Genotypic Characterization of Herpes Simplex Virus Type 1 Isolates in Immunocompromised Patients in Rio de Janeiro, Brazil

<div><p>Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that causes a variety of diseases, including an increased risk of developing more severe disease in HIV-infected individuals. In Brazil, there is no information about the molecular epidemiology of HSV-1 infection, especially in HIV-infected individuals. The aim of this study was to perform the genotypic characterization of HSV-1 among HIV-infected patients. A total of 214 serum samples from HIV-positive patients without HSV infection symptoms were enrolled in one of two reference hospitals for HIV infection managing in Rio de Janeiro. The gG and gI genes were analyzed by restriction fragment length polymorphism (RFLP) and full nucleotide sequencing of the US8 (1601 bp), UL44 (1996 bp), and UL23 (1244 bp) regions was performed. A total of 38.3% (82/214) and 32.7% (70/214) of the serum samples tested positive for gG and gI genes, respectively. RFLP analysis classified the HSV-1 as belonging to genotype A. Phylogenetic analysis of the Brazilian samples for the US8, UL44, and UL23 regions demonstrated that the nucleotide identity between Brazilian samples was higher than 97% for all genes. No acyclovir mutation was detected in the patients. The shedding of HSV in the serum samples from HIV-positive patients who were asymptomatic for HSV infection was detected in this work. This is the first report of molecular characterization of HSV-1 in Brazilian samples since there is no previous data available in the literature concerning the genotypic classification and stable distribution of Brazilian strains of HSV-1 in Rio de Janeiro, Brazil.</p></div

Primers used for amplification and sequencing of the UL23, UL44, and US8 genes of HSV-1.

<p>*F, forward; R, reverse; bp, base pairs.</p><p>Primers used for amplification and sequencing of the UL23, UL44, and US8 genes of HSV-1.</p

Characteristics and molecular information of HIV/HSV-positive samples.

<p>Characteristics and molecular information of HIV/HSV-positive samples.</p

The Bayesian phylogenetic tree constructed by using nucleotide sequence of UL44 (1996 bp).

<p>The GenBank accession number is shown for each sequence used. Posterior probabilities are shown at the branch label. Brazilian sequences are noted in red. Color code: Africa—Green, Asia—Blue, Europe—Pink, United States—purple, Brazil—red.</p

The Bayesian phylogenetic tree constructed by using nucleotide sequence of US8 coding region (1601 bp).

<p>The GenBank accession number is shown for each sequence used. Posterior probabilities are shown at the branch label. Brazilian sequences are noted in red. Color code: Africa—Green, Asia—Blue, Europe—Pink, United States—purple, Brazil—red.</p

Cynomolgus monkeys are successfully and persistently infected with hepatitis E virus genotype 3 (HEV-3) after long-term immunosuppressive therapy

<div><p>Epidemiological studies found that hepatitis E virus genotype 3 (HEV-3) infection was associated with chronic hepatitis and cirrhosis in immunocompromised patients. Our study aimed to investigate the relationship between the host immunosuppressive status and the occurrence of HEV-related chronic hepatitis. Here we describe a successful experimental study, using cynomolgus monkeys previously treated with tacrolimus, a potent calcineurin inhibitor immunosuppressant, and infected with a Brazilian HEV-3 strain isolated from naturally infected pigs. HEV infected monkeys were followed up during 160 days post infection (dpi) by clinical signs; virological, biochemical and haematological parameters; and liver histopathology. The tacrolimus blood levels were monitored throughout the experiment. Immunosuppression was confirmed by clinical and laboratorial findings, such as: moderate weight loss, alopecia, and herpes virus opportunistic infection. In this study, chronic HEV infection was characterized by the mild increase of liver enzymes serum levels; persistent RNA viremia and viral faecal shedding; and liver histopathology. Three out of four immunosuppressed monkeys showed recurrent HEV RNA detection in liver samples, evident hepatocellular ballooning degeneration, mild to severe macro and microvesicular steatosis (zone 1), scattered hepatocellular apoptosis, and lobular focal inflammation. At 69 dpi, liver biopsies of all infected monkeys revealed evident ballooning degeneration (zone 3), discrete hepatocellular apoptosis, and at most mild portal and intra-acinar focal inflammation. At 160 dpi, the three chronically HEV infected monkeys showed microscopic features (piecemeal necrosis) corresponding to chronic hepatitis in absence of fibrosis and cirrhosis in liver parenchyma. Within 4-months follow up, the tacrolimus-immunosuppressed cynomolgus monkeys infected with a Brazilian swine HEV-3 strain exhibited more severe hepatic lesions progressing to chronic hepatitis without liver fibrosis, similarly as shown in tacrolimus-immunosuppressed solid organ transplant (SOT) recipients. The cause-effect relationship between HEV infection and tacrolimus treatment was confirmed in this experiment.</p></div

