The term cell line refers to a group of cells that multiply on their own outside of an organism. Healthy human cells have finite life spans because they have internal controls that regulate how many divisions each cell can undergo. However some cancer cells are immortal, meaning they do not die after a set number of divisions, as a result of alterations that happen when cells become cancerous. That property of cancer cells makes them more durable than normal cells for scientific research. Many medical researchers use laboratory-grown human cancer cells as a model to understand how cells work and test theories on the causes and treatments of diseases. https://embryo.asu.edu/pages/hela-cells-50-years-good-bad-and-ugly-2002-john-r-masters
……….....................................................…
Animals have been used in industrialized production of human vaccines since vaccine farms were established to harvest cowpox virus from calves in the late 1800s. From that point, and through the first half of the 20th Century, most vaccines would continue to be developed with the use of animals, either by growing pathogens in live animals or by using animal cells.
Although many vaccines and anti-toxin products were successfully developed this way, using animals in vaccine development – particularly live animals – is not ideal. Research animals are costly and require extensive monitoring, both to maintain their health and to ensure the continued viability of the research. They may be carrying other bacteria or viruses that could contaminate the eventual vaccine, as with polio vaccines from the mid 20th century that were made with monkey cells and eventually found to contain a monkey virus called SV40, or Simian Virus 40. (Fortunately the virus was not found to be harmful to humans.) Moreover some pathogens, such as the chickenpox virus, simply do not grow well in animal cells.
Even when vaccine development is done using animal products and not live animals – such as growing influenza vaccine viruses in chicken eggs – development can be hindered or even halted if the availability of the animal products drops. If an illness were to strike the egg-producing chickens, for example, they might produce too few eggs to be used in the development of seasonal flu vaccine, leading to a serious vaccine shortage. (It’s a common misconception that influenza vaccines could be produced more quickly if grown in cell cultures compared to using embryonated chicken eggs. In fact growing the vaccine viruses in cell cultures would take about the same amount of time. However cell cultures do not have the same potential availability issues as chicken eggs.)
For these and other reasons using cell culture techniques to produce vaccine viruses in human cell strains is a significant advance in vaccine development.
Cell cultures involve growing cells in a culture vessel. A primary cell culture consists of cells taken directly from living tissue and never sub-cultivated, and may contain multiple types of cells such as fibroblasts, epithelial and endothelial cells.
A cell strain is a cell culture that contains only one type of cell in which the cells are normal and have a finite capacity to replicate. Cell strains can be made by taking subcultures from an original, primary culture until only one type remains. Primary cultures can be manipulated in many different ways in order to isolate a single type of cell; spinning the culture in a centrifuge can separate large cells from small ones, for example. An immortalized cell line is a cell culture of a single type of cell that can reproduce indefinitely. Normally cells are subject to the Hayflick Limit, a rule named for cell biologist Leonard Hayflick, PhD. Hayflick determined that a population of normal human cells will reproduce only a finite number of times before they cease to reproduce.
However some cells in culture have undergone a mutation, or they have been manipulated in the laboratory, so that they reproduce indefinitely. …Cell lines are not used to produce vaccine viruses.
Researchers can grow human pathogens like viruses in cell strains to attenuate them – that is, to weaken them. One way viruses are adapted for use in vaccines is to alter them so that they are no longer able to grow well in the human body. This may be done, for example, by repeatedly growing the virus in a human cell strain kept at a lower temperature than normal body temperature. In order to keep replicating, the virus adapts to become better at growing at the lower temperature, thus losing its original ability to grow well and cause disease at normal body temperatures. Later when it’s used in a vaccine and injected into a living human body at normal temperature it still provokes an immune response but can’t replicate enough to cause illness.
The first licensed vaccine made with the use of a human cell strain was the adenovirus vaccine used by the military in the late 1960s. Later other vaccines were developed in human cell strains, most notably the rubella vaccine developed by Stanley Plotkin, MD, at the Wistar Institute in Philadelphia.
In 1941 Australian ophthalmologist Norman Gregg first realized that congenital cataracts in babies were the result of their mothers being infected with rubella during pregnancy. Along with cataracts it was eventually determined that congenital rubella syndrome (CRS) could also cause deafness, heart disease, encephalitis, mental retardation and pneumonia among many other conditions. At the height of a rubella epidemic that began in Europe and spread to the United States in the mid-1960s Plotkin calculated that 1% of all births at Philadelphia General Hospital were affected by congenital rubella syndrome. In some cases women who were infected with rubella while pregnant terminated their pregnancies due to the serious risks from CRS.
