Determination of Intestinal Viral Loads and Distribution of Bovine Viral Diarrhea Virus, Classical Swine Fever Virus, and Peste Des Petits Ruminants Virus: A Pilot Study.

The aim of this pilot study was to determine viral loads and distribution over the total length, at short distances, and in the separate layers of the intestine of virus-infected animals for future inactivation studies. Two calves, two pigs, and two goats were infected with bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV), and peste des petits ruminants virus (PPRV), respectively. Homogenously distributed maximum BVDV viral loads were detected in the ileum of both calves, with a mean titer of 6.0 log10 TCID50-eq/g. The viral loads in colon and caecum were not distributed homogenously. In one pig, evenly distributed CSFV mean viral loads of 4.5 and 4.2 log10 TCID50-eq/g were found in the small and large intestines, respectively. Mucosa, submucosa, and muscular layer/serosa showed mean viral loads of 5.3, 3.4, and 4.0 log10 TCID50-eq/g, respectively. Homogenous distribution of PPRV was shown in the ileum of both goats, with a mean viral load of 4.6 log10 TCID50-eq/g. Mean mucosa, submucosa, and muscular layer/serosa viral loads were 3.5, 2.8, and 1.7 log10 TCID50-eq/g, respectively. This pilot study provides essential data for setting up inactivation experiments with intestines derived from experimentally infected animals, in which the level and the homogeneous distribution of intestinal viral loads are required.

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 viruses.

Strategies to control spread of highly pathogenic avian influenza (HPAI) viruses by wild birds appear limited, hence timely characterization of novel viruses is important to mitigate the risk for the poultry sector and human health. In this study we characterize three recent H5-clade 2.3.4.4 viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the two H5N8 viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, which showed that chickens are efficiently infected by (fecal) contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector.

Salt inactivation of classical swine fever virus and African swine fever virus in porcine intestines confirms the existing in vitro casings model.

Natural casings, to be used as sausage containers, are being traded worldwide and may be contaminated with contagious viruses. Standard processing of such natural casings is by salt treatment with a duration of 30 days before shipment. Since information is lacking about the efficacy of these virus inactivation procedures, an in vitro 3D collagen matrix model, mimicking natural casings, was developed previously to determine the efficacy of salt to inactivate specific viruses. To validate this model, a comparison in vivo experiment was performed using intestines of pigs experimentally infected with African swine fever virus (ASFV) and classical swine fever virus (CSFV). Decimal reduction (D) values, were determined at 4 °C, 12 °C, 20 °C and 25 °C. The standard salt processing procedure showed an efficient inactivation of ASFV and CSFV over time in a temperature dependent way. Dintestine values of both viruses, treated with the standard salt treatment, were in line with the Dcollagen values. It was concluded that these results underline the suitability of the 3D collagen matrix model to determine virus inactivation and to replace animal experiments. Furthermore, an increase in storage time for standard salt processed casings derived from CSFV endemic regions is highly recommended for an efficient inactivation of CSFV.

Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge.

Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a non-structural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies.

Rapid emergence of a virulent PB2 E627K variant during adaptation of highly pathogenic avian influenza H7N7 virus to mice.

BACKGROUND: Highly pathogenic avian influenza (HPAI) viruses pose a potential human health threat as they can be transmitted directly from infected poultry to humans. During a large outbreak of HPAI H7N7 virus among poultry in The Netherlands in 2003, bird to human transmission was confirmed in 89 cases, of which one had a fatal outcome. METHODS: To identify genetic determinants of virulence in a mammalian host, we passaged an avian H7N7/03 outbreak isolate in mouse lungs and evaluated the phenotype of the mouse-adapted variant in animal models and in vitro. RESULTS: Three passages in mouse lungs were sufficient to select a variant that was highly virulent in mice. The virus had a MLD50 that was >4.3 logs lower than that of its non-lethal parental virus. Sequence analysis revealed a single mutation at position 627 in PB2, where the glutamic acid was changed to a lysine (E627K). The mouse-adapted virus has this mutation in common with the fatal human case isolate. The virus remained highly pathogenic for chickens after its passage in mice. In ferrets, the mouse-adapted virus induced more severe disease, replicated to higher titers in the lower respiratory tract and spread more efficiently to systemic organs compared with the parental virus. In vitro, the PB2 E627K mutation had a promoting effect on virus propagation in mammalian, but not in avian cells. CONCLUSIONS: Our results show that the E627K mutation in PB2 alone can be sufficient to convert an HPAI H7N7 virus of low virulence to a variant causing severe disease in mice and ferrets. The rapid emergence of the PB2 E627K mutant during mouse adaptation and its pathogenicity in ferrets emphasize the potential risk of HPAI H7N7 viruses for human health.