Prevalence of intrathecal acyclovir resistant virus in herpes simplex encephalitis patients

Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR-associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real-time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients. Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients. Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV-sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients

Prevalence of Intrathecal Acyclovir Resistant Virus in Herpes Simplex Encephalitis Patients.

Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR-associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real-time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients. Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients. Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV-sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients

Immunity to TBEV Related Flaviviruses with Reduced Pathogenicity Protects Mice from Disease but Not from TBEV Entry into the CNS.

Tick-borne encephalitis virus (TBEV) is a leading cause of vector-borne viral encephalitis with expanding endemic regions across Europe. In this study we tested in mice the efficacy of preinfection with a closely related low-virulent flavivirus, Langat virus (LGTV strain TP21), or a naturally avirulent TBEV strain (TBEV-280) in providing protection against lethal infection with the highly virulent TBEV strain (referred to as TBEV-Hypr). We show that prior infection with TP21 or TBEV-280 is efficient in protecting mice from lethal TBEV-Hypr challenge. Histopathological analysis of brains from nonimmunized mice revealed neuronal TBEV infection and necrosis. Neuroinflammation, gliosis, and neuronal necrosis was however also observed in some of the TP21 and TBEV-280 preinfected mice although at reduced frequency as compared to the nonimmunized TBEV-Hypr infected mice. qPCR detected the presence of viral RNA in the CNS of both TP21 and TBEV-280 immunized mice after TBEV-Hypr challenge, but significantly reduced compared to mock-immunized mice. Our results indicate that although TBEV-Hypr infection is effectively controlled in the periphery upon immunization with low-virulent LGTV or naturally avirulent TBEV 280, it may still enter the CNS of these animals. These findings contribute to our understanding of causes for vaccine failure in individuals vaccinated with TBE vaccines

Viral thymidine kinase polymorphisms in clinical samples obtained from herpes simplex encephalitis patients.

<p>Viral thymidine kinase polymorphisms in clinical samples obtained from herpes simplex encephalitis patients.</p

Demographics and clinical characteristics of herpes simplex encephalitis patients.

<p>Demographics and clinical characteristics of herpes simplex encephalitis patients.</p

Location of identified amino acid substitutions in HSV thymidine kinase.

<p>Schematic representation of all identified amino acid substitutions in the thymidine kinase (TK) proteins of intrathecal HSV-1 (<b>A</b>) and HSV-2 (<b>B</b>) isolates obtained from herpes simplex encephalitis patients. Note that except for the D286E mutation in HSV-1 all amino acid substitutions are located outside of the functional domains of TK. Grey boxes depict highly conserved and functional domains, i.e. ATP-binding site (ATP), nucleotide binding sites (NBS) and a cysteine residue at position 336 and 337 for HSV-1 and HSV-2, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155531#pone.0155531.ref031" target="_blank">31</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155531#pone.0155531.ref032" target="_blank">32</a>]. TK amino acid locations are indicated according to HSV-1 reference strain 17 (GenBank accession number: JN555585.1) and HSV-2 reference strain HG52 (NCBI accession number: NC_001798.1). Regular font: natural polymorphisms; <b>bold</b>: new mutation; <u><b>bold and underlined</b></u>: mutation with unclear significance for ACV resistance.</p

Acyclovir susceptibility of HSV isolates obtained from mucocutaneous samples of herpes simplex encephalitis patients.

<p>Acyclovir susceptibility of HSV isolates obtained from mucocutaneous samples of herpes simplex encephalitis patients.</p