Following one such abortion the fetus was sent to Plotkin at the laboratory he had devoted to rubella research. Testing the kidney of the fetus, Plotkin found and isolated the rubella virus. Separately Leonard Hayflick (also working at the Wistar Institute at that time) developed a cell strain called WI-38 using lung cells from an aborted fetus. Hayflick found that many viruses, including rubella, grew well in the WI-38, and he showed that it proved to be free of contaminants and safe to use for human vaccines.
Plotkin grew the rubella virus he had isolated in WI-38 cells kept at 86°F (30°C), so that it eventually grew very poorly at normal body temperature. (He chose the low temperature approach following previous experiences with attenuating poliovirus.) After the virus had been grown through the cells 25 times at the lower temperature it was no longer able to replicate enough to cause illness in a living person but was still able to provoke a protective immune response. The rubella vaccine developed with WI-38 is still used throughout much of the world today as part of the combined MMR (measles, mumps and rubella) vaccine.
Although it has now been used in the United States for more than 30 years, Plotkin’s rubella vaccine was initially ignored by the U.S. Food and Drug Administration in favor of rubella vaccines developed using duck embryo cells and dog kidney cells. In the late 1960s there was concern in the country that a vaccine developed using human cells could be contaminated with other pathogens, though this concern was not supported by documented evidence. This is interesting in light of the discovery earlier in the decade that polio vaccines developed using primary monkey kidney cells were contaminated with simian viruses: this was one of the reasons researchers began using the normal human cell strain WI-38 in the first place. According to Hayflick, however, the main reason for using WI-38 was the fact that it could be stored in liquid nitrogen, reconstituted, and tested thoroughly before use for contaminating viruses. (None has ever been found in WI-38.) Primary monkey kidney cells could not be frozen and then reconstituted for testing as this would violate the concept of primary cells--originally the only class of cells allowed by the FDA to produce human virus vaccines.
After testing, Plotkin’s vaccine was first licensed in Europe in 1970 and was widely used there with a strong safety profile and high efficacy. In light of that data and of larger side effect profiles with the other two rubella vaccines, it was licensed in the United States in 1979 and replaced the rubella vaccine component that had previously been used for Merck’s MMR (measles, mumps, rubella) combination vaccine. In 2005 the CDC declared rubella eliminated from the United States, though the threat from imported cases remains. The World Health Organization declared the Americas free from rubella in 2015.
Groups that object to abortion have raised ethical questions about Plotkin’s rubella vaccine (and other vaccines developed with similar human cell strains) over the years.
Because of its position on abortion some members of the Catholic Church asked for its moral guidance on the use of vaccines developed using cell strains started with human fetal cells. This includes the vaccine against rubella as well as those against chickenpox and hepatitis A, and some other vaccines. The official position according to the National Catholic Bioethics Center is that individuals should, when possible, use vaccines not developed with the use of these human cell strains. However in the case where the only vaccine available against a particular disease was developed using this approach, the NCBC notes.
One is morally free to use the vaccine regardless of its historical association with abortion. The reason is that the risk to public health if one chooses not to vaccinate outweighs the legitimate concern about the origins of the vaccine. This is especially important for parents who have a moral obligation to protect the life and health of their children and those around them.
The NCBC does note that Catholics should encourage pharmaceutical companies to develop future vaccines without the use of these cell strains. To address concerns about fetal cells remaining as actual ingredients of the vaccines, however, they specifically note that fetal cells were used only to begin the cell strains that were used in the preparation of the vaccine virus:
Descendant cells are the medium in which these vaccines are prepared. The cell lines under consideration were begun using cells taken from one or more fetuses aborted almost 40 years ago. Since that time the cell lines have grown independently. It is important to note that descendant cells are not the cells of the aborted child. They never, themselves, formed a part of the victim's body.
In total only two fetuses, both obtained from abortions done by maternal choice, have given rise to the human cell strains used in vaccine development. Neither abortion was performed for the purpose of vaccine development.
