Reply to ”Questioning the Use of Zika Virus Injection in Dogs with Advanced-Stage Brain Tumors”
Authors: Carolini Kaid, Mayana Zatz
Journal: Molecular Therapy (2020)

To the editor: The questions and criticisms raised by Bienzle et al. are a misunderstanding of our results and aims of our report, which we are happy to clarify. Although we agree that there were minor errors, they certainly do not invalidate our observations and conclusions. We did not state that the three dogs had CNS tumors with a neural origin, but brain tumors, which could include tumors of extra-axial origin. Although some results are shown in the three cases, each case is unique and was presented separately, as case reports. In case 1, the meningioma diagnosed antemortem was confirmed in postmortem histopathological analysis. The intracranial lesion located in the frontal lobe was observed in the first MRI and, at 7 days post-injection (DPI), it appeared with a lower contrast in the magnetic resonance imaging (MRI) (Data S1). However, no tumor mass was observed in MRI images at 14 DPI, and the histopathological data showed no presence of tumor cells, precluding the diagnostic confirmation. The Pitbull dog was in the pre-coma state when brought to the clinic and was included to assess the safety of the study, as specified in the paper. In case 2, the right testicle had a Leydigoma and the left one a seminoma. However, just the right one showed severe inflammatory infiltrate, degeneration, and multifocal necrosis. If caused by Zika virus (ZIKV), it should have been observed in both testes. Thus, we suggested that the presence of Leydigoma tumor, rather than ZIKV, could be the cause of the right testis inflammation. The article mentioned by Bienzle et al. refers to the study of Ma et al.,1Ma W. Li S. Ma S. Jia L. Zhang F. Zhang Y. Zhang J. Wong G. Zhang S. Lu X. et al.Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice.Cell. 2016; 167: 1511-1524.e10Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar performed in knockout mice. However, in wild-type mice, “no abnormalities were observed at 8 DPI.” We believe that normal pet dogs are comparable to the wild-type mice, which presented no abnormalities in the genital tract after ZIKV administration. In humans, previous studies showed that the ZIKV replication in the testes was temporary, reversible, and caused no permanent damage to the reproductive function or fertility.2Oliveira D.B.L. Durigon G.S. Mendes É.A. Ladner J.T. Andreata-Santos R. Araujo D.B. Botosso V.F. Paola N.D. Neto D.F.L. Cunha M.P. et al.Persistence and Intra-Host Genetic Evolution of Zika Virus Infection in Symptomatic Adults: A Special View in the Male Reproductive System.Viruses. 2018; 10: 615Crossref Scopus (17) Google Scholar Importantly, male dogs can be castrated, avoiding any further complications. In case 3, the detection of less than 10% of ZIKV-positive cells was expected since meningioma is a CNS tumor with no neural origin. A new study confirmed that ZIKV was able to decrease 20% of cell viability in two meningioma human cell lines.3Zhu Z. Mesci P. Bernatchez J.A. Gimple R.C. Wang X. Schafer S.T. Wettersten H.I. Beck S. Clark A.E. Wu Q. et al.Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin αvβ5 Axis.Cell Stem Cell. 2020; 26: 187-204.e10Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The most important point here is to confirm that ZIKV induced an anti-tumoral effect recruiting the immune system without causing any damage in the normal CNS tissue. The alternative hypothesis suggested by Bienzle et al. is unlikely since it requires necrosis in the CORE region of the tumor mass with clinical worsening.4Lee S.Y. Ju M.K. Jeon H.M. Jeong E.K. Lee Y.J. Kim C.H. Park H.G. Han S.I. Kang H.S. Regulation of Tumor Progression by Programmed Necrosis.Oxid. Med. Cell. Longev. 2018; 2018 (3537471)Crossref Scopus (70) Google Scholar In cases 2 and 3, the most necrotic area was found in the tumor border, and the treatment improved overall survival with good quality of life of 150 and 80 days, respectively. The affirmation that “immunofluorescent images did not correlate with histopathology of the meningioma” is unjustified. For diagnosis confirmation, 10 different markers were used for histopathological analysis (Figure S2). All hemotoxylin and eosin staining, immunochemistry, and immunofluorescence images, were taken from random fields. Regarding the detection of ZIKV antigen, safety was widely discussed in the article and it was the first/main concern of the study. Viral presence was analyzed by RT-PCR and ZIKV antigen detection not only in unaffected CNS tissue, but also in non-CNS tissues. All three cases were included in this study after not responding to the standard-of-care therapies and glucocorticoid high dose administration failure. Dogs 2 and 3 received low glucocorticoid inflammatory doses (dexamethasone 0.3 mg/kg), resulting in a quick clinical improvement. Thus, the glucocorticoid fast response during ZIKV treatment is unlikely to represent a typical “short-term clinical improvement”. The viremia quantified in blood aimed to assess safety before the dog could return home. The approach used was the same reported by Hirsch et al.5Hirsch A.J. Smith J.L. Haese N.N. Broeckel R.M. Parkins C.J. Kreklywich C. DeFilippis V.R. Denton M. Smith P.P. Messer W.B. et al.Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues.PLoS Pathog. 2017; 13: e1006219Crossref PubMed Scopus (152) Google Scholar in rhesus macaques. Dog 1 presented a viremia of 4.4 copies/μL, below the detection limit (∼100 copies) measured with RT-PCR, the most sensitive technique. The blood viremia analysis was repeated until 9 DPI, and no viral particle was found. In short, the absence of peripheral viremia in case 1 supports the safety of the procedure, which was the main objective of the present study. We agree that our report cases represent a small original clinical trial, which strongly suggests a potential oncolytic effect of ZIKV injections. We show sufficient evidence to motivate the scientific community to investigate the ZIKV oncolytic effect mechanisms as a potential new therapy against brain tumors, for which there is no effective treatment for both dogs and humans. Before translating this therapy to the veterinary clinic, a larger clinical study must be done and the oncolytic mechanism of ZIKV must be better understood.