Evaluation of chickens infected with a recombinant virulent NDV clone expressing chicken IL4.

BACKGROUND: There is evidence that chicken IL4 (chIL4) functions similarly to its mammalian analogue by enhancing type 2 T helper (Th2) humoral immunity and promoting protection against parasitic infections; however, no studies have been performed to assess the effect of chIL4 on the pathogenesis of Newcastle disease (ND). To assess the role of chIL4 in velogenic NDV pathogenesis we created a vNDV infectious clone expressing chIL4. We hypothesized that co-expression of chIL4 during virus replication would result in decreased inflammation caused by the Th1 response and thereby increasing survival to challenge with vNDV. METHODS: To evaluate the effect of chIL4 during early infection with velogenic Newcastle disease virus (NDV) in chickens, recombinant NDV clones expressing either chIL4 (rZJ1-IL4) or a control expressing green fluorescent protein (rZJ1-GFP) were created by inserting an expression cassette in an intergenic region of the NDV genome. The pathogenesis of rZJ1-IL4 was assessed in 4-week-old specific pathogen free chickens. The extent of virus replication was evaluated by titration in mucosal secretions and immunohistochemistry in multiple tissues. Expression of chIL4 was confirmed in tissues using immunohistochemistry. RESULTS: Infection of birds with the rZJ1-IL4 resulted in successful viral replication in vivo and in vitro and generation of the chIL4 in tissues. All birds were clinically normal 2 DPI, with one bird in each group showing conjunctival swelling and enlarged spleens grossly. At 5 DPI, moderate or severe depression was observed in birds infected with rZJ1-GFP or rZJ1-IL4, respectively. Neurological signs and thymic atrophy were observed in one bird infected with rZJ1-IL4. Grossly, conjunctival swelling, mottled spleen and proventricular hemorrhages were observed at 5 DPI in one bird from each group. At 5 DPI, severe necrosis in the spleen, bursa and cecal tonsils were observed in birds infected with rZJ1-GFP, along with minimal evidence of chIL4 expression. In contrast, splenic atrophy, and moderate necrosis in the bursa and cecal tonsils were observed in birds infected with rZJ1-IL4. In addition, chIL4 signal was found in all tissues of rZJ1-IL4 birds at 5DPI. CONCLUSIONS: The production of chIL4 by a recombinant NDV strain resulted in the activation of the positive feedback loop associated with IL4 production. Insertion of chIL4 into NDV may decrease necrosis to lymphoid organs while increasing the severity of lymphoid atrophy and prolonged disease. However, with a low number of birds it is difficult to determine whether these results are significant to disease outcome.

Late development of pustular, erosive lesions in the muzzle of calves inoculated with Pseudocowpox virus.

We studied the pathogenesis of Pseudocowpox virus (PCPV), a zoonotic parapoxvirus associated with mucocutaneous lesions in cattle. Inoculation of calves with PCPV isolate SD 76-65 intranasally (n = 6) or transdermally in the muzzle (n = 2) resulted in virus replication and shedding up to day 13 post-infection (pi). No local or systemic signs were observed in inoculated calves up to day 20pi, when the clinical monitoring was discontinued. However, from days 28-34 pi, seven (7/8) inoculated calves underwent an asynchronous clinical course characterized by development of a few (one or two) to countless papulo-pustular, erosive-fibrinous and scabby lesions in the muzzle, in some cases extending to the lips and gingiva. In some animals, the lesions coalesced, forming extensive fibrinotic/necrotic and scabby plaques covering almost entirely the muzzle. The clinical course lasted 8-15 days and spontaneously subsided after day 42pi. Infectious virus and/or viral DNA were detected in swabs collected from lesions of 5/8 animals between days 34 and 42pi. Histological examination of fragments collected from the muzzle lesions of two affected calves (day 36pi) revealed marked epidermal hyperplasia and severe orthokeratotic and parakeratotic hyperkeratosis, covered by thick scabs. The epidermis showed multifocal areas of keratinocyte coalescing necrosis and mild multifocal vacuolar degeneration. Sera of inoculated calves at 50pi showed partial virus neutralization at low dilutions, demonstrating seroconversion. The delayed and severe clinical course associated with virus persistence in lesions are novel findings and contribute for the understanding of PCPV pathogenesis.

Identification of equine herpesvirus type 1 as cause of abortion in mares in Southern Brazil / Identificação do herpesvirus equino tipo 1 como causa de abortos em éguas no sul do Brasil