Two main human cell strains have been used to develop currently available vaccines, in each case with the original fetal cells in question obtained in the 1960s. The WI-38 cell strain was developed in 1962 in the United States, and the MRC-5 cell strain (also started with fetal lung cells) was developed, using Hayflick's technology, in 1970 at the Medical Research Center in the United Kingdom. It should be noted that Hayflick's methods involved establishing a huge bank of WI-38 and MRC-5 cells that, while not capable of infinitely replicating like immortal cell lines, will serve vaccine production needs for several decades in the future.
https://www.historyofvaccines.org/content/articles/human-cell-strains-vaccine-development
……………..........................................
Antoinette Cornelia van der Kuyl, Marion Cornelissen and Ben Berkhout* Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
The novel human retrovirus xenotropic murine leukemia virus-related virus (XMRV) is arguably the most controversial virus of this moment. After its original discovery in prostate cancer tissue from North American patients, it was subsequently detected in individuals with chronic fatigue syndrome from the same continent. However most other research groups, mainly from Europe, reported negative results. The positive results could possibly be attributed to contamination with mouse products in a number of cases, as XMRV is nearly identical in nucleotide sequence to endogenous retroviruses in the mouse genome. But the detection of integrated XMRV proviruses in prostate cancer tissue proves it to be a genuine virus that replicates in human cells, leaving the question: how did XMRV enter the human population? We will discuss two possible routes: either via direct virus transmission from mouse to human, as repeatedly seen for, e.g., Hantaviruses, or via the use of mouse-related products by humans, including vaccines. We hypothesize that mouse cells or human cell lines used for vaccine production could have been contaminated with a replicating variant of the XMRV precursors encoded by the mouse genome.
The xenotropic murine leukemia virus-related virus (XMRV) is undoubtedly the most controversial human virus since its first detection in human samples in 2006 (Urisman et al., 2006). XMRV infection still lacks a firm disease association, although the virus was originally isolated from prostate cancer tissue and subsequently detected in the blood of American patients with chronic fatigue syndrome (CFS; Lombardi et al., 2009), and in the respiratory tract of patients with or without a respiratory tract infection (Fischer et al., 2010). However irregular XMRV detection (Fischer et al., 2008, 2010; Lombardi et al., 2009; Groom et al., 2010; Switzer et al., 2010; van Kuppeveld et al., 2010) suggests that it is not likely to be a major causal factor. First we do not know whether the biological reservoir has been investigated thus far, as most studies focused exclusively on blood or prostate tissue (summarized in Table 1). Second some pathogenic retroviruses do not cause much of a viremia, and experimental infection of macaques suggests that this is also the case for XMRV (Sharma et al., 2010). In these monkeys virus inoculation resulted in a low transient plasma viremia, followed by a wide dissemination of replicating virus into various organs including spleen, lymph nodes and gastrointestinal tract. Third, sequence variation may exist, but such variant virus strains could be missed by the PCR primers used.
Whether or not the virus causes disease in humans (reviewed extensively by Silverman et al., 2010, see also comments by Coffin and Stoye, 2009, by Kearney and Maldarelli, 2010, by Kaiser, 2010 and the cautionary note by Weiss, 2010), and how and when XMRV entered the human population – as the first Gammaretrovirus to do so – remains unclear. To add to the ongoing discussion we would like to propose an alternative possible source for XMRV, human vaccines or other biological products that were produced in murine cells.
One of the most striking aspects of XMRV biology is the high sequence similarity to mouse chromosomal sequences that encode endogenous retroviruses. Initially this raised the speculation that contamination with mouse DNA could explain the presence of XMRV in human samples. However the absence of other mouse-derived sequences, combined with the ease of infection of human cells with XMRV in vitro (Stieler et al., 2010) and the detection of integrated proviruses in prostate cancer tissues (Dong et al., 2007; Kim et al., 2008) indicated that laboratory contamination with mouse products is not a likely explanation for the origin of XMRV, at least for some of these studies. If contamination does not provide an explanation, where does the virus come from and how did it end up in humans?
