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|Written by Administrator|
|Wednesday, 10 June 2009 12:00|
Porcine Epidemic Diarrhoea is an atypical Coronovirus affecting pigs of all ages and can be persistent on breeder farms through consecutive litters of piglets after weaning and after loss of their immunity from their mother’s milk. Oral infection results in viral replication in the epithelial cells of the small intestinal villi. Viruses are shed in the faeces and no other organs are affected.
Diarrhoea is the only symptom after a 3-4d incubation period and clinically resembles another Coronavirus TGEV (Transmissible Gastroenteritis) in many respects. Shortening of villi is observed and often necrosis of back muscle. Deliberate infection of pregnant sows can somewhat lower mortality rate but otherwise no vaccine is reportedly available according to Merck. Detection is by ELISA (www.merckvetmanual.com).
In Thailand, mortality in suckling piglets may approach 90% and a new Korean vaccine has failed to give any protection despite claimed success in Korea. Outbreaks have occurred since 2007 (Nitikanchana,2009).
Coronaviruses (CoVs) family Coronaviridae :order Nidovirales ; are a group of positive strand RNA viruses found worldwide in birds and mammals. CoVs are classed in 3 clusters. Group 1 is found in dogs (CCoV), cat (FCoV), humans (HCoV-229E) and pigs (PEDV, TGEV and PRCoV). Group 2 is found in cattle (BCoV), humans (HCoV-OC43), mouse (MHV), pigs (HEV), birds (SARS-CoV), and dogs (CRCoV). Group 3 are exclusively avian.
Groupings are based on antigenic cross-reactivity and other criteria. Coronaviruses are prone to high frequency mutations and have a potential to jump species barriers as demonstrated by SARS (Patel and Heldens, 2009). The Japanese live-attenuated vaccine J-vac ™ (Nisseiken) has been used in Korea to prevent PEDV but unfortunately has also a potential to revert to virulence. Also, RFLP studies have recently shown that the Korean field-isolates are more closely related to each other than the vaccine strain. Sequence analysis also indicates progressive point mutations in the field (Lee et. al., 2008).
Clinically, 3 viruses in pigs are likely to cause severe diarrhoea – TGEV, PEDV and PoRV-a (porcine rotavirus A) and a 3 DNA / 6 RNA multiplex PCR detection system has been recently described that also differentiates TGEV, PEDV and PoRV-a all of which are RNA viruses (Ogawa et. al.,2009). PEDV replicates in small intestinal villous enterocytes that subsequently undergo lysis causing massive atrophy to the small intestinal villi as well as reduced villus height. Reduced enzyme activity was also demonstrated for lactase, maltase, sucrase, leucine aminopeptidase N and alkaline phosphotase. It is thought that both cellular and enzymatic damage leads to the severe diarrhoea observed in PEDV (Jung, Ahn & Chae, 2006).
The genome of PEDV is 30kb in length and subgenomic mRNA are translated into 3 main viral proteins – the S or Spike protein (180-220 kDa), the M or Membrane protein (27-32 kDa) and the N or Nucleocapsid protein (55-58 kDa). The S protein is a glycoprotein involved in specific receptor binding, cell fusion and induction of neutralizing antibodies. The S protein comprises of S1 being the ‘head’ that is involved in antibody induction while the S2 is the ‘stem’ and is a transmembrane glycoprotein. Recently, 2 new epitopes SS2 and SS6 have been described and presumed to be part of the S1 domain (Sun et. al., 2008).
In another study, the monoclonal antibody 2C10 was found to have significant (58% neutralization at 1:400 dilution) neutralizing activity against a Korean strain of PEDV named KPEDV-9. In bio-panning the 7-mer phage peptide library with 2C10, there was a preponderance of 2 peptide motifs SHRLP(Y/Q)(P/V) that accounted for 70% and GPRPVTH accounting for 30%.
There is therefore some likelihood that the 2C10 monoclonal antibody recognizes a discontinuous epitope of PEDV with structural similarity to these 2 motifs that may be used as future DNA vaccine candidates (Cruz, Kim & Shin, 2006. Epidermal Growth Factor (EGF) has been shown to promote mucosal repair in pigs infected by PEDV. EGF binds to a specific receptor on the basolateral surface of enterocytes during mucosal damage and stimulates mitogenic activity. EGF administered to pigs with sterile saline was effective in promoting mucosal recovery 3days post-administration as evidenced histopathologically by epidermal cell proliferation and increased villus length (Jung et. al., 2008).
The effect of PEDV on the epithelia of the small intestines is catastrophic to the piglet’s host defence mechanisms as Paneth cells would also be destroyed. Paneth cells are the first line of defence against pathogens. They secrete AMPs (antimicrobial peptides) into the lumen of the crypts that serve to protect the other cells of the intestinal villus. These crypts also house stem-cells that serve to constantly replenish dead or damaged epithelial cells. Paneth cells in mice, rats and humans have been shown to produce α-Defensins although this has not yet been demonstrated in the pig (Ganz, 2003). Nevertheless, the AMPs cercropin P1 and PR-39 have been isolated in the small intestines of the pig (Boman et. al., 1993).