Foi realizado um estudo retrospectivo dos casos suspeitos de aborto por herpesvírus equino observados na região sul do Rio Grande do Sul entre 1978 e 2016. Foram revisados os protocolos de necropsia do Laboratório Regional de Diagnóstico da Faculdade de Veterinária da Universidade Federal de Pelotas resgatando-se os dados epidemiológicos, lesões macroscópicas e histológicas de cada caso. Foram observados dois surtos da enfermidade com prevalência entre 5,7% e 50% nos diferentes estabelecimentos, e dois casos individuais. Em todos os casos foram enviados fragmentos de órgãos fetais formolizados. Histologicamente, em todos os casos foram observados focos de necrose no fígado, pulmão e baço e presença de corpúsculos de inclusão acidofílico em hepatócitos, células epiteliais pulmonares e leucócitos. A imuno-histoquímica utilizando anticorpo policlonal comercial para herpesvirus equino-1 (EHV-1) revelou marcação positiva em todos os casos. Além disso, foi extraído DNA dos tecidos emblocados em parafina dos casos e submetidos à técnica de nested-PCR seguida de sequenciamento genômico dos amplicons em duas amostras. Estes achados indicam, que EHV-1 deve ser considerado como diagnóstico diferencial em casos de aborto em equinos no sul do Rio Grande do Sul.(AU)

Porcine epidemic diarrhea virus: An overview of current virological and serological diagnostic methods.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute, highly contagious, and severe enteric disease that leads to high mortality rates in suckling piglets. Therefore, accurate diagnosis of PEDV infection is critical for the implementation of control measures for the virus. Many diagnostic tests have been recently developed and are currently available for the detection of PEDV, its proteins or nucleic acid, including virus isolation, immunofluorescence (IF) or immunohistochemistry (IHC), polymerase chain reaction (PCR) and isothermal amplification assays. Additionally, several serological assays have been developed and are currently used for the detection of antibodies against PEDV. Molecular assays such as real-time reverse transcriptase-PCR (rRT-PCR) became the methods of choice for the diagnosis of PEDV infection, providing sensitive, specific and rapid detection of the virus RNA in clinical samples. Whereas serological assays have been widely used to monitor prior exposure to the virus and to evaluate the efficacy of novel vaccine candidates or vaccination strategies. Here we discuss the properties of current PEDV diagnostic assays and prospects for improving diagnostic strategies in the future.

African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Available data from vaccination/challenge experiments in pigs indicate that ASF protective immunity may be haemadsorption inhibition (HAI) serotype-specific. Recently, we have shown that two ASFV proteins, CD2v (EP402R) and C-type lectin (EP153R), are necessary and sufficient for mediating HAI serological specificity (Malogolovkin et al., 2015).. Here, using ASFV inter-serotypic chimeric viruses and vaccination/challenge experiments in pigs, we demonstrate that serotype-specific CD2v and/or C-type lectin proteins are important for protection against homologous ASFV infection. Thus, these viral proteins represent significant protective antigens for ASFV that should be targeted in future vaccine design and development. Additionally, these data support the concept of HAI serotype-specific protective immunity.

African swine fever virus CD2v and C-type lectin gene loci mediate serological specificity.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASF virus (ASFV) strain diversity and the viral antigens responsible for protection in the pig. Available data from vaccination/challenge experiments in pigs indicate ASF protective immunity is haemadsorption inhibition (HAI) serotype-specific. A better understanding of ASFV HAI serological groups and their diversity in nature, as well as improved methods to serotype ASFV isolates, is needed. Here, we demonstrated that the genetic locus encoding ASFV CD2v and C-type lectin proteins mediates HAI serological specificity and that CD2v/C-type lectin genotyping provides a simple method to group ASFVs by serotype, thus facilitating study of ASFV strain diversity in nature, and providing information necessary for eventual vaccine design, development and efficacious use.

Novel gammaherpesvirus functions encoded by bovine herpesvirus 6 (bovine lymphotropic virus).

The genus Macavirus of the subfamily Gammaherpesvirinae includes viruses that infect lymphoid cells of domestic and wild ruminants and swine, causing asymptomatic latent infections in reservoir hosts. Here, we describe the genome of bovine herpesvirus 6 (BoHV-6), a macavirus ubiquitous in healthy cattle populations. The BoHV-6 genome exhibited architecture conserved in macaviruses, including a repetitive H-DNA region and unique 141 kbp L-DNA region predicted to encode 77 genes. BoHV-6 encoded, in variable genomic regions, a novel complement of genes relative to other characterized macaviruses, probably contributing to distinctive aspects of BoHV-6 infection biology and host range. Most notably, BoHV-6 encoded the first herpesviral protein (Bov2.b2) similar to cellular ornithine decarboxylase, an enzyme that catalyses the first and rate-limiting step in the biosynthesis of polyamines. Bov2.b2 conceivably mediates a novel mechanism by which BoHV-6 promotes cell-cycle-dependent viral replication.

Orf virus ORFV121 encodes a novel inhibitor of NF-kappaB that contributes to virus virulence.

Orf virus (ORFV), the type member of the genus Parapoxvirus of the Poxviridae, has evolved novel strategies (proteins and/or mechanisms of action) to modulate host cell responses regulated by the nuclear factor-κB (NF-κB) signaling pathway. Here, we present data indicating that ORFV ORFV121, a gene unique to parapoxviruses, encodes a novel viral NF-κB inhibitor that binds to and inhibits the phosphorylation and nuclear translocation of NF-κB-p65. The infection of cells with an ORFV121 deletion mutant virus (OV-IA82Δ121) resulted in increased NF-κB-mediated gene transcription, and the expression of ORFV121 in cell cultures significantly suppressed NF-κB-regulated reporter gene expression. ORFV ORFV121 physically interacts with NF-κB-p65 in the cell cytoplasm, thus providing a mechanism for the inhibition of NF-κB-p65 phosphorylation and nuclear translocation. Notably, the deletion of ORFV121 from the viral genome markedly decreased ORFV virulence and disease pathogenesis in sheep, indicating that ORFV121 is a virulence determinant for ORFV in the natural host.