Direct transmission of viruses from wild rodents to humans is not uncommon, e.g., rodent Hantaviruses and Arenaviruses spread through excrement via aerosols and are able to infect non-rodent species, including humans (Hart and Bennett, 1999; Klein and Calisher, 2007; Charrel and de Lamballerie, 2010). Transmission of xenotropic murine leukemia viruses (X-MLV’s) to humans is possible as human cells do express the XPR1 protein that is able to function as receptor for xenotropic and polytropic murine retroviruses. The human XPR1 receptor protein shows a preference for xenotropic retroviruses but is also able to mediate infection of polytropic murine leukemia retroviruses (P-MLV’s; Tailor et al., 1999). Classical laboratory mice strains are hybrids between Mus musculus musculus, M. m. domesticus and M. m. castaneus, with around two-thirds of the genome coming from M. m. domesticus (Yang et al., 2007). X-MLV’s cannot (re-)infect most of the laboratory mouse strains due to polymorphisms in the XPR1 protein that disable xenotropic virus entry (Marin et al., 1999). Interestingly the XPR1 genotype that prohibits X-MLV entry was not found in wild-caught M. m. domesticus, suggesting that it is a rare allele (Baliji et al., 2010). Indeed extensive screening identified seven strains of laboratory mice strains containing a permissive allele, of which at least three were susceptible to X-MLV and XMRV in cell culture (Baliji et al., 2010). In addition the F/St mouse strain also produced infectious X-MLV together with a life-long viremia (Baliji et al., 2010). Many feral mice species, e.g., M. dunni and M. spretus, are also susceptible to infection with X-MLV’s (Battini et al., 1999; Marin et al., 1999; Tailor et al., 1999). Evidence on M. m. castaneus is conflicting, with some reporting a non-functional and others a susceptible XPR1 phenotype (Marin et al., 1999; Yan et al., 2010). The ability of XPR1 to function as a receptor for xenotropic viruses was found to depend on the identity of two amino acid residues (Marin et al., 1999).
Every mouse genome contains multiple copies of endogenous MLV and has thus the capacity to express viral RNA and possibly viral particles. Endogenous MLV transcription has been described for many tissues and several mouse strains. It remains unclear if and when virus particles are generated and whether these particles are actually excreted. Zoonotic transmission of these viruses could have occurred in the many million years that mice and men have shared the same environment. But current XMRV sequences isolated from human samples do closely mimic mouse genomic sequences, thus suggesting a low number of replication cycles since zoonotic transmission, which is thus likely to have occurred recently. The mutation rate of MLV’s is not different from other retroviruses (Sanjuan et al., 2010; although its replication rate may be low), implying that if the transmission had taken place a long time ago, more nucleotide substitutions should have become fixed. Phylogenetic sequence analysis however revealed very short branches for XMRV and the mouse xMERV sequences on chromosomes 7 and 9, indicating that very few mutations have occurred since transmission (Urisman et al., 2006; Fischer et al., 2008, 2010). In addition to the loci on chromosomes 7 and 9, a BLAST search using the NCBI sequence database1 retrieves loci on mouse chromosomes 4, 11, and 12 with a much higher (98–100%) sequence identity to XMRV-gag nucleotide sequences (e.g., GenBank accession numbers AC124739, AY349138, and AL627314). Blasting whole genome XMRV sequences recovers very similar sequences with large stretches of sequence identity on mouse chromosomes 4, 5, 13, and Y, especially for the 3′ end of the XMRV genome. These results suggest that the genome of human XMRV is present, albeit in two parts, in the mouse genome with effectively no nucleotide changes. Even in slowly evolving retroviruses like foamy viruses, 100% sequence identity is only seen in animals with close contact or humans that have been bitten by an infected primate, suggestive of direct transmission, while intraspecies variation is generally around 85–95% for the pol gene (Switzer et al., 2004; Calattini et al., 2006; Liu et al., 2008).
A recently described locus (Baliji et al., 2010) on chromosome 1 of M. musculus (GenBank accession number AC115959) contains a provirus that is 92% homologous to XMRV from the 22Rv1 cell line (GenBank accession number FN692043) over its complete genome length. This provirus, Bxv1, is mainly found in Japanese M. molossinus (a natural hybrid of M. castaneus and M. musculus) and is highly expressed in some laboratory mouse strains (Baliji et al., 2010). However the Bxv1 provirus is less likely to be the source of XMRV, as its similarity to XMRV is much lower than that of other murine loci.
