Hepatitis E Virus Genotype 3 among Hemodialysis Patients in Mexico: First Identification of Chronic Infection.

The global distribution of hepatitis E virus (HEV) is attributed to its capacity to spread through several routes of transmission; hemodialysis has gained increased amounts of attention in recent years. Although Mexico is considered a hyperendemic region for hepatitis E, no HEV surveillance is performed in the country. The frequency of HEV in hemodialysis (HD) patients has not been determined. Herein, we conducted a cross-sectional single-center analytical study including 67 serum samples from HD patients. Anti-HEV IgG and IgM antibodies and the viral genome were determined; partial regions within the HEV genome were sequenced for further phylogenetic analysis. Globally, 14.9% of the tested patients exhibited reactivity for IgG antibodies against HEV, and none showed reactivity to IgM. A total of 5.9% of the samples showed HEV genome amplification, and sequencing confirmed the identity of genotype 3; subsequent analysis of positive cases revealed two acute cases and chronic hepatitis E infection in one patient. Notably, the chronic patient was negative for anti-HEV IgG antibodies. Our findings highlight the importance of viral genome testing in HD patients and the need to establish guidelines for HEV detection in Mexico.

Tracing the History of Hepatitis E Virus Infection in Mexico: From the Enigmatic Genotype 2 to the Current Disease Situation.

Hepatitis E virus (HEV) is the major cause of acute viral hepatitis worldwide. This virus is responsible for waterborne outbreaks in low-income countries and zoonosis transmission in industrialized regions. Initially, considered self-limiting, HEV may also lead to chronic disease, and evidence supports that infection can be considered a systemic disease. In the late 1980s, Mexico became a hot spot in the study of HEV due to one of the first virus outbreaks in Latin America related to enterically transmitted viral non-A, non-B hepatitis. Viral stool particles recovered from Mexican viral hepatitis outbreaks represented the first identification of HEV genotype (Gt) 2 (Gt2) in the world. No new findings of HEV-Gt2 have been reported in the country, whereas this genotype has been found in countries on the African continent. Recent investigations in Mexico have identified other strains (HEV-Gt1 and -Gt3) and a high frequency of anti-HEV antibodies in animal and human populations. Herein, the potential reasons for the disappearance of HEV-Gt2 in Mexico and the advances in the study of HEV in the country are discussed along with challenges in studying this neglected pathogen. These pieces of information are expected to contribute to disease control in the entire Latin American region.

Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases.

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10⁻³ M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s⁻¹, and 1.2 +/- 0.105 × 104 M⁻¹s⁻¹, respectively. The L-asparaginase activity was reduced in the presence of Mn²âº, Zn²âº, Ca²âº, and Mg²âº metal ions for about 52% to 31%. In addition, we found that NH4⁺, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.

The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli.

A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a DeltarelA DeltaspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Omega element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-beta-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.

Regulation of transcription and activity of Rhizobium etli glutaminase A.

The present study determines the regulatory mechanisms that operate on Rhizobium etli glutaminase A. glsA gene expression levels were evaluated under several metabolic conditions by fusions of the glsA gene promoter and the transcriptional reporter cassette uidA2-aad. glsA expression was directly correlated to the glutaminase A activity found under the tested growth conditions, reaching its maximum level in the presence of glutamine and during exponential growth phase. Glutamine induces glsA expression. The influence of allosteric metabolites on glutaminase A activity was also determined. The purified enzyme was inhibited by 2-oxoglutarate and pyruvate, whereas oxaloacetate and glyoxylate modulate it positively. Glutaminase A is not inhibited by glutamate and is activated by ammonium. Glutaminase A participates in an ATP-consuming cycle where glutamine is continually degraded and resynthesized by glutamine synthetase (GS). GS and glutaminase A activities appear simultaneously during bacterial growth under different metabolic conditions and their control mechanisms are not reciprocal. Slight overproduction in glutaminase A expression causes a reduction in growth yield and a dramatic decrease in bacterial growth. We propose a model for regulation of glutaminase A, and discuss its contribution to glutamine cycle regulation.