“Deu Zika” in mice brain
For a long time, “deu zika” has been a very popular expression in Brazil to indicate something has gone wrong - often meant as a joke. For example, as a Brazilian, I could say “deu zika” when referring to the Germany X Brazil World Cup 2014 football match. Ironically, after the games, Brazilians discovered an actual virus named Zika existed and was circulating in the country. No longer funny to hear, Zika virus (ZIKV) soon became an international public health concern for unborn babies. The Latin American health authorities, World Health Organization (WHO), and Center for Disease Control and Prevention (CDC, USA) reported that the new circulating virus was able to infect pregnant women and affect the foetal development. However, the latter claim had to be formally supported with experimental evidence. Now, scientists have been able to achieve this by infecting animal models, such as mice, with the virus. Here we discuss the findings of two independent research groups from United States and Brazil.
When new viruses are discovered, they are often named after the place where they were first identified. Zika is a forest in Uganda (East Africa), where ZIKV was first isolated in 1947. Unlike the Ebola virus, which is named after a river in Central Africa, ZIKV can be transmitted by Aedes mosquitoes - the same type of mosquitoes that transmit dengue virus. The new virus was not considered a threat as most human cases were not associated with severe symptoms. However, in 2007, ZIKV caused an outbreak in the Pacific Islands. In 2013, ZIKV hit Brazil and spread to other countries in Latin America. The human cases from 2007 onwards have indicated an ability of the virus to infect the brain of unborn babies. Some of these babies were born with a smaller head size than average, which is classified as “microcephaly” and associated with growth restriction and miscarriage or spontaneous abortion.
Scientists now wanted to confirm whether the virus can indeed cause all of these phenomena in the brain. One way of investigating this is through laboratory experimentation using animals. This is an important step because it means any candidate drugs or vaccines against the virus can be tested for their ability to block virus replication and so prevent damage to the developing foetus.
Mice are often used as models to study human diseases because they are mammals, like humans, and so share a lot of our biology, and because they are easy to manipulate genetically - we know how to change their genes. And this is exactly what Michael Diamond and his colleagues in the USA did to develop their mother-to-foetus or “in utero” transmission model of ZIKV infection.
When a virus tries to infect a cell it faces a hostile environment. The frontline of the immune system, called the innate immune response, defends cells against invading viruses. Viruses that infect us have, by definition, evolved to overcome these initial defences.
But this first line of defence differs between different animals - a virus that has specifically adapted to overcome our human defences may be unable to overcome mouse defences or defence systems in other animals. Indeed, this is the case for ZIKV and so in order to develop a working mouse model for the disease, it was necessary to eliminate their frontline defences.
Researchers achieved this by either deleting one of the innate immune genes in the mice or by blocking an important anti-viral signalling pathway. With their defences lowered, the mice were now susceptible to ZIKV infection and could be used to study viral transmission from pregnant mice to their developing young.
The scientists observed that their ZIKV infection model in mice shared a lot of features with the human disease. For example, they found ZIKV in the placenta, suggesting a route by which the virus crosses from mother to child, and cell death in the brain of the developing foetus.
Although the results of this study alone did not demonstrate definitively that the cell death in the foetal brain is directly caused by the virus, subsequent studies from Brazil and China have extended these observations by looking at cell death in specific neural cell subtypes.
In Brazil, a study led by Patricia Beltrão-Braga used the Brazilian ZIKV strain currently circulating in South America. They were able to recapitulate the findings of Michael Diamond and colleagues in the USA but without changing the genes of the mice to lower their defences against Zika virus. They achieved this by using a different strain of mice that have naturally weakened defences.
Patricia Beltrão-Braga and colleagues compared the Brazilian ZIKV strain to an older African strain and concluded that only the Brazilian strain was associated with death of neural cells. However, the team only compared the two viruses in vitro and did not test the effect of the African strain on foetal development in pregnant mice. This would be a revealing experiment as, unlike the Brazilian strain, the African strain has never been associated with microcephaly in newborns.
