For over 50 years the RNA genome of picornaviruses (illustrated below for poliovirus) was thought to be translated into a single, long polyprotein. All this time a very small upstream open reading frame (uORF) has gone undetected – until now.
The TWiVaniellosÂ discuss a thermostable poliovirus empty capsid vaccine, and twoÂ cell genes that act as a switchÂ between entry and clearance of picornavirus infection.
You can find TWiV #425 at microbe.tv/twiv, or listen below.
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On episode #374 of the science show This Week in Virology,Â the TWiVniks consider the roleÂ of a cell enzyme that removesÂ a protein linked to the 5′-end of the picornavirus genome, and the connection between malaria, Epstein-Barr virus, and endemic Burkitt’s lymphoma.
You can find TWiV #374 at microbe.tv/twiv.
On episode #365 of the science show This Week in Virology,Â Vincent, Alan, and Kathy trace the feudÂ over genome editing, a new virus discovered in human blood, and the origins of hepatitis A virus.
You can find TWiV #365 at www.microbe.tv/twiv.
Foot-and-mouth disease virus (FMDV) infects cloven-hoofed animals such as cattle, pigs, sheep, goats, and many wild species. The disease caused by this virus is a substantial problem for farmers because infected animals cannot be sold. Transgenic pigs have now been producedÂ whichÂ express a short interfering RNA (siRNA) and consequently have reduced susceptibility to infection with FMDV.
FMDV is classified in the picornavirus family which also contains poliovirus and rhinoviruses. The virus is highly contagious and readily spreads long distances via wind currents, and among animals by aerosols and contact with farm equipment. Infection causes a high fever and blisters in the mouth and on the feet – hence the name of the disease. When outbreaks occur, they are economically devastating. The 2001 FMDV outbreak in the United Kingdom was stopped by mass slaughter of all animals surrounding the affected areas – an estimated 6,131,440 – in less than a year.
Vaccines against the virus can be protective but they are not an optimal solution. One problem is that antigenic variation of the virus may thwart protection. In addition, countries free of FMDV generally do not vaccinate because this practice would make the animals seropositive andÂ prevent their export (it is not possible to differentiate between antibodies produced by natural infection versus immunization). Furthermore, if there were an outbreak of foot-and-mouth disease in such countries, the rapid replication and spread of the virus would make vaccination ineffective – hence culling of animals as described above is required. Clearly other means of protecting animals against FMDV are needed.
Synthetic short interfering RNAs (siRNA) have been shown to block viral replication in cell culture and in animals. To achieve such inhibition, short synthetic RNAs complementary to viral sequences are produced in cells. Upon infection, these siRNAs combine with the cellular RNA-induced silencing complex (RISC) which then targets the viral RNA for degradation.
To determine if siRNA could be used to protect pigs from foot-and-mouth disease, a complementary viral sequence was first identified that blocks FMDV replication in cell culture by ~97%. A vector containing this siRNA sequence was then used to produce transgenic pigs. Such animals not only express the antiviral siRNA, but as the encoding vector is present in germ cells, it is passed on to progeny pigs.
Expression of the siRNA was confirmed in a variety of transgenic pig tissues, including heart, lung, spleen, liver, kidney, and muscle. In fibroblasts produced from transgenic pigs, virus replication was reduced 30 fold. When transgenic pigs were inoculated intramuscularly with FMDV, none of the animals developed signs of disease such as fever or blisters of the feet and nose. In contrast, control non-transgenic pigs developed high fever and lesions. Viral RNA levels in the blood of transgenic pigs were 100-fold lower than in control animals. At 10 days post-infection no viral RNA was detected in heart, lung, spleen, liver, kidney, and muscle, while high levels were observed in these organs fromÂ non-transgenic controls.
These results show that siRNAs can protect transgenic pigs from FMDV induced disease. An important question that must be answered is whether transgenic pigs still contain enough virus to transmit infection to other animals. In addition, siRNAs are short – 21 nucleotides – and a mutation in the viral genome can block their inhibitory activity. Therefore it would be important to determine if mutations arise in the FMDV genome that lead to resistance to siRNAs.
Even if transgenic siRNA pigs do not transmit infection, and viral resistance does not arise, I am not sure that consumers are ready to accept such genetically modified animals.
Rhinovirus is theÂ most frequent cause of the common cold, and the virus itself is quite common: there are over 160 types, classified into 3 species. The cell receptor has just been identified for the rhinovirus C species, which can cause more severe illness than members of the A or B species: it is cadherin-related family member 3.
Because viruses are obligate intracellular parasites, the genome must enter a cell before new particles can be made. The first step in this process is binding of the virus particle to a receptor on the plasma membrane. Two different membrane proteins serve as receptors for members of rhinovirus A and B species: intracellular adhesion molecule 1, and low-density lipoprotein receptor (illustrated).
It has not been possible to propagate species C rhinoviruses in conventional cell cultures, which has hampered research on how the virus replicates. The lack of a cell culture system required a different approach to identifying a cell receptor for this virus. It was known that the virus replicates in primary organ or cell cultures derived from sinus tissue, but not in a variety of epithelial and transformed cell lines (e.g. HeLa cells). In silico comparison of gene expression profiles revealed 400 genes that are preferentially expressed in virus-susceptible cells. This list was narrowed down to 12 genes that encode plasma membrane proteins. A subset of these genesÂ were introduced into cells and tested for the ability to serve as aÂ rhinovirus C receptor. Introduction of the gene encoding cadherin-related family member 3 (CDHR3) into HeLa cells allowed rhinovirus C binding and infection.
The cadherin family comprises cell surface proteins that are involved in cell-cell communication. The exact cell function of CDHR3 is not known, but the protein is found in human lung, bronchial epithelium, and cultured airway epithelial cells. A mutation in the gene encoding this protein is associated with wheezing illness and asthma in children. This mutation, which causes a change from cysteine to tyrosine at amino acid 529, was found to increase virus binding and virus replication inÂ HeLa cells that synthesize CDHR3. It will be important to determine if this amino acid change increases rhinovirus C replication in humans, thereby leading to more serious respiratory illness.
The CDHR3 gene was used to establishÂ a stable HeLa cell line that produces the receptor and which can be infected with species C rhinoviruses. This cell line will be useful for illuminating the details of viral replication in cells, which has so far been elusive due to lack of a susceptible and permissive cell line. It may also be possible to produce transgenic mice with the human CDHR3 gene, which could serve as a model for studying rhinovirus C pathogenesis. Transgenic mice that produce the receptor for the related polioviruses, CD155, are a model for poliomyelitis.