Xenotropic murine leukemia virus-related virus is actually a recombinant virus, resembling polytropic-endogenous sequences for the 5′ half up to approximately the middle of the pol gene and xenotropic-endogenous sequences for the 3′ half of the genome, which includes the env gene (see: Courgnaud et al., 2010). This recombination event is likely to have occurred in the mouse before transmission to humans. At least one recombinant provirus, Bxv1, is already found in the M. musculus genome (Baliji et al., 2010). This locus is heterogeneous in subspecies of M. musculus, suggesting that it represents a recent integration. Recombination rates are high for all retroviruses because they package two copies of the RNA genome in virions, which drives subsequent mixing of sequences during the reverse transcription process. Recombination also enables the generation of replication-competent viruses from defective endogenous proviruses. Recombination can also extend the viral host range (cell type and/or host species). A virus carrying a xenotropic env gene is more infectious for human cells as the human XPR1 protein has a preference for xenotropic murine envelope proteins over polytropic ones. The replication-competent endogenous cat retrovirus RD-114 is an example of a recombinant virus expressed from endogenous sequences. It combines FcEV gag–pol genes (FcEV is an endogenous retrovirus of cats) and a BaEV env gene (BaEV is an endogenous retrovirus of African monkeys) (van der Kuyl et al., 1999). RD-114 is expressed by all species of the genus Felis but not in other felines, and probably originates from a cross-species transmission of BaEV, followed by a recombination event and subsequent germ-line integration.
Detection rates of XMRV in populations are extremely variable, with 0–67% positivity in patients and 0–3.7% in healthy controls (Fischer et al., 2008, 2010; Lombardi et al., 2009; Groom et al., 2010; Switzer et al., 2010; van Kuppeveld et al., 2010), suggesting that virus prevalence and thus exposure could vary significantly with geographic location. Although the virus could possibly be transmitted from feral mice to humans in a natural setting, followed by a rapid dissemination in the human population, the high XMRV sequence similarity on two continents would suggest an alternative transmission route. Likely sources of XMRV are mouse-derived products. Some mouse genomes encode complete copies of X-MLV’s with at least 92% similarity to XMRV; segments with even higher homology are present on other locations, and could result in novel recombinant viruses. So, X-MLV’s that closely resemble XMRV could then be produced from these loci and virions could be excreted from mouse tissue or cell cultures.
MLV’s, including xenotropic sequences, are actively transcribed in mouse brain (Kwon et al., 2008), and mice can produce virus particles of different MLV classes (Ribet et al., 2008). In vivo recombination between endogenous and exogenous polytropic MLV’s has also been reported, resulting in viable viral offspring capable of infecting a variety of species (Evans et al., 2009). The Bxv1 locus in M. musculus molossinus is an example of an endogenous xenotropic/polytropic recombinant MLV that is expressed and gives rise to a life-long viremia in laboratory mice of the F/St strain (Baliji et al., 2010).
Although there was no evidence of X-MLV transmission to human embryonic stem cells expressing XPR1 after cocultivation with murine cells expressing X-MLV particles in a single report (Amit et al., 2005), this does not imply that transmission may not have occurred on another occasion. The prostate carcinoma cell line 22Rv1 is a popular research tool because it contains approximately 10 integrated copies of the XMRV provirus and it produces infectious virus (Knouf et al., 2009). The origin of the 22Rv1 cell line may represent a recent transmission case as a carcinoma was grafted in nude mice to establish this permanent cell line (Sramkoski et al., 1999). The complete 22Rv1 provirus has 99% sequence similarity with other XMRV isolates (Paprotka et al., 2010). Possibly the 22Rv1 carcinoma cells were infected with XMRV by mouse cells surrounding the tumor graft (Knouf et al., 2009).
One of the most widely distributed biological products that frequently involved mice or mouse tissue, at least up to recent years, are vaccines, especially vaccines against viruses. Some, for instance vaccines against rabies virus (Plotkin and Wiktor, 1978), yellow fever (YF) virus (Frierson, 2010), and Japanese encephalitis (JE) virus (Inactivated Japanese Encephalitis Virus Vaccine, 1993), consisted of viruses that were cultured on mouse brains. Such vaccines were in use from 1931 (YF vaccine) until now (JE vaccine, licensed in Japan since 1954). For rabies virus early vaccines were mainly of goat or sheep nerve tissue origin. In addition, suckling mouse brain-derived rabies virus vaccines were used in South America and France (Plotkin and Wiktor, 1978). No mouse-derived rabies vaccine was ever licensed in the USA (Dennehy, 2001). Live-attenuated YF vaccines were originally also grown on mouse brain, but an YF vaccine grown on chicken eggs (named 17D) became available in 1937 and was since the vaccine of choice in the America’s. In 1962 contamination of the 17D vaccine with oncogenic avian leukosis virus was detected both in England and in the USA, but fortunately no excess of cancer incidence among vaccines was reported (Frierson, 2010). In France the mouse brain-derived YF vaccine was discontinued as late as 1982.
