Comparable Infection Level and Tropism of Measles Virus and Canine Distemper Virus in Organotypic Brain Slice Cultures Obtained from Natural Host Species

Measles virus (MV) and canine distemper virus (CDV) are closely related members of the family Paramyxoviridae, genus Morbillivirus. MV infection of humans and non-human primates (NHPs) results in a self-limiting disease, which rarely involves central nervous system (CNS) complications. In contrast, infection of carnivores with CDV usually results in severe disease, in which CNS complications are common and the case-fatality rate is high. To compare the neurovirulence and neurotropism of MV and CDV, we established a short-term organotypic brain slice culture system of the olfactory bulb, hippocampus, or cortex obtained from NHPs, dogs, and ferrets. Slices were inoculated ex vivo with wild-type-based recombinant CDV or MV expressing a fluorescent reporter protein. The infection level of both morbilliviruses was determined at different times post-infection. We observed equivalent infection levels and identified microglia as main target cells in CDV-inoculated carnivore and MV-inoculated NHP brain tissue slices. Neurons were also susceptible to MV infection in NHP brain slice cultures. Our findings suggest that MV and CDV have comparable neurotropism and intrinsic capacity to infect CNS-resident cells of their natural host species.

Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs). IE1 interacts directly with PML, the key protein of PML-NBs, through its core domain and disrupts the dot-like multiprotein complexes thereby abrogating the antiviral effects. Here we present the crystal structures of the human and rat cytomegalovirus core domain (IE1CORE). We found that IE1CORE domains, also including the previously characterized IE1CORE of rhesus CMV, form a distinct class of proteins that are characterized by a highly similar and unique tertiary fold and quaternary assembly. This contrasts to a marked amino acid sequence diversity suggesting that strong positive selection evolved a conserved fold, while immune selection pressure may have fostered sequence divergence of IE1. At the same time, we detected specific differences in the helix arrangements of primate versus rodent IE1CORE structures. Functional characterization revealed a conserved mechanism of PML-NB disruption, however, primate and rodent IE1 proteins were only effective in cells of the natural host species but not during cross-species infection. Remarkably, we observed that expression of HCMV IE1 allows rat cytomegalovirus replication in human cells. We conclude that cytomegaloviruses have evolved a distinct protein tertiary structure of IE1 to effectively bind and inactivate an important cellular restriction factor. Furthermore, our data show that the IE1 fold has been adapted to maximize the efficacy of PML targeting in a species-specific manner and support the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV.

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU’s) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

Molecular Characterisation of a Novel and Highly Divergent Passerine Adenovirus 1

Wild birds harbour a large number of adenoviruses that remain uncharacterised with respect to their genomic organisation, diversity, and evolution within complex ecosystems. Here, we present the first complete genome sequence of an atadenovirus from a passerine bird that is tentatively named Passerine adenovirus 1 (PaAdV-1). The PaAdV-1 genome is 39,664 bp in length, which was the longest atadenovirus to be sequenced, to the best of our knowledge, and contained 42 putative genes. Its genome organisation was characteristic of the members of genus Atadenovirus; however, the novel PaAdV-1 genome was highly divergent and showed the highest sequence similarity with psittacine adenovirus-3 (55.58%). Importantly, PaAdV-1 complete genome was deemed to contain 17 predicted novel genes that were not present in any other adenoviruses sequenced to date, with several of these predicted novel genes encoding proteins that harbour transmembrane helices. Subsequent analysis of the novel PaAdV-1 genome positioned phylogenetically to a distinct sub-clade with all others sequenced atadenoviruses and did not show any obvious close evolutionary relationship. This study concluded that the PaAdV-1 complete genome described here is not closely related to any other adenovirus isolated from avian or other natural host species and that it should be considered a separate species.

