Detection of Zaire ebolavirus in swine: Assay development and optimization

Ebolaviruses (family Filoviridae , order Mononegavirales ) cause often fatal, haemorrhagic fever in primates including humans. Pigs have been identified as a species susceptible to Reston ebolavirus (RESTV) infection, with indicated transmission to humans in the Philippines; however, their role during Ebola outbreaks in Africa needs to be clarified. To perform surveillance studies, detection of ebolavirus requires a prerequisite validation of viral RNA and antibody detection methods in swine samples. These diagnostic tests also need to be suitable for deployment to low‐level containment laboratories. In this study, we developed a set of tests for detection of antibodies against Zaire ebolavirus (EBOV ) in swine. Recombinant EBOV nucleoprotein was produced using a baculovirus expression system for indirect ELISA development. Evaluation of this assay was performed using laboratory and field samples, achieving a diagnostic specificity of 99%. Importantly, the indirect ELISA was able to detect antibodies to EBOV at 7 dpi, 3 days earlier than virus neutralization tests (VNT ). The format of the VNT in this work was modified to a microtitre plaque reduction neutralization assay (miPRNT ) complemented with immunostaining to provide a more rapid and highly specific assay. Finally, a confirmatory immunoblot assay was generated to supplement the indirect ELISA results.     

Evidence of exposure to henipaviruses in domestic pigs in Uganda

Hendra virus (HeV) and Nipah virus (NiV), belonging to the genus Henipavirus, are among the most pathogenic of viruses in humans. Old World fruit bats (family Pteropodidae) are the natural reservoir hosts. Molecular and serological studies found evidence of henipavirus infection in fruit bats from several African countries. However, little is known about the potential for spillover into domestic animals in East Africa, particularly pigs, which served as amplifying hosts during the first outbreak of NiV in Malaysia and Singapore. We collected sera from 661 pigs presented for slaughter in Uganda between December 2015 and October 2016. Using HeV G and NiV G indirect ELISAs, 14 pigs (2%) were seroreactive in at least one ELISA. Seroprevalence increased to 5.4% in October 2016, when pigs were 9.5 times more likely to be seroreactive than pigs sampled in December 2015 (p = 0.04). Eight of the 14 ELISA‐positive samples reacted with HeV N antigen in Western blot. None of the sera neutralized HeV or NiV in plaque reduction neutralization tests. Although we did not detect neutralizing antibodies, our results suggest that pigs in Uganda are exposed to henipaviruses or henipa‐like viruses. Pigs in this study were sourced from many farms throughout Uganda, suggesting multiple (albeit rare) introductions of henipaviruses into the pig population. We postulate that given the widespread distribution of Old World fruit bats in Africa, spillover of henipaviruses from fruit bats to pigs in Uganda could result in exposure of pigs at multiple locations. A higher risk of a spillover event at the end of the dry season might be explained by higher densities of bats and contact with pigs at this time of the year, exacerbated by nutritional stress in bat populations and their reproductive cycle. Future studies should prioritize determining the risk of spillover of henipaviruses from pigs to people, so that potential risks can be mitigated.     

Genetically similar hepatitis E virus strains infect both humans and wild boars in the Barcelona area,Spain, and Sweden

