**5. Genetic studies of halophilic viruses**

The genetic material that makes up the DNA of all organisms consist of four different nucleotides: Guanine, Adenine, Cytosine and Thymine. In the instance of RNA, the thymine is replaced with uracil. The natural pairing that takes place in double stranded DNA is that guanine pairs with cytosine through three hydrogen bonds and adenine binds with thymine through 2 hydrogen bonds. This hydrogen bonding plays an important role in not only the structure of DNA but also in the stability of the DNA and its resistance to denaturation. Genetic studies are a great way to not only better understand the organism in which you are studying but it is a great comparison tool to view similarities between both organisms of the same species and organisms from differing species. Studying genomics also allows scientists to form a road map of sorts that can help indicate which part of an organisms' genetic information is responsible for different actions and characteristics. Genetic studies of halophiles for example, help provide insight into how these organisms are able to survive in such harsh and hypersaline environments. Over the years, many different software programs have been designed to house known genetic information and to give scientists access to this information. One such program is called GAAS or "genome abundance and relative size". This program was originally developed to examine aquatic viruses on a global level. Viruses can be made up of both DNA and RNA that can be single stranded or double stranded. Viruses however, do not contain their own replication machinery and must utilize the machinery of their host to produce viral progeny. The GAAS program previously mentioned suggests that a large number of marine viruses have single stranded DNA as their genetic information type. However, most genomes of sequenced halophilic viruses appeared to be made up of double stranded DNA. According to GAAS the average size of the halophilic viral genomes were 51-263 kbps and the size of the viral genome appeared to be smaller when compared to that of its host. Another important aspect to take into consideration when studying genomics is whether or not portions of an organism's genome is "GC rich". Meaning that there is a higher percentage of guanine and cytosine nucleotide pairs in comparison to adenine and thymine base pairs. This information is important because due to the higher number of bonds between guanine and cytosine, it is thought that areas that are rich in these base pairs are considered to be more stable. Hence, there might be a higher GC concentration in certain areas within a organism's genome that encode for proteins that are vital for survival. If a protein is needed for survival, this would mean

that specific portion of the genome would be conserved across most organisms within that species. When studying halophilic viruses and their GC content, it was observed that species with a specific GC content appeared to cluster together [9].

### **6. Examples of halophilic viruses**

One of the first halophilic viruses to be analyzed at the molecular level was the virus ɸH. This temperate virus is known to infect archaea, specifically *Halobacterium salinarum*. ɸH's DNA is made up of double stranded DNA that is approximately 59kbp long and has a GC content percentage of 65% [9]. When it's genetic material was compared to that of it's host, there were very few similarities. Once infection with this virus takes place, the virus exists are a prophage in a closed circular state. This virus' structure was also observed to be very reliant on the salt concentration of its environment. Further investigation of this halophilic virus led to the discovery of 8 different variants, termed ɸH1- ɸH8 [9]. Another virus to be isolated from *Halobacterium salinarum* is the halovirus ɸN. ɸN has a icosahedral morphology with a non-contractile tail. Its DNA is made up linear double stranded DNA and is 56 kbps long with a GC content of 70% [28]. This virus is also known to carry out a virulent lifestyle.

The next two halophilic viruses were both isolated from a saltern in Australia and are named HF1 and HF2. These two viruses have quite a few characteristics in common. They both have the tailed capsid morphology and belong to the viral family *myoviridae*. Both of their genetic materials consist of double stranded DNA and they have the same 55.8% GC content present. Both viruses are also very sensitive to low NaCl concentrations and if there is no magnesium present, a minimum of 2 M NaCl is required for them to be active. Also, when these viruses infect their host they carry out a persistent infection type lifestyle. While these viruses appear to be very similar, there is a small difference in their genome size as well as differences in the halophilic prokaryotes they can infect. HF1 has a 76kbp sized genome and has three known halophilic hosts: *haloferax*, *halobacterium* and *haloarcula*. HF2 on the other hand, has a 77kbp long genome and is only known to be able to infect *Halobacterium sacchoravorum*. Further investigation into the genome of HF2 revealed that it does have some similarities between the genome of a well-known mesophilic bacteriophage. It's genome also exhibited some mosaicism which leads scientists to think that it's genetic material originated from a variety of different organisms that are not necessarily of halophilic viral origin.