Genetic Mutations in the LTR Region of SRLV Viruses in Capra ibex

The lentivirus (genus of the retroviruses family) can integrate a significant amount of viral cDNA into the DNA of the host cell and can efficiently infect dividing cells. They are able to spill over from their natural host species to induce new infections and pathologies among hosts of new species. This defines the crossing of species barrier that originates emergent viruses causing emergent diseases. The transmission of lentiviruses was observed between different species (domestic & wild). The small ruminant lentiviruses (SRLV) transmission is accompanied by genetic mutations in the genome of the virus. The study investigated the genetic mutations that accompany the infection and adaptation of SRLV to the new host. Genetic mutations were studied by amplifying and sequencing the Long Terminal Repeat (LTR) region.Blood samples were taken from Capra ibex living in the French Alps. Sera were tested using a commercially available ELISA. Peripheral blood mononuclear cells (PBMC) isolated on a Ficoll gradient were cultured in a macrophage differentiation medium to obtain monocyte-derived macrophage (MDM) monolayers for virus isolation. DNAs from non-cultured PBMC were used as templates for the PCR amplification of proviral DNA. PCR products (270 nt) were cloned and sequenced. Sequences were analysed using ClustalW.The alignments of the LTR fragment show three types of nucleotide mutations: replacement, addition, and deletion of nucleotide. Sequence analysis shows that the TATA box and the poly (A) site were highly conserved. The divergence of the LTR region between sequences obtained varied by 0.3 - 5.7 %. These differences were also shown by the phylogenetic tree. It can be seen that proviruses from the Capra ibex sequences are a closely related group, quite distinct from the reference sequence.

Vaccine Candidate Brucella melitensis 16MΔvjbR Is Safe in a Pregnant Sheep Model and Confers Protection

ABSTRACT As a natural host species for Brucella melitensis, pregnant sheep offer an ideal model to evaluate vaccine candidates for safety. B. melitensis strain Rev. 1 has been used almost exclusively to prevent brucellosis in small ruminants, but it causes abortions when given to pregnant animals. To evaluate the comparative safety of the candidate Brucella melitensis 16MΔvjbR, pregnant sheep (n = 6) were vaccinated subcutaneously with 1 × 1010 CFU/ml of 16MΔvjbR or 1 × 109 CFU/ml Rev. 1 at a highly susceptible stage of gestation (approximately 70 days). 16MΔvjbR resulted in only 1 abortion (1 of 6) compared with 4 of 6 (66.7%) abortions in the Rev. 1 cohort. The placenta was evaluated by culture to determine if vaccination resulted in colonization. As another measure of safety, effects of B. melitensis on the fetus/offspring (vertical transmission) was evaluated by culture and histopathology of fetal tissues to determine if vaccination prevented infection of the fetus. Vaccination with 16MΔvjbR resulted in less vertical transmission than Rev. 1. To determine if vaccination was efficacious and could reduce tissue colonization in sheep, the same cohort of sheep were challenged 5 weeks postpartum by conjunctival inoculation with 1 × 107 CFU/ml B. melitensis. Protection was similar between Rev. 1 and 16MΔvjbR, with no statistical difference in colonization in the target organs. Overall, the 16MΔvjbR vaccine was considered safer than Rev. 1 based on a reduced number of abortions and limited infection in the offspring. Future experiments are needed to further refine the vaccine dose to increase the safety margin and to evaluate protection in pregnant ewes. IMPORTANCE Brucellosis is one of the most commonly reported zoonotic disease with a worldwide distribution. Of the 12 Brucella species, Brucella melitensis is considered the most virulent and causes reproductive failure (abortions/stillbirths) in small ruminants, which can spread the disease to other animals or to humans. Vaccination of small ruminants is a key measure used to protect both human and animal health. However, the commercially available live-attenuated vaccine for Brucella melitensis Rev. 1 retains virulence and can cause disease in animals and humans. In order to evaluate the safety and efficacy in sheep, we vaccinated pregnant sheep with 16MΔvjbR. Our results indicate that 16MΔvjbR was safer for use during pregnancy, provided a similar level of protection as Rev. 1, and could be considered an improved candidate for future vaccine trials.

Into the Deep (Sequence) of the Foot-and-Mouth Disease Virus Gene Pool: Bottlenecks and Adaptation during Infection in Naïve and Vaccinated Cattle

Foot-and-mouth disease virus (FMDV) infects hosts as a population of closely related viruses referred to as a quasispecies. The behavior of this quasispecies has not been described in detail in natural host species. In this study, virus samples collected from vaccinated and non-vaccinated cattle up to 35 days post-experimental infection with FMDV A24-Cruzeiro were analyzed by deep-sequencing. Vaccination induced significant differences compared to viruses from non-vaccinated cattle in substitution rates, entropy, and evidence for adaptation. Genomic variation detected during early infection reflected the diversity inherited from the source virus (inoculum), whereas by 12 days post infection, dominant viruses were defined by newly acquired mutations. Mutations conferring recognized fitness gain occurred and were associated with selective sweeps. Persistent infections always included multiple FMDV subpopulations, suggesting distinct foci of infection within the nasopharyngeal mucosa. Subclinical infection in vaccinated cattle included very early bottlenecks associated with reduced diversity within virus populations. Viruses from both animal cohorts contained putative antigenic escape mutations. However, these mutations occurred during later stages of infection, at which time transmission is less likely to occur. This study improves upon previously published work by analyzing deep sequences of samples, allowing for detailed characterization of FMDV populations over time within multiple hosts.

