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Herpes simplex virus type 1 (HSV-1), an enveloped DNA virus, causes a variety of infections in humans. Primary infection usually occurs during childhood and subsequent to the initial outbreak, the virus enters the peripheral nervous system, residing there permanently in a latent state of infection; it is reactivated by the proper stimulus and causes recurrence of symptoms. Serious infection with HSV-1 can also lead to life-threatening encephalitis and ocular infections that result in corneal inflammation and scarification [24, 26]. Immunocompromised individuals and the recipients of organ transplantations are at high risk for increased severity of HSV-1 infection [6, 9]. In addition, HSV-1 has been shown to be a factor for spreading human immunodeficiency virus and causing severe diseases in AIDS patients [20, 21].
Currently, most of the treatments for HSV are based on nucleoside analogs of guanine, for example, acyclovir (ACV) is specifically phosphorylated by viral thymidine kinase in infected cells [17, 24]. However, widespread use of ACV has shown HSV develops resistance to ACV through mutations in genes coding for thymidine kinase or for DNA polymerase [4, 10, 11]. Thus, some immunocompromised patients and organ transplant recipients with recurrent HSV lesions develop resistance to ACV after repeated treatments [20, 21]. Therefore, it is important to develop new antiviral drugs with different mechanisms of action which can substitute for, or complement, acyclovir.
New types of antiviral agents from natural sources, especially those that possess high efficacy on resistant mutant viral strains and low toxicity to the host, are considered to be the most promising. One such candidate is cyanovirin-N (CV-N), a 101-amino acid protein (11 kDa) with known three-dimensional structure, that was originally isolated from an aqueous extract of the cyanobacterium Nostoc ellipsosporum [4]and later produced recombinantly in Escherichia coli [12]as an active agent against HIV. The recombinant CV-N is identical to natural CV-N in structure and bioactivity. CV-N contains two sequence repeats, 50 and 51 amino acids long, which exhibit significant similarity and equivalently positioned disulfide bonds. No similarity with any other proteins thus far deposited in published databases has been reported [2, 3, 13].
In our previous study, we have produced the purified and renatured recombinant CV-N protein in Escherichia coli with high efficiency [15]. In this report, we describe the activity of CV-N against HSV-1 in vitro, and further show the ability of CV-N to treat HSV-1 infection in mice.
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The MTT assay showed that CV-N had little cytotoxic effect on the proliferation of Vero cells, with a CC50 value of 359.03±0.56 μg/mL. The trypan blue exclusion assay indicated the viable cell numbers in the CV-N treated and untreated cells were the same, confirming that the CV-N had little toxic effects on the cells.
To explore whether the antiviral activity of CV-N was due to virucidal activity, we evaluated the TCID50 of HSV-1 after treatment with various concentrations of CV-N for 2 h. The result indicated the TCID50 of HSV-1 was the same in all groups treated with various concentrations of CV-N (0μg/mL, 6.25μg/mL, 12.50 μg/mL, 25μg/mL, 50μg/mL, 100μg/mL, 200μg/mL), all the cells showed typical CPE of HSV-1, suggesting that CV-N had no virucidal effect on HSV-1.
When CV-N were added after infection, CV-N showed significant inhibitory effect on HSV-1, with a treatment index (TI) of 158.86, and mild inhibitory effect on HSV-1 when added before infection, with a TI of 11.90 (Table 1).
Table 1. Mode of action of CV-N on HSV-1
To further confirm whether CV-N could repress HSV-1 DNA replication, the number of copies of HSV-DNA was detected by real-time FQ-PCR. The results showed that the copies of HSV-1 DNA significantly decreased with the treatment of CV-N from 2 h to 18 h and the inhibitory rate on HSV-DNA increased in a time-dependent manner (Table 2).
Table 2. The inhibition of CV-N on HSV-1 DNA replication
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The symptoms of mice herpes encephalitis appeared 3-5d post infection varying greatly depending on the dosages of drugs. In the untreated virus control group, the mice showed a tendency to restless, convulsions, tremor, behavior changes and weight loss; even more drowsiness and paralysis were observed 2-3d post infection. On d5, animals of this group began to die, and by d7, all of them had died. In the 0.5mg/kg CV-N treated group, the mice began to show the typical symptoms 3-4 d post infection. On 5 d, animals in this group began to die, and by 8 d, all of them had died. In the 5mg/kg CV-N treated group, the mice suffered light symptoms and the survival days were more than 9d. However 10mg/kg CV-N treated mice had very light symptoms and all survived more than 14d. Compared with the untreated control group, 10 mg/kg CV-N was effective in treating encephalitis in mice (Fig. 1).
Figure 1. Treatment of CV-N on herpes encephalitis in mice. Kunming mice were infected intracerebrally with HSV-1 (10-4 TCID50/mL). CV-N were administered intraperitoneally in a 500μL inoculum volume at 2h, 3d, 5d & 7d post infection respectively. Control animals were treated with normal saline instead of CV-N.
As shown microscopically, in the viral control group and the 0.5mg/kg CV-N treated group, the brain cells showed typical herpes simplex encephalitis pathological changes, such as nerve cell swelling, pyknosis of the nucleolus, edema of the perivascular space and intraventricular hemorrhaging. In the 5mg/kg CV-N treated group and 10mg/kg CV-N treated group however, the brain cells did not have visible changes, except for slight inflammation (Fig. 2).
Figure 2. Pathological changes in brain tissue of herpes encephalitis in mice (HE staining). A: HSV-1 infected mice treated with normal saline: severe swelling of the nerve cells, pyknosis nucleolus and perivascular edema. B: HSV-1 infected mice treated with low dose of CV-N(0.5mg/kg): mild swelling of the nerve cells and perivascular edema. C: HSV-1 infected mice treated with median dose of CV-N (5mg/kg): cells have no visible changes, except slight inflammation. D: HSV-1 infected mice treated with high dose of CV-N (10mg/kg): cells have no visible changes, except slight inflammation.