Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR.

Since 2014, porcine epidemic diarrhea virus (PEDV) has reemerged in Europe. RT-PCR methods have been described for the detection of PEDV, but none have been validated according to a norm. In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. The method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and RT-qPCR detection. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. The analytical and diagnostic specificities and sensitivities of this RT-qPCR were 100% in this study. A LoD of 50 genome copies/5 µl of extract from fecal matrices spiked with virus or RNA transcript and 100 genome copies/5 µl of extract from jejunum matrices spiked with virus were obtained. The Lower LQ (LLQ) was 100 genome copies/5 µl and the Upper LQ (ULQ) 108 copies/5 µl. This method is the first, validated according a norm for PEDV and may serve as a global reference method to harmonize detection and quantification of PEDV viral RNA in both field and experimental settings.

PCV2 co-infection does not impact PRRSV MLV1 safety but enhances virulence of a PRRSV MLV1-like strain in infected SPF pigs.

Co-infection by a type 1 modified live vaccine-like strain (MLV1-like) of porcine reproductive and respiratory syndrome virus (PRRSV) and a type 2 porcine circovirus (PCV2) was identified on a French pig farm with post-weaning multisystemic wasting syndrome (PMWS). An in vivo experiment was set up to characterize the virulence level of the MLV1-like strain compared with the parental MLV1 strain, and to assess the impact of PCV2 co-infection on the pathogenicity of both PRRSV strains. Six groups of six pigs each were inoculated only with either one of the two PRRSV strains or with PCV2, or co-inoculated with PCV2 and MLV1 or PCV2 and MLV1-like strains. Six contact pigs were added to each inoculated group to assess viral transmission. The animals were monitored daily for 35 days post-inoculation for clinical symptoms. Blood and nasal swabs were sampled twice a week, and tissue samples were collected during necropsy for viral quantification. Compared to MLV1-infected pigs, animals infected with the MLV1-like strain had increased PRRSV viremia and nasal shedding, a higher viral load in the tonsils, and lymph node hypertrophy at microscopic level. PCV2 co-infection did not influence clinical, virologic or transmission parameters for MLV1, but co-infected MLV1-like/PCV2 pigs had the most severe lung lesions, the highest viremia in contact animals and the highest transmission rate. Our study demonstrated that the MLV1 strain tested was safe when co-inoculated with PCV2 in piglets. However, co-infection by the MLV1-like strain and PCV2 resulted in increased virulence compared with that due to a single infection.

Impact of porcine circovirus type 2 (PCV2) infection on hepatitis E virus (HEV) infection and transmission under experimental conditions.

Hepatitis E virus is a zoonotic pathogen for which pigs have been identified as the main reservoir in industrialised countries. HEV infection dynamics in pig herds and pigs are influenced by several factors, including herd practices and possibly co-infection with immunomodulating viruses. This study therefore investigates the impact of porcine circovirus type 2 (PCV2) on HEV infection and transmission through experimental HEV/PCV2 co-infection of specific-pathogen-free pigs. No statistical difference between HEV-only and HEV/PCV2-infected animals was found for either the infectious period or the quantity of HEV shed in faeces. The HEV latency period was shorter for HEV/PCV2 co-infected pigs than for HEV-only infected pigs (11.6 versus 12.3 days). Its direct transmission rate was three times higher in cases of HEV/PCV2 co-infection than in cases of HEV-only infection (0.12 versus 0.04). On the other hand, the HEV transmission rate through environmental accumulation was lower in cases of HEV/PCV2 co-infection (4.3·10-6 versus 1.5·10-5 g/RNA copies/day for HEV-only infected pigs). The time prior to HEV seroconversion was 1.9 times longer in HEV/PCV2 co-infected pigs (49.4 versus 25.6 days for HEV-only infected pigs). In conclusion, our study shows that PCV2 affects HEV infection and transmission in pigs under experimental conditions.

Lessons learnt from a porcine epidemic diarrhea (PED) case in France in 2014: Descriptive epidemiology and control measures implemented.

An acute epidemic of porcine epidemic diarrhea (PED) has affected the USA since 2013 and spread all around the world. In France, the immune status of the pig population against PED virus (PEDV) was expected to be low due to the absence of circulation of the virus since the 80's and a compulsory notification of PED was set up in 2014. Here, we reported the first case of a PED outbreak in December 2014 in the North of France after a long absence of the disease, the monitoring of the excretion and the control measure implementation. The isolated strain in France in December 2014 was a PEDV "S-InDel" strain which was close to the "S-InDel" German PEDV strain isolated in May 2014. The individual shedding duration of PEDV in feces was estimated around 20 days for pigs of different ages. Biosecurity measures implemented allowed the limitation of PEDV spread to fattening and farrowing rooms without dissemination to the nursery block. Using strict biosecurity measures, direct shipment of infected fatteners to the slaughterhouse, strict decontamination protocols with a quarantine of 6 weeks for replacement gilts without voluntary contamination helped PEDV fade out within the herd and avoided the spread to other herds. PEDV presence in manure was investigated as well as the inactivation treatment of the virus present in the liquid manure. An increase to a pH 12 of liquid manure by liming led to the absence of PEDV detection by RT-PCR after seven days.

The expression level of gC1qR is down regulated at the early time of infection with porcine circovirus of type 2 (PCV-2) and gC1qR interacts differently with the Cap proteins of porcine circoviruses.

Porcine circoviruses (PCV) are small, non-enveloped single-stranded DNA-viruses. Porcine circovirus type 2 (PCV-2) is the causal agent of post-weaning multisystemic wasting syndrome (PMWS) whereas porcine circovirus of type 1 (PCV-1) is non- pathogenic. gC1qR is a membrane-located receptor of the complement protein subunit C1q and interacts with PCV capsid proteins. The mechanisms associated with the triggering of PMWS are not well known and gC1qR may have a role in the life cycle and eventually in the pathogenicity of PCV. The objectives of this study were to determine the level of expression of gC1qR during early PCV-2 infection, to determine the region of PCV-2 capsid protein (Cap) required for the interaction with gC1qR and to evaluate the interaction of gC1qR with Cap proteins of different PCV strains. The results indicate that gC1qR transcripts are downregulated in the tonsils and the tracheo-bronchial lymph nodes of piglets infected by PCV-2 at the early time of the infection. The N-terminal amino acids (a.a. 1-59) of PCV-2b Cap, an arginine rich region, are involved in the interaction with gC1qR. Porcine gC1qR interacts with Cap proteins of two pathogenic viral strains, PCV-2a and PCV-2b, while interaction has been observed with only one Cap protein of two investigated strains of PCV-1. The amino acids 30 and 49 of PCV-1Cap, solely, were not responsible of the difference of interaction observed. We have also shown that gC1qR interacts strongly with PCV-2Caps and PCV-1 GER Cap. This result suggests that the different interaction of gC1qR with PCV Cap proteins may have an impact on the pathogenicity of the PCV.

Complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in december 2014.

We report the first and only case of a porcine epidemic diarrhea (PED) outbreak occurring in December 2014 in northern France, and we show using the full-length genome sequence of the French PED virus (PEDV) isolate that it was a PEDV indel strain close to German PEDV strains recently isolated.