Hepatitis E virus (HEV) is a hepatotropic virus, endemic in Europe where it infects humans and animals, with domestic pigs and wild boars as main reservoirs. The number of HEV‐infected cases with unknown source of infection increases in Europe. There are human HEV strains genetically similar to viruses from domestic pigs, and zoonotic transmission via consumption of uncooked pork meat has been shown. Due to continuous growth of the wild boar populations in Europe, another route may be through direct or indirect contacts with wild boars. In the Collserola Natural Park near Barcelona, Spain, the wild boars have spread into Barcelona city. In Sweden, they are entering into farmlands and villages. To investigate the prevalence of HEV and the risk for zoonotic transmissions, the presence of antibodies against HEV and HEV RNA were analysed in serum and faecal samples from 398 wild boars, 264 from Spain and 134 from Sweden and in sera from 48 Swedish patients with HEV infection without known source of infection. Anti‐HEV was more commonly found in Spanish wild boars (59% vs. 8%; p < 0.0001) while HEV RNA had similar prevalence (20% in Spanish vs. 15% in Swedish wild boars). Seven Swedish and three Spanish wild boars were infected with subtype 3f, and nine Spanish with subtype 3c/i. There were three clades in the phylogenetic tree formed by strains from wild boars and domestic pigs; another four clades were formed by strains from humans and wild boars. One strain from a Spanish wild boar was similar to strains from chronically infected humans. The high prevalence of HEV infections among wild boars and the similarity between wild boar HEV strains and those from humans and domestic pigs indicate that zoonotic transmission from wild boar may be more common than previously anticipated, which may develop into public health concern.     

Belgian Wildlife as Potential Zoonotic Reservoir of Hepatitis E Virus

Hepatitis E is an acute human liver disease in healthy individuals but may become chronic in immunocompromised patients. It is caused by the hepatitis E virus (HEV) and can have a zoonotic origin, particularly in high‐income countries. In this study, 383 sera from wild boars were selected for serology; for virological analyses, 69 sera and 61 livers from young wild boars were used. A total of 189 and 235 sera of, respectively, red deer and roe deer were collected for serological analysis. For virological analyses, 84 and 68 sera and 29 and 27 livers from, respectively, red and roe deer were sampled. An apparent seroprevalence of 34% (95% CI 29.71–39.46) was found in wild boars, of 1% (95% CI 0–2.4) in red deer and 3% (95% CI 0.8–4.2) in roe deer. To assess the ELISA screening prevalence, Western blot (WB) analyses were carried out, a receiver operating characteristic curve analysis was performed and different scenarios with varying ELISA specificities relative to WB were analysed. Seroprevalence remained high whatever the scenario in the wild boar population. In wild boar, 4 of 69 sera and 4 of 61 livers were detected as positive for HEV RNA. All sequences obtained from sera belonged to genotype HEV‐3. HEV RNA, belonging to genotype HEV‐3, was detected in one of 29 red deer livers. Wild boar can be considered as a host reservoir of the virus in Belgium. However, in contrast to the epidemiological role played by them in other countries, the low prevalence in deer makes these species an unlikely reservoir. This evidence needs further investigation to determine in which situation deer can serve as reservoir. These results also raise the question of the dynamics of HEV infection between wild fauna, domestic pigs and humans.     

Chlamydiales in Guinea‐pigs and Their Zoonotic Potential

The aim was to detect and characterize chlamydial infections in guinea‐pigs (GP) with ocular disease, study their pathogenicity and zoonotic potential and to test for the presence of Acanthamoebae spp. in GP eyes and to investigate whether they could act as vectors for Chlamydia‐like organisms. Overall 126 GP, of which 77 were symptomatic, were screened by clinical examination, cytology, gross pathology, histology, immunohistochemistry, polymerase chain reaction (PCR) and bacteriology. A new Chlamydiaceae‐specific intergenic spacer rRNA gene PCR, designed to amplify this segment linking the 16S and 23S regions, was performed. DNA samples were also received from one owner including samples of his cat and rabbit. Guinea‐pigs: 48 of 75 symptomatic, but only 11 of 48 asymptomatic GP were positive by PCR for Chlamydophila caviae guinea‐pig inclusion conjunctivitis (GPIC) (P < 0.0001). Eighteen of 75 or 15/48, respectively, were positive for DNA from Chlamydia‐like organisms. Acanthamoebae‐DNA could be found in two GP, of which one was symptomatic. Owner, cat and rabbit: Samples of all three species were positive by PCR for C. caviae GPIC and the owner's one‐day disposable contact lenses showed a positive PCR result for the Chlamydia‐like organism Parachlamydia acanthamoebae. No Acanthamoebae‐DNA could be detected. This study is the first to describe Chlamydia‐like organisms in GP and to detect C. caviae GPIC in human, cat and rabbit. Therefore, C. caviae GPIC could pose a zoonotic potential. We believe that the finding of C. caviae GPIC in species other than GP is probably not unique.     