It is very likely therefore that Paneth cells in pigs produce AMPs (also lysozymes and possibly Defensins) and the effect of PEDV on these cells would result in catastrophic histological changes that could contribute greatly to high mortality. It had been suggested over 3 decades ago that Paneth cells also play a role in viral inactivation (Erlandsen et. al., 1974). Lately, there is now overwhelming evidence that Defensins has a major role in viral inactivation. Their modes of antiviral activity include the blocking of viral entry by heparan sulphate (a glycosaminoglycan) interaction, blocking cell-to-cell spreading, specific receptor blocking including the blocking of viral entry by interfering with glycoprotein receptors, by viral envelope interference, cellular membrane interactions and host cell stimulation.
Picture showing Paneth Cells and Stem Cells on a villus
The farmed pig Sus domestica has been selectively bred over many generations and is a direct descendent of wild pigs. Wild pigs have been observed to behave as non-selective scavengers that may also eat dead flesh. In the course of evolution, wild pigs would have been exposed to viruses from the flesh of other mammals and it is thought that they would have evolved a viable first line of defence within the intestinal tract, probably in the form of Paneth cells.
Chimpanzees have evolved in a similar way in that they may from time to time switch from their normally vegetarian diet to eat the flesh of even other chimpanzees. In the course of evolution, they have active DEFT genes that produce antiviral Defensins. Human beings, on the other hand, have a point-mutation whereby a premature stop-codon aborts translation of our DEFT genes and may have led to our susceptibility to HIV-1 that Chimpanzees are tolerant of. Through the process of domestication, Sus domestica may also have lost certain potentially antiviral genes.
To control PEDV, we propose to express a set of 3 or more antiviral genes as a Chimeric AntiMicrobial Peptide or ChAMP fusion protein. We intend to use genes that block the Spike protein S so as to interfere with entry and fusion; and also interfere with the integration of the viral genome with that of the piglet’s crypt enterocytes by blocking integrase enzyme activity; and finally also to block viral mRNA transcription at the ribosomal level so that the viral Membrane protein M and the viral Nucleocapsid protein N cannot be successfully transcribed.
It is our professional opinion that a pipeline of recombinant antiviral drugs can be made for PEDV and this will prove more effective than vaccines due to the high frequency mutations common to Coronaviruses (Patel and Heldens, 2009; Lee et.al.,2008). Furthermore, it may be possible to repair the mucosal epithelium that is the target of PEDV (Jung, Ahn & Chae,2006) by also ‘stringing-together’ an EGF-expressing gene (Jung et. al., 2008) that will help with repair as well as stem-cell stimulation to replace virus-infected Paneth and crypt enterocytes.
The same strategy employed against PEDV can also be used against PSSRV with some minor modifications. This new family of recombinant antiviral biologics may be changed periodically amongst cohorts to prevent build-up of viral resistance at the farm-level.
As Specific Pathogen Free (SPF) pork is being offered 30-50% higher prices (Nitikanchana, 2009) for export to Japan; the same ChAMP-based technology may be used to eliminate pig viruses in the 5th month of the rearing cycle with subsequent confirmation using multiplex PCR for 3 DNA and 6 RNA viruses (Ogawa et. al.,2009).
Those that are PCR negative may then be transferred to a biosecure ‘finishing facility’ for its final 6th month to undergo a withdrawal period for the ChAMP drugs. The successful completion of the ‘finishing period’ can be established by ELISA or HPLC. Finally, as a last test before export certification, PCR or RT-PCR may be done 48-72 hrs prior to slaughter.
The advantage of this ChAMP-based technology for ‘instant-SPF’ pork is that the high cost of holding pigs in expensive biosecure facilities is only for its 6th and final month as opposed to its entire 6 month rearing cycle. In the end, the result is the same as the final PCR or RT-PCR test will confirm whether or not each individual pig is indeed Specific Pathogen Free for a given list of pig viruses.
Figure 1 The arterivirus particle: (Left) A diagram of the Porcine respiratory and reproductive syndrome virus (PRRSV) particle. The diameter of the virion is ~ 55 nm. Although the proteins encoded by each of the 3 ORFs (ORFs 2 to 7) of PRRSV have been shown to be components of the virion, the glycoproteins encoded by ORFs 3 and 4 have not yet been identified as structural components of the other arteriviruses. The ORF5 protein is the major virion glycoprotein and forms heterodimers with M. The ORF 2, 3, and 4 glycoproteins are minor components. (Right) A negatively stained electron micrograph of extracellular virions of Lactate dehydrogenase-elevating virus (LDV). Surface patterns observed on virions are indicated by the arrow. Bar represents 50 nm.
|Last Updated on Friday, 10 July 2009 09:07|