The first and only haloalkaliphile type virus known to date is the tailed virus, ɸCh1. This virus is made up of linear double stranded DNA and is 58.5 kbps long with a GC content of 62%. This host works by invading host and integrating itself into the hosts genome. From there the virus is known to carry out a temperate infection lifestyle and requires the molarity of NaCl to be at least 2 M in order to remain active. While scientists were further investigating ɸCh1, this led to the discovery of the methyltransferase gene and its corresponding protein MɸCh1. It is also interesting to note that the protein products produced from this particular virus app appear to be acidic with isoelectric points ranging from 3.3–5.2 [9].

The next two halophilic viruses to be discussed were the first spindle shaped or lemon shaped halophilic viruses discovered. Naturally these two viruses do have a lot of similarities as well. They both are believed to be distantly related to the *fuselloviridae* family but they were given their own new family classification within the *salterproviridae* family [9]. They also share a common host, *Haloarcula hispanica*, which they both infect in a chronic and persistent fashion [9]. They are also noted to be not strictly lytic and are not lysogenic. Their genetic material is made

### *Viruses of Extremely Halophilic Prokaryotes DOI: http://dx.doi.org/10.5772/intechopen.96720*

up of double stranded DNA with a 39–40% GC content percentage [9]. An interesting feature of both viruses is also that they are able to encode for their own DNA polymerase which aides in their DNA replication within the host cell [27]. There is a slight difference in the sizes of these viruses, His-1 being 15 kbps long and His-2 being 16kbp long [9]. Another interesting discovery when further investigation the virus His-2, transfection experiments were carried out and this seemed to lead to broadening this viruses host range [9]. His-2 after the transfection experiments was then able to infect not only *Haloarcula hispanica* but also several *Haloferax*, *Halorubrum*, *Haloterrigena turkmenica*, and *Natrialba asiatica* species [9].

This next halophilic virus has been one of the most extensively studied viruses, SH1. This virus was isolated from a salt lake in Australia and it has a spherical morphology with a layered shell surrounded by a protein capsid that also has spikes [9, 24]. This structure is similar to other well-known mesophilic and hyperthermophilic phages such as PRD-1 – a gram negative phage, Bam35 – a gram positive phage, PBCV-1 – a algal virus and STIV – a hyperthermophilic virus [9]. Its genome is a total of 30kbp long and is made up of double stranded DNA [9]. This virus is known to have two different hosts, *Halorubrum sodomense* and *Haloarcula hispanica.*

HRPV-1 is the shortest halophilic virus known measuring at only 7 kbps long. Its genetic material is made up single stranded DNA with a GC content percentage of 54.2% [9]. It is also pleomorphic in morphology and is surrounded by a lipid envelope [9]. Its hosts consist of species within the *Halorubrum* genus. Another example of a short halophilic virus is the virus HHPV-1. It has a genome that is 8kbp in length with a GC content percentage of 56% [9]. Its structural morphology is also pleomorphic, and its DNA is a circular double stranded DNA [9]. The host for this virus is the archaeon *Haloarcula hispanica* [9].

Another identified halophilic virus is the spherical virus, SNJ1. This isolate was collected from its hosts within the *Natrinema* species [28]. It's genetic material is composed of circular double stranded DNA and is approximately 16.3 kbps long [25, 28]. Within its genetic sequence, 48.8–69.7% of that sequence is of GC content [28]. While this virus' genome has been further investigated it was noted that it is actually identical to a known plasmid pHH205 [29]. Another halophilic virus also isolated from species within the *Natrinema* genus is SNJ2. This virus is the first virus to be pleomorphic in regard to morphology that also carries out a temperate lifestyle [26]. The genetic material that makes up SNJ2 is a discontinuous double stranded circular DNA that is 16.9 kbps long with a GC content of 59.1% [30]. Further research into this virus suggests that the amount of SNJ2 viral particles present is dependent on the presence of the virus SNJ1 within the host. This is because SNJ1 is thought to act similarly to a plasmid, without which, ample amounts of SNJ2 is not produced [26].