Hepatitis E virus in sheep in Italy

Hepatitis E virus (HEV) is the leading cause of human enterically transmitted viral hepatitis occurring around the world both as outbreaks and as sporadic cases. The accumulating literature indicates that domestic pigs and wild boars are the main reservoirs of genotype 3 and genotype 4 for human infections in industrialized countries. However, the recent identification of HEV from various animal species poses additional potential concerns for HEV zoonotic infection. In this study, the role of sheep as potential host of hepatitis E virus (HEV) was investigated. By screening 192 sheep from seven farms located in Abruzzo Region (Southern Italy), HEV‐specific antibodies were detected in the sera of 41 animals (21.3%) whilst the RNA of HEV, genotype 3, was detected in 20 faecal (10.4%) and three serum samples (1.6%). Upon sequence analyses of a partial ORF2 gene region of eight HEV positive samples, the sheep sequences all grouped together within HEV genotype 3 subtype c, being most closely related to HEV strains identified in goat and wild boar from Abruzzo. This is the first study that demonstrates, serologically and molecularly, the presence of HEV in sheep population in a European country.     

Specific detection and differentiation of tick‐borne encephalitis and West Nile virus induced IgG antibodies in humans and horses

Tick‐borne encephalitis virus (TBEV) and West Nile virus (WNV) are important arthropod‐borne zoonotic flaviviruses. Due to the emergence of WNV in TBEV‐endemic regions co‐circulation of both viruses is increasing. Flaviviruses are structurally highly similar, which leads to cross‐reacting antibodies upon infection. Currently available serological assays for TBEV and WNV infections are therefore compromised by false‐positive results, especially in IgG measurements. In order to discriminate both infections novel diagnostic methods are needed. We describe an ELISA to measure IgG antibodies specific for TBEV and WNV, applicable to human and horse sera. Mutant envelope proteins were generated, that lack conserved parts of the fusion loop domain, a predominant target for cross‐reacting antibodies. These were incubated with equine and human sera with known TBEV, WNV or other flavivirus infections. For WNV IgG, specificities and sensitivities were 100% and 87.9%, respectively, for horse sera, and 94.4% and 92.5%, respectively, for human sera. TBEV IgG was detected with specificities and sensitivities of 95% and 96.7%, respectively, in horses, and 98.9% and 100%, respectively, in humans. Specificities increased to 100% by comparing individual samples on both antigens. The antigens could form the basis for serological TBEV‐ and WNV‐assays with improved specificities.     

Could Bat Cell Temperature and Filovirus Filament Length Explain the Emergence of Ebola Virus in Mammals? Predictions of a Thermodynamic Model

The host reservoir of Zaire ebolavirus (EBOV) remains elusive. One suggestion is that EBOV emerges in mammals when the precursor virus jumps from mayflies (or other riverine insects) to insectivorous bats. However, this does not fit with the current view that filoviruses cannot infect arthropods. Here, it is first argued that the evidence that arthropods are refractory is not definitive. Second, it is proposed that a combination of filovirus filament length and the high temperature (~42°C) experienced by an insect virus ingested by a flying bat, together with the large number of insects eaten by bats (e.g. during an ephemeral mass emergence of mayflies), facilitate jumping the species barrier. The length of a filovirus filament is related to the number of genome copies (GC). Predictions from a preliminary thermodynamic model developed here suggest that filament length could greatly affect EBOV infectivity to mammalian cells with infectivity peaking for filaments of a certain length. Importantly, the infectivity to mammals of even short filaments may be more than one million‐fold higher than that for the single GC virion. Third, it is proposed that at the high temperature within the bat, the phospholipid phosphatidylserine in the virus envelope promotes filament formation through fusion of single GC particles within the ingested insect, thus hugely increasing their infectivity to bats. Forth, according to the thermodynamic model, increasing the temperature from 27°C (insect cell temperature at average air temperature in Guinea, West Africa) to 42°C (bat) could increase the affinity of the filaments for bat cells by 1–2 orders of magnitude, while having no effect on the binding affinity of the single GC virions. The thermodynamic model developed here is supported by the counterintuitive observation that high glycoprotein densities on the EBOV surface reduce its infectivity in contrast to other viruses such as HIV.     