Experimental evidence from different research groups has thus demonstrated ZIKV infection can interfere in the foetus development in mice. So far this has only been observed in genetically modified animals (engineered to reduce their ability to block virus replication) or in animals with naturally low resistance to the virus. (Although in the latter case the scientists used an incredibly high amount of virus to infect the animals.) Mice cannot recapitulate ZIKV infection exactly as it happens in humans per se, but the mouse models described above gave a good indication that ZIKV has the ability to cross the placental barrier and infect foetuses. Therefore, these animal models could be explored as additional systems for drug testing and vaccine development.
Technical overview in brief
Miner et al. 2016 (Diamond’s group) infected Ifnar1 KO female mice crossed with wild-type (wt) males with a French Polynesian strain of ZIKV. Alternatively, they used wild-type mice associated with monoclonal antibodies against Ifnar1. A high viral load was found in the placenta in comparison to the dam serum. As a consequence, virus RNA was detected in mouse foetuses found with growth restriction amongst other features, but not microcephaly. They used dengue virus (DENV) as control.
Cugola et al. 2016 (Beltrão-Braga’s group) infected pregnant SJL and C57BL/6 mice with high titers of a South American strain of ZIKV. Only SJL mice developed major symptoms of infection, such as growth restriction and intrauterine growth restriction (IUGR). They did not use any other viruses as controls in these experiments. Furthermore, they demonstrated that human neurospheres and brain organoids showed marked ZIKV replication and CASP3 activation. As controls, they used ZIKV MR-766 (Uganda) strain and Yellow fever virus.
Together, both studies alongside with Li et al. 2016 results generate a great complementary evidence that ZIKV can cross the placenta and cause IUGR in mice models. Specially, Diamond’s group performed next-generation sequencing to rule out the presence of other pathogens in their virus inoculum. However, microcephaly was not an isolated observation. Also, there is no formal evidence that other viruses, such as ZIKV MR-766 and/or neurotropic flaviviruses, could cause similar phenomena in those models. If proven, the latter would strengthen the claim for using immunodeficient mice to explore ZIKV mechanisms as they happen in humans.
Antonio Gregorio Dias Jr. (writer and blog editor) & Layal Liverpool (writer)
References:
Cugola et al. The Brazilian Zika virus strain causes birth defects in experimental models. Nature 534: 267-273, 2016.
Li et al. Zika virus disrupts neural progenitor development and leads to microcephaly in mice. Cell Stem Cell 19: 1-7, 2016.
Miner et al. Zika virus infection during pregnancy in mice caused placental damage and fetal demise. Cell 165: 1081-1091, 2016.
When new viruses are discovered, they are often named after the place where they were first identified. Zika is a forest in Uganda (East Africa), where ZIKV was first isolated in 1947. Unlike the Ebola virus, which is named after a river in Central Africa, ZIKV can be transmitted by Aedes mosquitoes - the same type of mosquitoes that transmit dengue virus. The new virus was not considered a threat as most human cases were not associated with severe symptoms. However, in 2007, ZIKV caused an outbreak in the Pacific Islands. In 2013, ZIKV hit Brazil and spread to other countries in Latin America. The human cases from 2007 onwards have indicated an ability of the virus to infect the brain of unborn babies. Some of these babies were born with a smaller head size than average, which is classified as “microcephaly” and associated with growth restriction and miscarriage or spontaneous abortion.
Scientists now wanted to confirm whether the virus can indeed cause all of these phenomena in the brain. One way of investigating this is through laboratory experimentation using animals. This is an important step because it means any candidate drugs or vaccines against the virus can be tested for their ability to block virus replication and so prevent damage to the developing foetus.
Mice are often used as models to study human diseases because they are mammals, like humans, and so share a lot of our biology, and because they are easy to manipulate genetically - we know how to change their genes. And this is exactly what Michael Diamond and his colleagues in the USA did to develop their mother-to-foetus or “in utero” transmission model of ZIKV infection.
When a virus tries to infect a cell it faces a hostile environment. The frontline of the immune system, called the innate immune response, defends cells against invading viruses. Viruses that infect us have, by definition, evolved to overcome these initial defences.