Gender, age, sex, and body weight of the cynomolgus monkeys used in this study.

<p>Gender, age, sex, and body weight of the cynomolgus monkeys used in this study.</p

Dynamic of HEV RNA measured by quantitative reverse transcription PCR qRT-PCR.

<p>Immunocompetent monkeys (G1) were inoculated with HEV-3; tacrolimus-treated monkeys inoculated with HEV-3 (G2) and tacrolimus-treated monkeys (G3). qRT-PCR assay were performed on RNA extracted from sera, liver biopsies and faeces samples. The viral load results are show in: <b>(A)</b> sera, <b>(B)</b> liver biopsies, and <b>(C)</b> faeces.</p

Phylogenetic analysis of Hepatitis E virus strains from human and animal samples.

<p>The Bayesian phylogenetic tree was constructed by using concatenated partial nucleotide sequence of ORF1 and ORF2 (546 nt) of HEV. For each sequence used, the GenBank accession number, animal species from which it was isolated and the corresponding genotype are shown. The tree was rooted at midpoint. Posterior probabilities (pp) are shown at the branch label. Numerical value ≥ 0.7 indicates the pp replicates that supported the interior branch. Newly described HEV sequences in this study are indicated. Scale bar indicates evolutionary distance of 0.2 substitutions per position in the sequence.</p

Chronological analysis of histopathological features of liver biopsies from immunocompetent cynomolgus monkeys infected with HEV (G1), monkeys previously treated with tacrolimus and infected with HEV (G2) and monkeys only treated with tacrolimus (G3).

<p><b>Hematoxylin-eosin (H&E)–stained paraffin section. (G1A)</b> Lobular architecture disorganization, ballooning degeneration of the hepatocytes with lytic necrosis. Acidophilic clumps (arrow) are located in a perinuclear position (Mallory bodies). <b>(G1B)</b> The liver cytoarchitecture was modified by ballooning and lytic necrosis of hepatocytes. Lipid droplets and inflammatory cell infiltration were observed. <b>(G1C)</b> Hepatic parenchyma shows significantly fat droplets deposition, mixture of macrosteatosis and microsteatosis. <b>(G1D)</b> Focal collection of lymphocytes and macrophages localized in the pericentral area. Ballooned hepatocytes and lytic necrosis were noted. <b>(G2A)</b> Irregular distribution pattern of hepatocytes, ballooning ad cytolitic necrosis associated with fatty changes. <b>(G2B)</b> Normal liver architecture. Lymphohistiocytic infiltration of portal liver tract. Microvesicular steatosis is also present. <b>(G2C)</b> Disarray of the cytoarchitecture of the parenchyma. Hepatocytes exhibiting ballooning, lytic necrosis and steatosis. Note glycogen accumulation in hepatocyte nuclei. <b>(G2D)</b> Interface hepatitis surrounded by micro and macrosteatosis. <b>(G3A)</b> Normal hepatic venule. Hepatocytes plates shows regular distribution. <b>(G3B)</b> Lobular disarray and hepatocellular ballooning predominantly distributed in zone 3. <b>(G3C)</b> Significative and strong diffusely distributed mixture of hepatocellular macrosteatosis and microsteatosis in all zones from zone 1 to 3. <b>(G3D)</b> Hepatocytes cords converging towards portal tract. Minimal fat droplets deposition in the hepatocyte cytoplasm.</p