Domestic pigs are susceptible to experimental infection with non-human primate-derived Reston virus without the need for adaptation.

Domestic pigs are a critical component of the food supply and one of the most commonly raised production animals. Pork consumption has driven the intensification of pig production expanding into environments conducive to increased emergence and spread of infectious diseases, including the spillover of pathogens into human populations. One of these emerging viruses, Reston virus (RESTV), is an enigma among the Orthoebolavirus genus in that its lack of human pathogenicity is in stark contrast to the high virulence associated with most other ebolaviruses. RESTV is, however, associated with outbreaks of highly lethal hemorrhagic disease in non-human primates (NHP), as well as poorly understood clinical manifestations of mixed virulence and lethality in naturally and experimentally infected domestic pigs. Our results show it is possible for RESTV derived from an NHP to infect domestic pigs resulting in a spectrum of disease, from asymptomatic to severe respiratory distress. Further, we report on the first experimental transmission of RESTV between infected pigs and a co-housed, naïve animal, as well as the first report of the successful use of group oral fluids for the detection of RESTV RNA and virus-specific IgA antibodies.

Susceptibility of Domestic Swine to Experimental Infection with Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent that causes coronavirus disease, has been shown to infect several species. The role of domestic livestock and associated risks for humans in close contact with food production animals remains unknown for many species. Determining the susceptibility of pigs to SARS-CoV-2 is critical to a One Health approach to manage potential risk for zoonotic transmission. We found that pigs are susceptible to SARS-CoV-2 after oronasal inoculation. Among 16 animals, we detected viral RNA in group oral fluids and in nasal wash from 2 pigs, but live virus was isolated from only 1 pig. Antibodies also were detected in only 2 animals at 11 and 13 days postinoculation but were detected in oral fluid samples at 6 days postinoculation, indicating antibody secretion. These data highlight the need for additional livestock assessment to determine the potential role of domestic animals in the SARS-CoV-2 pandemic.

The Biosafety Level 4 Zoonotic Laboratory Network (BSL4ZNet): Report of a workshop on live animal handling.

The Biosafety Level 4 Zoonotic Laboratory Network (BSL4ZNet) was established in 2016, to provide a means of communication and support for the global high-containment laboratory community. Its working groups focus on international response, institutional cooperation and knowledge sharing, scientific excellence and training. In the latter role, BSL4ZNet sponsored its first international workshop in February 2018, held at the USDA National Centers for Animal Health, Ames, Iowa, USA, focused on necropsy procedures in high-containment laboratories. A second workshop, in November 2018, was held at the National Microbiology Laboratories (CFIA/PHAC) in Winnipeg, Canada, and focused on decontamination. A third workshop, held at the Australian Animal Health Laboratory in Geelong, Australia, in February 2019, was devoted to handling methods and ethical concerns for live animals in high-containment laboratories. The third workshop brought together 12 laboratorians from seven partner organizations in Australia, Canada, Germany, the United Kingdom and the United States. It included both discussion-based and hands-on training sessions on animal welfare, animal models, site-specific infrastructure constraints, health monitoring and humane endpoints, sampling procedures, and carcass disposal. This report summarizes the inception, development, and structure of the BSL4ZNet, and highlights the aims and results of the Geelong workshop.

Protection against henipaviruses in swine requires both, cell-mediated and humoral immune response.

Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus, within the family Paramyxoviridae. Nipah virus has caused outbreaks of human disease in Bangladesh, Malaysia, Singapore, India and Philippines, in addition to a large outbreak in swine in Malaysia in 1998/1999. Recently, NiV was suspected to be a causative agent of an outbreak in horses in 2014 in the Philippines, while HeV has caused multiple human and equine outbreaks in Australia since 1994. A swine vaccine able to prevent shedding of infectious virus is of veterinary and human health importance, and correlates of protection against henipavirus infection in swine need to be better understood. In the present study, three groups of animals were employed. Pigs vaccinated with adjuvanted recombinant soluble HeV G protein (sGHEV) and challenged with HeV, developed antibody levels considered to be protective prior to the challenge (titers of 320). However, activation of the cell-mediated immune response was not detected, and the animals were only partially protected against challenge with 5×10(5) PFU of HeV per animal. In the second group, cross-neutralizing antibody levels against NiV in the sGHEV vaccinated animals did not reach protective levels, and with no activation of cellular immune memory, these animals were not protected against NiV. Only pigs orally infected with 5×10(4) PFU of NiV per animal were protected against nasal challenge with 5×10(5) PFU of NiV per animal. This group of pigs developed protective antibody levels, as well as cell-mediated immune memory. Peripheral blood mononuclear cells restimulated with UV-inactivated NiV upregulated IFN-gamma, IL-10 and the CD25 activation marker on CD4(+)CD8(+) T memory helper cells and to lesser extent on CD4(-)CD8(+) T cells. In conclusion, both humoral and cellular immune responses were required for protection of swine against henipaviruses.