Although being the most effective means to prevent infectious diseases and to save lives, serious contamination problems involving vaccines have occurred (Pastoret, 2010). Contamination with unrelated viruses such as the presence of hepatitis B virus (HBV) in YF vaccine preparations stemming from the use of human serum for stabilization, and simian virus 40 (SV40) and foamy viruses through the use of monkey cell cultures (Pastoret, 2010). Some vaccine viruses are inactivated before use, hopefully also inactivating any contaminating virus particles, but the contaminating virus may be more stable than the vaccine virus. For instance, SV40 is highly resistant to inactivation (Murray, 1964). Endogenous retroviruses constitute a distinct class of contaminating viruses, as these viruses are encoded by all cells of a certain species and therefore cannot be avoided even through rigorous screening (Miyazawa, 2010).
Contamination with endogenous avian leukosis viruses is a major problem for vaccine viruses grown in chicken embryos or chicken embryonic fibroblasts (Hussain et al., 2003). Infectious cat endogenous RD-114 virus has been found in several veterinary vaccines produced in cat cell cultures (Miyazawa et al., 2010; Yoshikawa et al., 2010).
Apart from vaccines, other mouse-derived biologicals could have been a source of XMRV in the human population. Monoclonal antibodies present a modern treatment for many cancers and other diseases including cardiovascular disease, psoriasis, and auto-immune disorders (for a review see: Stern and Herrmann, 2005). The first monoclonal antibody, OKT3 (to be used against transplant rejection), was approved by the FDA in 1986. The market for monoclonal antibody therapy has been expanding rapidly after the year 2000. Initially murine antibodies produced by the hybridoma technique were used (Kohler and Milstein, 1975), but these have been largely abandoned because of (sometimes severe) allergic reactions. The murine antibodies were often replaced by humanized antibodies mainly produced in transgenic mice. Monoclonal antibodies generated in mice could possibly be polluted by XMRV and related viruses. Platinum Taq polymerase from Invitrogen Corporation, prepared using mouse monoclonal antibodies, is known to be frequently contaminated with mouse DNA, which can generate false-positive PCR amplifications in combination with X-MLV or XMRV primers (Erlwein et al., 2010a). It is less likely that monoclonal antibodies from mice are a major source of XMRV in the human population as they are in use only recently, but they could provide a future supply of mouse-derived viruses. Although monoclonals are treated with detergents before use in patients, virus inactivation may not be complete, especially as protein function should be conserved. And if retroviral particles containing RNA genomes are copurified with the antibody proteins, the absence of mouse DNA may give a false impression of safety.
It is possible that XMRV particles were present in virus stocks cultured in mice or mouse cells for vaccine production, and that the virus was transferred to the human population by vaccination. The sequence homogeneity of all XMRV isolates known today suggests that only a single or very few transmissions have occurred, which is consistent with the proposed vaccination route. Nowadays vaccine batches are carefully checked with sensitive PCR assays for the presence of contaminating retroviruses, but this screening was not performed in the early years of vaccination (Trijzelaar, 1993 see also Miyazawa et al., 2010). Apart from vaccines, other biological products have been generated using mice or mouse cells. Alternatively laboratory contamination with a mouse-derived virus of cell lines used for, e.g., vaccine production could have occurred (Hartley et al., 2008; Takeuchi et al., 2008; Stang et al., 2009). The virus could then unintentionally have been transmitted to the human population.
Nowadays many vaccine strains are grown in human diploid cell lines (Fletcher et al., 1998), which are susceptible to MLV infection. A recent report detected other MLV-related sequences in CFS patients and healthy controls from North America (Lo et al., 2010), suggesting that more MLV strains may have been transmitted to the human population, possibly in a similar fashion. However solid evidence that these polytropic MLV sequences represent replicating virus is currently lacking.