Detection of Foot‐and‐mouth Disease Virus RNA and Capsid Protein in Lymphoid Tissues of Convalescent Pigs Does Not Indicate Existence of a Carrier State

A systematic study was performed to investigate the potential of pigs to establish and maintain persistent foot‐and‐mouth disease virus (FMDV) infection. Infectious virus could not be recovered from sera, oral, nasal or oropharyngeal fluids obtained after resolution of clinical infection with any of five FMDV strains within serotypes A, O and Asia‐1. Furthermore, there was no isolation of live virus from tissue samples harvested at 28–100 days post‐infection from convalescent pigs recovered from clinical or subclinical FMD. Despite lack of detection of infectious FMDV, there was a high prevalence of FMDV RNA detection in lymph nodes draining lesion sites harvested at 35 days post‐infection, with the most frequent detection recorded in popliteal lymph nodes (positive detection in 88% of samples obtained from non‐vaccinated pigs). Likewise, at 35 dpi, FMDV capsid antigen was localized within follicles of draining lymph nodes, but without concurrent detection of FMDV non‐structural protein. There was a marked decline in the detection of FMDV RNA and antigen in tissue samples by 60 dpi, and no antigen or viral RNA could be detected in samples obtained at 100 dpi. The data presented herein provide the most extensive investigation of FMDV persistence in pigs. The overall conclusion is that domestic pigs are unlikely to be competent long‐term carriers of infectious FMDV; however, transient persistence of FMDV protein and RNA in lymphoid tissues is common following clinical or subclinical infection.     

Evidence of porcine circovirus‐like virus P1 in piglets with an unusual congenital tremor

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.     

Recombinant Protein p30 for Serological Diagnosis of African Swine Fever by Immunoblotting Assay

This article is devoted to the development and evaluation of the immunoblotting test system for serological diagnosis of African swine fever (ASF), based on the highly purified recombinant p30 of ASF virus (ASFV) strain Stavropol 01/08 (Stavropol 2008), representative of the ASFV currently circulating in the Russian Federation. The main project stages are as follows: (i) cloning of the central hydrophilic region of the ASFV gene CP204L (p30) into a prokaryotic vector; (ii) expression and chromatographic purification of the recombinant product p30 with thioredoxin and poly‐histidine site (p30e1_TrxA_6xHis); (iii) development of the immunoblotting test system (Rec p30‐IB) using the highly purified recombinant p30; and (iv) evaluation of Rec p30‐IB using sera and organ samples from domestic pigs and wild boars experimentally or naturally infected by ASFV. Testing of the Rec p30‐IB showed the diagnostic specificity and sensitivity of the assay to be 98.75% and 100.00%, respectively. High sensitivity of the Rec p30‐IB allowed the detection of ASFV‐specific antibodies in samples of organs of the immune system and blood sera, collected from domestic pigs and wild boars, starting from 6 to 8 days post‐infection, regardless of virus virulence, seroimmunotype and geographic origin of the samples (East Europe, South Europe, West Europe, Central and south‐east Africa).     

Are tree squirrels involved in the circulation of flaviviruses in Italy?

