important implications for future development of ORFV-vectored vaccines for swine.

Pathogenesis in lambs and sequence analysis of putative virulence genes of Brazilian orf virus isolates.

The parapoxvirus orf virus (ORFV) is the agent of contagious ecthyma, an ubiquitous mucocutaneous disease of sheep and goats that may present variable clinical presentations. We herein studied the pathogenesis of ORFV infection in lambs and analyzed three putative virulence genes of four Brazilian ORFV isolates. Lambs inoculated in the labial commissures with each ORFV isolate (n=4, viral titer 10(5.6) TCID50/ml) developed classical orf lesions, characterized by a progressive course of erythema/macules, vesicles, pustules and proliferative scabs. Lesions lasted an average of 22.9 days (18-26) and virus shedding was detected for approximately 24.6 days (18-30). Two isolates (SV269/11 and SV820/10) produced more severe, long-lasting lesions resulting in highest clinical scores. Lambs inoculated with isolate SV581/11 developed lesions markedly milder (lower clinical scores [p<0.05]) and more limited than the other groups. Virus shedding by SV581/11 group, however, lasted similarly or even longer than the other groups. Sequence analysis of three virulence genes (VEGF, VIR and IL-10v) revealed amino acid deletions and mutations in VEGF and IL-10v genes of SV581/11 and SV252/11, the isolate(s) producing milder lesions. Additionally, the VEGF gene of isolate SV581/11 presented the lowest amino acid identity with the other isolates and with ORFV standard strain OV-IA82. Thus, these results demonstrate that ORFV isolates may display differential virulence in lambs and these differences might be associated with genetic changes in putative virulence genes.

Vacina experimental produzida em cultivo celular confere proteção parcial contra o ectima contagioso em ovinos

Contagious ecthyma, also known as orf, is a debilitating disease of sheep and goats caused by the parapoxvirus, orf virus (ORFV). Vaccination has been used with relative success to reduce the losses caused by the disease, yet the current vaccines contain virulent virus, are empirically produced through skin scarification of live lambs, and present questionable efficacy. Therefore, the present study aimed at developing and testing an experimental ORFV vaccine produced in tissue culture. The ORFV strain IA-82 was submitted to 21 passages in BHK-21 cells and then used to immunize lam bs (n=30) through skin scarification of the internal face of the hind limb. Vaccination produced localized pustules and scabs lesions in 16 out of 30 animals, indicating an adequate replication of the vaccine virus. Ninety days after vaccination, vaccinated (n=16) and control lambs (n=16) were inoculated with a virulent ORFV strain (106,9TCID50/ml) in the labial commissure. Vaccinated and control lambs developed typical orf lesions, characterized by hyperemia, vesicles, pustules and scab formation. Nonetheless, vaccinated animals developed milder lesions compared to controls and the clinical scores were significantly lower (p<0.05) between days 10 and 22 post-challenge. In addition, the mean duration of clinical disease was significantly reduced in vaccinated animals (p<0.05). Furthermore, vaccinated animals excreted much less virus (p<0.05) and for a significantly shorter period of time than did the controls (13 days versus 22 days, p<0.001). These results demonstrate partial protection by the experimental vaccine and, upon improvement of immunization and protection indices, are promising towards the use of tissue culture-based ORFV vaccines.

Genital immunization of heifers with a glycoprotein Edeleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate

Weiss M., Vogel F.S.F., Martins M., Weiblen R., Roehe P.M., Franco A.C. & Flores E.F. 2010. Genital immunization of heifers with a glycoprotein E-deleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a

Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions.

Three dog shelters in Rio Grande do Sul were investigated for associations between the occurrence of respiratory viruses and shelter environmental conditions. Nasal secretions randomly collected during the cold season were tested via PCR, and this data collection was followed by nucleotide sequencing of the amplicons. In shelter #1 (poor sanitary and nutritional conditions, high animal density and constant contact between dogs), 78% (58/74) of the nasal samples were positive, 35% (26/74) of which were in single infections and 44% (32/74) of which were in coinfections. Shelters #2 and #3 had satisfactory sanitary and nutritional conditions, outdoors exercise areas (#2) and animal clustering by groups (#3). In shelter #2, 9% (3/35) of the samples were positive for Canine parainfluenza virus (CPIV), and 6% (2/35) were positive for Canid herpesvirus 1 (CaHV-1). In shelter #3, 9% (7/77) of the samples were positive for Canine adenovirus type 2 (CAdV-2), and 1% (1/77) were positive for Canine distemper virus (CDV). The amplicon sequences (CPIV and CDV nucleoprotein gene; CAdV-2 E3 gene; CaHV-1 glycoprotein B gene) showed 94–100% nucleotide identity with GenBank sequences. Our results demonstrate that CPIV, CAdV-2 and CDV are common in dog shelters and that their frequencies appear to be related with environmental and nutritional conditions. These results indicate the need for control/prevention measures, including vaccination and environmental management, to minimize these infections and improve dog health.