Xenotropic murine leukemia virus-related virus was found in samples from CFS patients in North America but not in Europe. The virus was detected in prostate cancer tissue from patients on both continents. There is a single report with negative results from China (Hong et al., 2010), and a single report with one positive sample from Mexico (Martinez-Fierro et al., 2010) but none from other areas of the world, leaving many questions about the true distribution of XMRV in humans. Prevalence of XMRV from North American studies varies between 3.7 and 67% in four studies with two other studies reporting negative results (one in CFS patients and healthy controls (Switzer et al., 2010), and one in HIV-infected patients receiving antiretroviral therapy and untreated men at risk for HIV infection (Kunstman et al., 2010)). In Europe XMRV was detected in two studies from Germany (Fischer et al., 2010) and in one from the Netherlands (Verhaegh et al., 2010) but not in the UK (Erlwein et al., 2010b; Groom et al., 2010), France (Jeziorski et al., 2010), Denmark (Maric et al., 2010), and two other studies from the Netherlands (Cornelissen et al., 2010; van Kuppeveld et al., 2010), although the nature of the samples analyzed differed between studies. Table 1 summarizes the results from these studies.
Xenotropic murine leukemia virus-related virus sequences from Germany and North America exhibit very little nucleotide divergence, suggesting that they descended from a common ancestor relatively recently. A close inspection of the phylogenetic trees obtained with XMRV-gag sequences (Fischer et al., 2008, 2010) suggests that XMRV sequences from the USA are closer to the common ancestor than German XMRV sequences, although the trees are not optimal due to high sequence conservation. Being closer to the most recent common ancestor (MRCA) is suggestive of an older virus. Possibly XMRV was transmitted from mice to men in the USA and soon after this event introduced into Germany. Germany had close connections with the USA after World War II, with large numbers of military personnel (and their families) stationed in Germany from 1945 till present times. In 2006 there were still 57,080 American army employees distributed over more than 200 locations in Germany, mainly in the south and west of the country2. US military personnel are highly vaccinated, e.g., virtually all recruits were vaccinated with YF vaccine in 1941–1942 after the outbreak of World War II (Frierson, 2010). A massive outbreak of jaundice, with at least 26,000 cases in the Western region of the USA, was due to the use of human serum contaminated with HBV in the vaccine (see Frierson, 2010). Recently massive smallpox vaccination of the US army personnel has been carried out (Grabenstein and Winkenwerder, 2003). XMRV-infected Americans could subsequently have introduced the virus into Germany.
The combined results suggest (1) that XMRV was recently transmitted from mice to humans either from a single source or at least from a single (sub) species of mice, and (2) that all XMRV-positive individuals known today were infected with this newly emerged virus only recently, as a very high sequence identity is normally only seen after a direct retrovirus transmission.
Whatever the mechanism of XMRV cross-species transmission from mouse in humans the possible spread from human to human forms a major health threat. Sexual transmission was initially proposed (Hong et al., 2009), but XMRV was not detected in seminal plasma from HIV-infected men (Cornelissen et al., 2010). The detection of XMRV fragments in the respiratory tract (Fischer et al., 2010) suggests that the virus may be transmitted by saliva, although RNA concentrations were low. Transmission through saliva, mainly by biting, has been reported for most retrovirus genera, including ecotropic MLV’s (Portis et al., 1987). Another major threat is transmission through blood products as infectious virus has been cultured from blood cells (Lombardi et al., 2009).
Up till now all patients with detectable XMRV have been adults, the majority of them middle-aged or older (mean ± 55 years). A study in 142 children with a diversity of pathologies, including respiratory diseases in France revealed no XMRV infections in that age group (Jeziorski et al., 2010), although the incidence of XMRV in France is not known. Another study in autistic children from the USA and Italy was also negative for XMRV (Satterfield et al., 2010). XMRV can likely be acquired at any age and then probably establishes a chronic, latent infection like other retroviruses. Therefore the age of XMRV-infected individuals does not provide an unambiguous clue about when XMRV entered the human population.
In conclusion the most likely mode of XMRV transmission points to mouse-derived biological products, but it cannot formally be excluded that the virus was once transferred from feral mice to humans. The latter scenario is less likely as it would imply that a very rapid spread in human population must have occurred to explain its presence on two continents. In this scenario the extreme sequence similarity among XMRV genomes, both between and within individuals, would argue that the virus replicates at very low levels. Among biological products, vaccines that were produced in mice or mouse cells are possible candidates that warrant further inspection. If XMRV was introduced in human population through the use of biologicals, a background level of the virus in the human population, possibly varying with geography or age group, would be expected. Such a low level presence would then also explain the (absence of) detection of the virus in different studies as well as its controversial association with disease. https://www.frontiersin.org/articles/10.3389/fmicb.2010.00147/full
……………………...................................….