West Nile virus (WNV ), Usutu virus (USUV ) and tick‐borne encephalitis virus (TBEV ) are emerging zoonotic flaviviruses (family Flaviviridae ), which have circulated in Europe in the past decade. A cross‐sectional study was conducted to assess exposure to these antigenically related flaviviruses in eastern grey squirrels (Sciurus carolinensis ) in Italy. Seventeen out of 158 (10.8%; CI _95%: 5.9‐15.6) squirrels’ sera tested through bELISA had antibodies against flaviviruses. Specific neutralizing antibodies to WNV , USUV and TBEV were detected by virus neutralization tests. Our results indicate that tree squirrels are exposed to Culex and tick‐borne zoonotic flaviviruses in Italy. Moreover, this study shows for the first time USUV and TBEV exposure in grey squirrels, broadening the host range reported for these viruses. Even though further studies are needed to define the real role of tree squirrels in the epidemiology of flaviviruses in Europe, this study highlights that serology could be an effective approach for future investigations aimed at broadening our knowledge about the species exposed to these zoonotic infections.     

Presence of Hepatitis E Virus in a RED Deer (Cervus elaphus) Population in Central Italy

Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free‐living red deer (Cervus elaphus) population in central Italy by detecting virus‐specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti‐HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.     

Development and evaluation of swine vesicular disease isotype‐specific antibody ELISAs based on recombinant virus‐like particles

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot‐and‐mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false‐positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus‐like particles (VLP) and an SVD‐specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD‐VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD‐specific, without cross‐reactivity to other vesicular diseases. A panel of 16 SVD‐positive reference sera was evaluated using the SVD‐VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD‐VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD‐VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD‐VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false‐positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false‐positive samples and the use of time‐consuming virus neutralization tests, with benefit for international trade in swine and related products.     

Susceptibility of Pigs to Zoonotic Hepatitis E Virus Genotype 3 Isolated from a Wild Boar

In Europe, zoonotic hepatitis E virus (HEV) genotype 3 strains mainly circulate in humans, swine and wild boar. The aim of this study was to investigate the potential transmission of a wild boar originating HEV strain (WbHEV) to swine by intravenous or oral inoculation and to study the consequences of infection of a WbHEV strain, a WbHEV strain previously passaged in a pig and a swine HEV strain after oral inoculation. Firstly, an intravenous infection was performed for which five piglets were divided into two groups with three pigs inoculated with a WbHEV field strain and two pigs inoculated with a HEV‐negative swine liver homogenate. All pigs were necropsied 8, 9 and 10 days post‐inoculation. Secondly, an oral infection of 56 days was performed on 12 piglets divided into four groups inoculated with a WbHEV strain, a WbHEV strain previously passaged in swine, a swine HEV strain or a HEV‐negative swine liver homogenate. After intravenous inoculation, HEV RNA was detected in serum, bile, liver, spleen, duodenum, jejunum, colon, lung, gastro‐hepatic lymph nodes and faeces in all infected piglets. After oral inoculation, HEV RNA was detected in serum, bile, liver, gastro‐hepatic lymph nodes and faeces. Most of HEV‐inoculated pigs became seropositive at day 15. This study provides experimental evidence of early viral spread throughout the organism after intravenous infection with a WbHEV strain and supports the notion that such a zoonotic strain could be transmitted via the natural faecal–oral route of infection between wild boar and pigs but also between pigs.     

