Molecular characterization of type 1 porcine reproductive and respiratory syndrome viruses (PRRSV) isolated in the Netherlands from 2014 to 2016.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a devastating pig disease present all over the world. The remarkable genetic variation of PRRSV, makes epidemiological and molecular analysis of circulating viruses highly important to review current diagnostic tools and vaccine efficacy. Monitoring PRRS viruses supports modern herd management by explaining the source of found viruses, either internally or externally from the herd. No epidemiological or molecular study has been published on circulating PRRS-viruses in the Netherlands, since the early nineties. Therefore, the objective of this study is to investigate circulating PRRS-viruses in the Netherlands in 2014, 2015 and 2016 on a molecular level by sequencing ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7. The results demonstrate that the 74 PRRSV strains belong to PRRSV-1, but the diversity among strains is high, based on nucleotide identity, individual ORF length and phylogenetic trees of individual ORFs. Furthermore, the data presented here show that the phylogenetic topology of some viruses is ORF dependent and suggests recombination. The identity of the strain of interest might be misinterpreted and wrong conclusions may be drawn in a diagnostic and epidemiological perspective, when only ORF5 is analyzed, as performed in many routine sequencing procedures.

Porcine epidemic diarrhea virus (PEDV) introduction into a naive Dutch pig population in 2014.

Porcine epidemic diarrhea virus (PEDV) is the highly contagious, causative agent of an economically important acute enteric disease in pigs of all ages. The disease is characterized by diarrhea and dehydration causing mortality and growth retardation. In the last few decades, only classical PEDV was reported sporadically in Europe, but in 2014 outbreaks of PEDV were described in Germany. Phylogenetic analysis showed a very high nucleotide similarity with a variant of PEDV that was isolated in the US in January 2014. The epidemiological situation of PEDV infections in the Netherlands in 2014 was unknown and a seroprevalence study in swine was performed. In total, 838 blood samples from sows from 267 farms and 101 samples from wild boars were collected from May till November 2014 and tested for antibodies against PEDV by ELISA. The apparent herd prevalence of 0.75% suggests that PEDV was not circulating on a large scale in the Netherlands at this time. However, in November 2014 a clinical outbreak of PEDV was diagnosed in a fattener farm by PCR testing. This was the first confirmed PEDV outbreak since the early nineties. Sequence analyses showed that the viruses isolated in 2014 and 2015 in the Netherlands cluster with recently found European G1b strains. This suggests a one event introduction of PEDV G1b strains in Europe in 2014, which made the Netherlands and other European countries endemic for this type of strains since then.

Field vaccinated chickens with low antibody titres show equally insufficient protection against matching and non-matching genotypes of virulent Newcastle disease virus.

Newcastle disease (ND) is a severe threat to the poultry industry and is caused by virulent strains of Newcastle disease virus (NDV). Many countries maintain a vaccination policy, but NDV is rapidly evolving as shown by the discovery of several new genotypes in the last decades. We tested the efficacy of the currently used classical commercial ND vaccine based on the genotype II strain VG/GA, applied under standard field conditions, against outbreak strains. Field vaccinated broilers were challenged with four different viruses belonging to genotype II, V or VII. A large proportion of field vaccinated broilers showed suboptimal immunity and the protection level against early and recent NDV isolates was dramatically low. Furthermore, there were no significant differences in protection afforded by a genotype II vaccine against a genotype II virus challenge compared to a challenge with viruses belonging to the other genotypes. This study suggests that the susceptibility of vaccinated poultry to NDV infection is not the result of vaccine mismatch, but rather of poor vaccination practices.

Adaptation of novel H7N9 influenza A virus to human receptors.

The emergence of the novel H7N9 influenza A virus (IAV) has caused global concerns about the ability of this virus to spread between humans. Analysis of the receptor-binding properties of this virus using a recombinant protein approach in combination with fetuin-binding, glycan array and human tissue-binding assays demonstrates increased binding of H7 to both α2-6 and α2-8 sialosides as well as reduced binding to α2-3-linked SIAs compared to a closely related avian H7N9 virus from 2008. These differences could be attributed to substitutions Q226L and G186V. Analysis of the enzymatic activity of the neuraminidase N9 protein indicated a reduced sialidase activity, consistent with the reduced binding of H7 to α2-3 sialosides. However, the novel H7N9 virus still preferred binding to α2-3- over α2-6-linked SIAs and was not able to efficiently bind to epithelial cells of human trachea in contrast to seasonal IAV, consistent with its limited human-to-human transmission.

A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs, virus shedding and seroconversion.

The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison, the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical signs were observed only in the FL-Herts-infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection. However, only the AV324/96-infected and rgAV324-infected birds, and not the FL-Herts-infected birds, shed virus in the cloaca. The AV324/96-infected pigeons showed higher mean antibody titres than the rgAV324-infected birds, whereas the antibody titres of the FL-Herts-infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons.

Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence.

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein--a well known virulence determinant of NDV--but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.

The viral replication complex is associated with the virulence of Newcastle disease virus.

Virulent strains of Newcastle disease virus ([NDV] also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.

Virulence of pigeon paramyxovirus type 1 does not always correlate with the cleavability of its fusion protein.

Some pigeon paramyxovirus type 1 (PPMV-1) strains exhibit low virulence in chickens, despite their fusion (F) protein's multi-basic cleavage site. To elucidate the molecular basis of the low pathogenicity of these strains, we constructed an infectious full-length cDNA clone of PPMV-1 strain AV324. This strain is non-virulent for chickens, although its F protein contains the typical virulence motif (112)RRKKRF(117). By using reverse genetics, we exchanged the F genes of AV324 and a virulent Newcastle disease virus (NDV) strain (Herts) and evaluated the recovered chimeric viruses for their pathogenicity in 1-day-old chickens and in embryonated eggs. Our results show that the F protein of AV324, and probably those of similar PPMV-1 strains, are functionally not different from those of virulent NDV strains and that the difference in pathogenicity must be determined by other factors.

Efficacy of intradermally administrated E2 subunit vaccines in reducing horizontal transmission of classical swine fever virus.

To investigate if intradermal (ID) vaccination and intramuscular (IM) vaccination result in a comparable reduction of horizontal transmission of classical swine fever virus (CSFV), two registered E2 subunit marker vaccines were examined. Vaccine A was a water-in-oil emulsion containing the E2 glycoprotein originating from the Alfort/Tübingen strain and vaccine B was a water-oil-water emulsion containing the E2 glycoprotein originating from the Brescia strain. Eight groups, of ten pigs each, were vaccinated with either vaccine A or B, intramuscularly (IM) or intradermally (ID). Two different vaccination-challenge intervals were used for each vaccine. Furthermore, one group was vaccinated with a tenfold ID dose of vaccine A and one non-vaccinated group served as a control group. Five pigs from each group were challenged with the moderately virulent CSFV strain Paderborn, while the remaining five pigs served as contacts. Using vaccine A, full transmission to all contact pigs in both ID vaccinated groups occurred. No virus transmission was observed when IM vaccinated pigs were challenged 14 days post-vaccination (14dpv) whereas only one out of five contact pig became infected when they were challenged 10dpv. Using vaccine B no virus transmission was observed when pigs were ID or IM vaccinated and challenged 10dpv. When challenged 3dpv full transmission occurred in the ID vaccinated group, whereas four out of five contact pigs became infected in the IM vaccinated group. This result indicates that ID vaccination does not result in better protection against horizontal CSFV transmission compared to IM vaccination, for the vaccines studied.