But this first line of defence differs between different animals - a virus that has specifically adapted to overcome our human defences may be unable to overcome mouse defences or defence systems in other animals. Indeed, this is the case for ZIKV and so in order to develop a working mouse model for the disease, it was necessary to eliminate their frontline defences.
Researchers achieved this by either deleting one of the innate immune genes in the mice or by blocking an important anti-viral signalling pathway. With their defences lowered, the mice were now susceptible to ZIKV infection and could be used to study viral transmission from pregnant mice to their developing young.
The scientists observed that their ZIKV infection model in mice shared a lot of features with the human disease. For example, they found ZIKV in the placenta, suggesting a route by which the virus crosses from mother to child, and cell death in the brain of the developing foetus.
Although the results of this study alone did not demonstrate definitively that the cell death in the foetal brain is directly caused by the virus, subsequent studies from Brazil and China have extended these observations by looking at cell death in specific neural cell subtypes.
In Brazil, a study led by Patricia Beltrão-Braga used the Brazilian ZIKV strain currently circulating in South America. They were able to recapitulate the findings of Michael Diamond and colleagues in the USA but without changing the genes of the mice to lower their defences against Zika virus. They achieved this by using a different strain of mice that have naturally weakened defences.
Patricia Beltrão-Braga and colleagues compared the Brazilian ZIKV strain to an older African strain and concluded that only the Brazilian strain was associated with death of neural cells. However, the team only compared the two viruses in vitro and did not test the effect of the African strain on foetal development in pregnant mice. This would be a revealing experiment as, unlike the Brazilian strain, the African strain has never been associated with microcephaly in newborns.
Experimental evidence from different research groups has thus demonstrated ZIKV infection can interfere in the foetus development in mice. So far this has only been observed in genetically modified animals (engineered to reduce their ability to block virus replication) or in animals with naturally low resistance to the virus. (Although in the latter case the scientists used an incredibly high amount of virus to infect the animals.) Mice cannot recapitulate ZIKV infection exactly as it happens in humans per se, but the mouse models described above gave a good indication that ZIKV has the ability to cross the placental barrier and infect foetuses. Therefore, these animal models could be explored as additional systems for drug testing and vaccine development.
Technical overview in brief
Miner et al. 2016 (Diamond’s group) infected Ifnar1 KO female mice crossed with wild-type (wt) males with a French Polynesian strain of ZIKV. Alternatively, they used wild-type mice associated with monoclonal antibodies against Ifnar1. A high viral load was found in the placenta in comparison to the dam serum. As a consequence, virus RNA was detected in mouse foetuses found with growth restriction amongst other features, but not microcephaly. They used dengue virus (DENV) as control.
Cugola et al. 2016 (Beltrão-Braga’s group) infected pregnant SJL and C57BL/6 mice with high titers of a South American strain of ZIKV. Only SJL mice developed major symptoms of infection, such as growth restriction and intrauterine growth restriction (IUGR). They did not use any other viruses as controls in these experiments. Furthermore, they demonstrated that human neurospheres and brain organoids showed marked ZIKV replication and CASP3 activation. As controls, they used ZIKV MR-766 (Uganda) strain and Yellow fever virus.
Together, both studies alongside with Li et al. 2016 results generate a great complementary evidence that ZIKV can cross the placenta and cause IUGR in mice models. Specially, Diamond’s group performed next-generation sequencing to rule out the presence of other pathogens in their virus inoculum. However, microcephaly was not an isolated observation. Also, there is no formal evidence that other viruses, such as ZIKV MR-766 and/or neurotropic flaviviruses, could cause similar phenomena in those models. If proven, the latter would strengthen the claim for using immunodeficient mice to explore ZIKV mechanisms as they happen in humans.
Antonio Gregorio Dias Jr. (writer and blog editor) & Layal Liverpool (writer)
References:
Cugola et al. The Brazilian Zika virus strain causes birth defects in experimental models. Nature 534: 267-273, 2016.
Li et al. Zika virus disrupts neural progenitor development and leads to microcephaly in mice. Cell Stem Cell 19: 1-7, 2016.
Miner et al. Zika virus infection during pregnancy in mice caused placental damage and fetal demise. Cell 165: 1081-1091, 2016.
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