Identification of a novel Afipia species isolated from an Indian flying fox.

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.

Characterization of the twin-arginine transport secretome in Sinorhizobium meliloti and evidence for host-dependent phenotypes.

The twin-arginine transport (Tat) system is essential for cell viability in Sinorhizobium meliloti and may play a role during the development of root nodules. Utilizing an in vivo recombination strategy, we have constructed 28 strains that contain deletions in predicted Tat substrates. Testing of these mutations for symbiotic proficiency on the plant hosts alfalfa and sweet clover shows that some of these mutations affect associations with these hosts differentially.

The twin arginine transport system appears to be essential for viability in Sinorhizobium meliloti.

The twin arginine transport (Tat) system is responsible for transporting prefolded proteins to the periplasmic space. The Tat pathway has been implicated in many bacterial cellular functions, including motility, biofilm formation, and pathogenesis and symbiosis. Since the annotation of Sinorhizobium meliloti Rm1021 genome suggests that there may be up to 94 putative Tat substrates, we hypothesized that characterizing the twin arginine transport system in this organism might yield unique data that could help in the understanding of twin arginine transport. To initiate this work we attempted a targeted mutagenesis of the tat locus. Despite repeated attempts using a number of different types of media, the attempts at mutation construction were unsuccessful unless the experiment was carried out in a strain that was merodiploid for tatABC. In addition, it was shown that a plasmid carrying tatABC was stable in the absence of antibiotic selection in a tat deletion background. Finally, fluorescence microscopy and live/dead assays of these cultures show a high proportion of dead and irregularly shaped cells, suggesting that the loss of tatABC is inversely correlated with viability. Taken together, the results of this work provide evidence that the twin arginine transport system of S. meliloti appears to be essential for viability under all the conditions that we had tested.

A locus necessary for the transport and catabolism of erythritol in Sinorhizobium meliloti.

In this work we have genetically defined an erythritol utilization locus in Sinorhizobium meliloti. A cosmid containing the locus was isolated by complementation of a transposon mutant and was subsequently mutagenized using Tn5 : : B20. The locus was found to consist of five transcriptional units, each of which was necessary for the utilization of erythritol. Genetic complementation experiments using genes putatively annotated as erythritol catabolic genes clearly showed that, of the 17 genes at this locus, six genes are not necessary for the utilization of erythritol as a sole carbon source. The remaining genes encode EryA, EryB, EryC and TpiB as well as an uncharacterized ABC-type transporter. Transport experiments using labelled erythritol showed that components of the ABC transporter are necessary for the uptake of erythritol. The locus also contains two regulators: EryD, a SorC class regulator, and SMc01615, a DeoR class regulator. Quantitative RT-PCR experiments showed that each of these regulators negatively regulates its own transcription. In addition, induction of the erythritol locus was dependent upon EryD and a product of erythritol catabolism. Further characterization of polar mutations revealed that in addition to erythritol, the locus contains determinants for adonitol and l-arabitol utilization. The context of the mutations suggests that the locus is important for both the transport and catabolism of adonitol and l-arabitol.

Formate-dependent autotrophic growth in Sinorhizobium meliloti.

It was found that S. meliloti strain SmA818, which is cured of pSymA, could not grow on defined medium containing only formate and bicarbonate as carbon sources. Growth experiments showed that Rm1021 was capable of formate/bicarbonate-dependent growth, suggesting that it was capable of autotrophic-type growth. The annotated genome of S. meliloti Rm1021 contains three formate dehydrogenase genes. A systematic disruption of each of the three formate dehydrogenase genes, as well as the genes encoding determinants of the Calvin-Benson-Bassham, cycle was carried out to determine which of these determinants played a role in growth on this defined medium. The results showed that S. meliloti is capable of formate-dependent autotrophic growth. Formate-dependent autotrophic growth is dependent on the presence of the chromosomally located fdsABCDG operon, as well as the cbb operon carried by pSymB. Growth was also dependent on the presence of either of the two triose-phosphate isomerase genes (tpiA or tpiB) that are found in the genome. In addition, it was found that fdoGHI carried by pSymA encodes a formate dehydrogenase that allows Rm1021 to carry out formate-dependent respiration. Taken together, the data allow us to present a model of how S. meliloti can grow on defined medium containing only formate and bicarbonate as carbon sources.