Immunogenicity of ORFV-based vectors expressing the rabies virus glycoprotein in livestock species

The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV∆024RABV-G or ORFV∆121RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV∆024RABV-G and ORFV∆121RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV∆121RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV∆024RABV-G-immunized animals, indicating a higher immunogenicity of ORFVΔ121-based vectors in these animal species.

Glycoprotein-G-gene-based molecular and phylogenetic analysis of rabies viruses associated with a large outbreak of bovine rabies in southern Brazil

A large outbreak of hematophagous-bat-associated bovine rabies has been occurring in Rio Grande do Sul (RS), the southernmost Brazilian state, since 2011, with official estimates exceeding 50,000 cattle deaths. The present article describes a genetic characterization of rabies virus (RABV) recovered from 59 affected cattle and two sheep, from 56 herds in 16 municipalities (2012-2016). Molecular analysis was performed using the nucleotide (nt) and predicted amino acid (aa) sequences of RABV glycoprotein G (G). A high level of nt and aa sequence identity was observed among the examined G sequences, ranging from 98.4 to 100%, and from 97.3 to 100%, respectively. Likewise, high levels of nt and aa sequence identity were observed with bovine (nt, 99.8%; aa, 99.8%) and hematophagous bat (nt, 99.5%; aa, 99.4%) RABV sequences from GenBank, and lower levels were observed with carnivore RABV sequences (nt, 92.8%; aa, 88.1%). Some random mutations were observed in the analyzed sequences, and a few consistent mutations were observed in some sequences belonging to cluster 2, subcluster 2b. The clustering of the sequences was observed in a phylogenetic tree, where two distinct clusters were evident. Cluster 1 comprised RABV sequences covering the entire study period (2012 to 2016), but subclusters corresponding to different years could be identified, indicating virus evolution and/or introduction of new viruses into the population. In some cases, viruses from the same location obtained within a short period grouped into different subclusters, suggesting co-circulation of viruses of different origins. Subcluster segregation was also observed in sequences obtained in the same region during different periods, indicating the involvement of different viruses in the cases at different times. In summary, our results indicate that the outbreaks occurring in RS (2012 to 2016) probably involved RABV of different origins, in addition to a possible evolution of RABV isolates within this period.

Respiratory signs, fever and lymphopenia in calves inoculated with Brazilian HoBi-like pestiviruses

Hobi-like viruses (HobiPeV) comprise a novel, recently classified species of bovine pestiviruses, originally identified in commercial fetal bovine serum of Brazilian origin and, subsequently, isolated from diseased animals in several countries. Although frequently isolated from clinical cases, most HobiPeV isolates failed to reproduce overt disease in cattle upon experimental inoculation. Herein, we describe the outcome of experimental infection of four to six months-old seronegative calves with two Brazilian HobiPeV isolates. Calves inoculated intranasally with isolate SV478/07 developed viremia between days 2 and 9 post-inoculation (pi) and shed virus in nasal secretions up to day 11pi. These animals presented hyperthermia (day 7 to 10-11 pi) and lymphopenia from days 4 to 8pi. Clinically, all four calves developed varied degrees of apathy, anorexia, mild to moderate respiratory signs (nasal secretion, hyperemia), ocular discharge and pasty diarrhea in the days following virus inoculation. In contrast, calves inoculated with isolate SV757/15 presented only hyperthermia (days 3 to 10-11 pi) and lymphopenia (days 4-8 pi), without other apparent clinical signs. In these animals, viremia was detected up to day 9 pi and virus shedding in nasal secretions lasted up to day 12-14 pi. Both groups seroconverted to the inoculated viruses, developing virus neutralizing (VN) titers from 320 to 5120 at day 28pi. These results extend previous findings that experimental infections of calves with HobiPeV are predominantly mild, yet they also indicate that field isolates may differ in their ability to cause disease in susceptible animals.