Emerging infectious diseases and xenotransplantation

A total of 335 infectious diseases was reported in the global human population between 1940 and 2004, the majority of which were caused by zoonotic pathogens [ 1 ]. Although viral pathogens constitute only 25%, some have spread worldwide with most starting from Central Africa. These include human immunodeficiency virus (HIV) causing acquired immunodeficiency syndromes (AIDS), chikungunya virus and West Nile virus, which also cause severe diseases in humans. HIV‐1 and HIV‐2, for example, are the result of trans‐species transmission from non‐human primates [ 2 ] to humans sometime in the last century. The spread of two henipaviruses causing fatal diseases in horses, pigs and humans has been observed in Asia and Australia, and although these viruses represent transspecies transmissions from bats, secondary transmissions from pigs to humans have also occurred. These and many other examples of emerging infectious diseases call for strong safety considerations in the field of xenotransplantation. Whereas known viruses can easily be eliminated from donor pigs, strategies should be developed to detect new zoonotic pathogens. In addition, all pigs carry porcine endogenous retroviruses (PERVs) in their genome. Two of these, PERV‐A and PERV‐B, as wells as recombinant PERV‐A/C are able to infect human cells. The greatest threat appears to come from the recombinant PERV‐A/C viruses as they appear to have an increased infectivity [ 3 , 4 ]. An increase in PERV expression was not observed in multitransgenic pigs expressing DAF, TRAIL and HLAE, generated to prevent immune rejection [ 5 ]. Our laboratory has developed a variety of strategies to prevent PERV transmission following xenotransplantation: (i) selection of animals that do not harbour PERV‐C genomes in order to prevent recombination, (ii) selection of PERV‐A and PERV‐B low‐producers [ 6 ], (iii) development of an antiviral vaccine to protect xenotransplant recipients [ 7 ] and (iv) generation of transgenic pigs in which PERV expression is inhibited via RNA interference. Inhibition of PERV expression using either synthetic small interfering (si) RNA or short hairpin (sh) RNA was demonstrated in PERV infected human cells [ 8 ], in primary pig cells [ 9 ] and in all transgenic piglets born [ 10 ]. A second generation of pigs expressing PERV‐specific siRNA is now under study and experiments have been started to introduce multiple shRNA. Supported by Deutsche Forschungsgemeinschaft, DFG, DE729/4.     

Molecular detection and phylogenetic analysis of hepatitis E virus strains circulating in wild boars in south‐central Italy

Hepatitis E virus (HEV) is a zoonotic pathogen with a worldwide distribution, and infects several mammalian species, including pigs and wild boars, which are recognized as its natural reservoirs. The virus causes a usually self‐limiting liver disease with a mortality rate generally below 1%, although mortality rates of 15%–25% have been recorded in pregnant woman. Chronic infections can also occur. The prevalence of HEV has been extensively studied in wild boars and pigs in northern Italy, where intensive pig herds are predominantly located. In contrast, few data have been collected in south‐central Italy, where small pig herds are surrounded by large regional parks populated with heterogeneous wild fauna. In this study, 291 liver samples from wild boars caught in south‐central Italy were analysed with the molecular detection of viral RNA. Our results confirm the circulation of HEV in these animals, with a mean prevalence of 13.7% (40 of 291). A nucleotide sequence analysis showed that the HEV strains were highly conserved within the same geographic areas. The wild boar HEV strains belonged to the HEV‐3c subtype, which is frequently described in wild boars, and to an uncommon undefined subtype (HEV‐3j‐like).The viral prevalence detected is concerning because it could represent a potential risk to hunters, meat workers and consumers of wild boar liver and derivative products. The hypothesized inter‐species transmission of HEV to pigs and the possibility that the virus maintains its virulence in the environment and the meat chain also present potential risks to human health, and warrant further investigations in the near future.     

Pathogenicity and Molecular Characterization of Emerging Porcine Reproductive and Respiratory Syndrome Virus in Vietnam in 2007

In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the ‘porcine high fever disease’ that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV‐2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7–99.4% identity to Chinese strain HUN4, 68–69% identity to strain VR‐2332 and 58–59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re‐isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re‐isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.     

Similar frequency of Porcine circovirus 3 (PCV‐3) detection in serum samples of pigs affected by digestive or respiratory disorders and age‐matched clinically healthy pigs

Porcine circovirus 3 (PCV‐3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV‐3 infection in serum samples from animals suffering from post‐weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV‐3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4 week‐ to 4 month‐old pigs clinically diagnosed with respiratory (n = 129) or digestive (n = 126) disorders. Serum samples of age‐matched healthy animals (n = 60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous‐necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV‐3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV‐3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV‐3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV‐3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.