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.

A nuclear inhibitor of NF-kappaB encoded by a poxvirus.

Poxviruses have evolved various strategies to inhibit cytoplasmic events leading to activation of the nuclear factor κB (NF-κB) signaling pathway, with individual viruses often encoding multiple NF-κB inhibitors. Here, the novel orf virus (ORFV)-encoded protein ORFV002 was shown to inhibit nuclear events regulating NF-κB transcriptional activity. ORFV002 expression in cell cultures significantly decreased wild-type-virus-, tumor necrosis factor alpha (TNF-α)-, and lipopolysaccharide (LPS)-induced NF-κB-mediated gene expression. Expression of ORFV002 in cells, while not affecting phosphorylation or nuclear translocation of NF-κB-p65, markedly decreased TNF-α- and wild-type-virus-induced acetylation of NF-κB-p65, a p300-mediated nuclear modification of NF-κB-p65 that regulates its transactivating activity. ORFV002 was shown to colocalize and interact with NF-κB-p65, and expression of ORFV002 in cell cultures resulted in a reduced interaction of NF-κB-p65 with p300, suggesting that ORFV002 interferes with NF-κB-p65/p300 association. Deletion of ORFV002 from the OV-IA82 genome had no significant effect on ORFV pathogenesis in sheep, indicating that ORFV002 is nonessential for virus virulence in the natural host. This represents the first description of a nuclear inhibitor of NF-κB encoded by a poxvirus.

A novel inhibitor of the NF-{kappa}B signaling pathway encoded by the parapoxvirus orf virus.

The parapoxvirus orf virus (ORFV) is a pathogen of sheep and goats that has been used as a preventive and therapeutic immunomodulatory agent in several animal species. However, the functions (genes, proteins, and mechanisms of action) evolved by ORFV to modulate and manipulate immune responses are poorly understood. Here, the novel ORFV protein ORFV024 was shown to inhibit activation of the NF-kappaB signaling pathway, an important modulator of early immune responses against viral infections. Infection of primary ovine cells with an ORFV024 deletion mutant virus resulted in a marked increase in expression of NF-kappaB-regulated chemokines and other proinflammatory host genes. Expression of ORFV024 in cell cultures significantly decreased lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB-responsive reporter gene expression. Further, ORFV024 expression decreased TNF-alpha-induced phosphorylation and nuclear translocation of NF-kappaB-p65, phosphorylation, and degradation of IkappaBalpha, and phosphorylation of IkappaB kinase (IKK) subunits IKKalpha and IKKbeta, indicating that ORFV024 functions by inhibiting activation of IKKs, the bottleneck for most NF-kappaB activating stimuli. Although ORFV024 interferes with activation of the NF-kappaB signaling pathway, its deletion from the OV-IA82 genome had no significant effect on disease severity, progression, and time to resolution in sheep, indicating that ORFV024 is not essential for virus virulence in the natural host. This represents the first description of a NF-kappaB inhibitor encoded by a parapoxvirus.

Acute and latent infection by bovine herpesvirus type 5 in experimentally infected goats.

The ability of alphaherpesviruses to infect different ruminant species may have important implications for control/eradication efforts. Serological data indicate that goats may be naturally infected with bovine herpesviruses. To investigate the susceptibility of goats to bovine herpesvirus-5 (BoHV-5), 3-4-month-old kids were inoculated intranasally with each of three Brazilian BoHV-5 isolates (G1, n=8; G2, n=5; G3, n=5). The acute infection was characterized by virus shedding in nasal secretions for up to 14 days (titers up to 10(5.97)TCID(50)/mL), mild respiratory signs and conjunctivitis. All animals seroconverted to BoHV-5, developing virus neutralizing (VN) titers from 4 to 32 to the homologous viruses. At day 60 post inoculation (pi), two animals from each group were euthanized for tissue collection and the remaining goats were submitted to dexamethasone administration (0.4 mg kg(-1) for 5 days). Dexamethasone treatment resulted in virus reactivation in 9 out of 12 animals, as ascertained by virus shedding and/or by increase in VN titers. Virus shedding was detected in 8/12 animals and lasted from 1 to 9 days. Latent viral DNA was detected by PCR in the olfactory bulb and/or trigeminal ganglia of 6/6 goats euthanized at day 60 pi and in 12/12 animals euthanized 40 days post-dexamethasone. These results show that young goats are susceptible to BoHV-5 and may shed virus upon reactivation of latent infection. Thus, it is reasonable to expect that goats raised in close contact with cattle in areas where BoHV-5 is endemic may be infected and therefore should be considered potential reservoirs of the virus.