Into the deep (sequence) of the foot-and-mouth disease virus gene pool: bottlenecks and adaptation during infection in naïve and vaccinated cattle

Foot-and-mouth disease virus (FMDV), like many RNA viruses, infects hosts as a population of closely related viruses referred to as a quasispecies. The behavior of this quasispecies has not been described in detail over the full course of infection in a natural host species. In this study, virus samples taken from vaccinated and non-vaccinated cattle up to 35 days post experimental infection with FMDV A24-Cruzeiro were analyzed by deep-sequencing. Vaccination induced significant differences compared to viruses from non-vaccinated cattle. in virus substitution rates, entropy, and evidence for adaptation. Genomic variation detected during early infection was found to reflect the diversity inherited from the source virus (inoculum), whereas by 12 days post infection (dpi) dominant viruses were defined by newly acquired mutations. In most serially sampled cattle, mutations conferring recognized fitness gain occurred within numerous genetic backgrounds, often associated with selective sweeps. Persistent infections always included multiple FMDV subpopulations, suggesting independently maintained foci of infection within the nasopharyngeal mucosa. Although vaccination prevented disease, subclinical infection in this group was associated with very early bottlenecks which subsequently reduced the diversity within the virus population. This implies an added consequence of vaccination in the control of foot-and-mouth disease. Viruses sampled from both animal cohorts contained putative antigenic escape mutations. However, these mutations occurred during later stages of infection, at which time transmission between animals is less likely to occur.ImportancePreparedness and control of foot-and-mouth disease virus have substantial, yet distinct implications in endemic and free regions. Viral evolution and emergence of novel strains are of critical concern in both settings. The factors that contribute to the asymptomatic carrier state, a common form of long-term FMDV infection in cattle and other species, are important but not well-understood. This experimental study of foot-and-mouth disease virus in cattle explored the evolution of the pathogen through detailed sampling and analytical methods in both vaccinated and non-vaccinated hosts. Significant differences were identified between the viruses subclinically infecting vaccinated animals and those causing clinical disease in the non-vaccinated cohort. These results can benefit vaccination programs and contribute to the understanding of persistent infection of cattle.

Identification of the Coding-Complete Genome of Cycas Necrotic Stunt Virus in Transcriptomic Data Sets of Alfalfa (Medicago sativa)

We present evidence here that alfalfa (Medicago sativa L.) can be a natural host species for a new strain of Cycas necrotic stunt virus (CNSV), for which the name CNSV-A (alfalfa) is proposed. Prior to this report, the virus has not been identified in alfalfa.

Reliable and Standardized Animal Models to Study the Pathogenesis of Bluetongue and Schmallenberg Viruses in Ruminant Natural Host Species with Special Emphasis on Placental Crossing

Starting in 2006, bluetongue virus serotype 8 (BTV8) was responsible for a major epizootic in Western and Northern Europe. The magnitude and spread of the disease were surprisingly high and the control of BTV improved significantly with the marketing of BTV8 inactivated vaccines in 2008. During late summer of 2011, a first cluster of reduced milk yield, fever, and diarrhoea was reported in the Netherlands. Congenital malformations appeared in March 2012 and Schmallenberg virus (SBV) was identified, becoming one of the very few orthobunyaviruses distributed in Europe. At the start of both epizootics, little was known about the pathogenesis and epidemiology of these viruses in the European context and most assumptions were extrapolated based on other related viruses and/or other regions of the World. Standardized and repeatable models potentially mimicking clinical signs observed in the field are required to study the pathogenesis of these infections, and to clarify their ability to cross the placental barrier. This review presents some of the latest experimental designs for infectious disease challenges with BTV or SBV. Infectious doses, routes of infection, inoculum preparation, and origin are discussed. Particular emphasis is given to the placental crossing associated with these two viruses.