21 December 2010 Since the first observations that human retrovirus XMRV is associated with prostate cancer and chronic fatigue syndrome (CFS), new studies have been carried out to determine the role of the virus in these diseases. The results have been conflicting: XMRV (and related retroviruses) have been found in some patients but not in others. Whether laboratory contamination could explain the origin of XMRV has been considered by four independent research groups.
In a study of Japanese patients with prostate cancer or CFS the investigators found that control samples were positive when examined by PCR for XMRV sequences. They traced the problem to a component of a PCR kit that contained a mouse monoclonal antibody – produced in mouse cells, it likely was contaminated with murine viral nucleic acids. This PCR kit was also used to identify polytropic murine retroviruses in the blood of CFS patients.
The results of two studies demonstrate that clinical samples that test positive for XMRV may also be contaminated with mouse nucleic acids. DNA from peripheral blood was tested for XMRV by PCR using primers specific for the viral gag gene. Samples determined to be PCR positive (19/36 healthy volunteers; 2/112 CFS patients) always contained intracisternal A particle (IAP) sequences. IAPs are endogenous retrovirus-like mobile elements, and because they are present at 1000 copies in the mouse genome they can be readily detected by PCR. The authors conclude that positive results obtained with their XMRV gag PCR assay are due to contamination of human samples with mouse DNA.
What is the source of mouse DNA in the human samples included in these studies? Contamination might have occurred during blood collection, isolation of peripheral blood mononuclear cells (PBMC) or when DNA is prepared from PBMC. The authors note that fetal bovine serum and phosphate buffered saline, common laboratory reagents used for cell culture, appear to be involved. It is perhaps not surprising that fetal bovine serum could be contaminated with mouse DNA – after all it is known to contain bacteriophages which are acquired during slaughter of cattle. https://www.virology.ws/2010/12/21/is-xmrv-a-laboratory-contaminant/comment-page-2/
…………………...........................
8-10-20 The emergence of pathogenic virus infections like influenza and HIV have created an urgent need for new vaccines.
Virus-based vaccines are made in living cells (cell substrates). Some manufacturers are investigating the use of new cell lines to make vaccines. The continual growth of cell lines ensures that there is a consistent supply of the same cells that can yield high quantities of the vaccine.
In some cases the cell lines that are used might be tumorigenic, that is, they form tumors when injected into rodents. Some of these tumor-forming cell lines may contain cancer-causing viruses that are not actively reproducing. Such viruses are hard to detect using standard methods. These latent, or "quiet," viruses pose a potential threat since they might become active under vaccine manufacturing conditions. Therefore to ensure the safety of vaccines our laboratory is investigating ways to activate latent viruses in cell lines and to detect the activated viruses, as well as other unknown viruses, using new technologies. We will then adapt our findings to detect viruses in the same types of cell substrates that are used to produce vaccines. We are also trying to identify specific biological processes that reflect virus activity.
These methods will enable FDA scientists to help manufacturers to determine whether their specific cell substrate is safe to use for vaccine production. The methods our laboratory are developing and testing will help to ensure the production of safe and effective vaccines in two ways: 1) FDA will be able to develop testing guidelines for manufacturers who use new cell substrates for producing vaccines; and 2) FDA will publish the new methods it develops in peer-reviewed scientific journals, thus making them readily accessible to all manufacturers.
We are also evaluating the risk of retrovirus infections in humans. (Retroviruses are RNA viruses that use an enzyme called reverse transcriptase (RT) to replicate; RNA is the de-coded form of DNA). Simian foamy virus (SFV) can be transmitted from nonhuman primates (e.g., monkeys) to humans. Although there is no evidence that SFV causes disease, the virus can remain in a lifelong quiet state in the DNA after infection. Moreover two individuals in Africa were recently found to be infected with both HIV-1 and SFV. Therefore it is important to determine if SFV poses a threat to human health and to understand how the virus spreads in order to create strategies for controlling human infections. Such work will also help FDA to develop a new policy regarding blood donation by individuals working with nonhuman primates and implementing formal safety guidelines for people working with SFV-infected animals. We are also investigating the consequences of dual SFV and HIV-1 infection in the monkey model. https://www.fda.gov/vaccines-blood-biologics/biologics-research-projects/investigating-viruses-cells-used-make-vaccines-and-evaluating-potential-threat-posed-transmission
No comments:
Post a Comment