Clinical, virological and epidemiological characterization of dengue outbreak in Myanmar, 2015.

Hospital-based surveillance was conducted at two widely separated regions in Myanmar during the 2015 dengue epidemic. Acute phase serum samples were collected from 332 clinically diagnosed dengue patients during the peak season of dengue cases. Viremia levels were measured by quantitative real-time PCR and plaque assays using FcγRIIA-expressing and non-FcγRIIA-expressing BHK cells to specifically determine the infectious virus particles. By serology and molecular techniques, 280/332 (84·3%) were confirmed as dengue patients. All four serotypes of dengue virus (DENV) were isolated from among 104 laboratory-confirmed patients including two cases infected with two DENV serotypes. High percentage of primary infection was noted among the severe dengue patients. Patients with primary infection or DENV IgM negative demonstrated significantly higher viral loads but there was no significant difference among the severity groups. Viremia levels among dengue patients were notably high for a long period which was assumed to support the spread of the virus by the mosquito vector during epidemic. Phylogenetic analyses of the envelope gene of the epidemic strains revealed close similarity with the strains previously isolated in Myanmar and neighboring countries. DENV-1 dominated the epidemic in 2015 and the serotype (except DENV-3) and genotype distributions were similar in both study sites.

Retrospective seroepidemiological study of chikungunya infection in South Asia, Southeast Asia and the Pacific region.

Chikungunya virus (CHIKV) and Ross River virus (RRV) of the genus Alphavirus, family Togaviridae are mainly transmitted by Aedes mosquitoes and the symptoms they cause in patients are similar to dengue. A chikungunya (CHIK) outbreak re-emerged in several Asian countries during 2005-2006. This study aimed to clarify the prevalence of CHIKV infection in suspected dengue patients in six countries in South Asia and Southeast Asia. Seven hundred forty-eight serum samples were from dengue-suspected patients in South Asia and Southeast Asia, and 52 were from patients in Fiji. The samples were analysed by CHIKV IgM capture ELISA, CHIKV IgG indirect ELISA and focus reduction neutralization test against CHIKV or RRV. CHIK-confirmed cases in South Asia, particularly Myanmar and Sri Lanka, were 4·6%, and 6·1%, respectively; and in Southeast Asia, particularly Indonesia, the Philippines and Vietnam, were 27·4%, 26·8% and 25·0%, respectively. It suggests that CHIK was widely spread in these five countries in Asia. In Fiji, no CHIK cases were confirmed; however, RRV-confirmed cases represented 53·6% of suspected dengue cases. It suggests that RRV is being maintained or occasionally entering from neighbouring countries and should be considered when determining a causative agent for dengue-like illness in Fiji.

Japanese encephalitis.

Japanese encephalitis (JE) is an inflammation of the central nervous system in humans and animals, specifically horses and cattle. The disease, which can sometimes be fatal, is caused by the flavivirus Japanese encephalitis virus (JEV), of which there are five genotypes (genotypes 1, 2, 3, 4 and 5). The transmission cycle of the virus involves pigs and wild birds as virus amplifiers and mosquitoes as vectors for transferring the virus between amplifying hosts and to dead- end hosts, i.e. humans, horses and cattle. In horses and cattle the disease is usually asymptomatic, but when clinical signs do occur they include fever, decreased appetite, frothing at the mouth, rigidity of the legs and recumbency, and neurological signs, such as convulsive fits, circling, marked depression and disordered consciousness. In pigs, it can cause abortion and stillbirths. At present, the virus is detected in a wide area covering eastern and southern Asia, Indonesia, northern Australia, Papua New Guinea and Pakistan. JEV RNA has also been detected in Italy, first in dead birds in 1997 and 2000 and then in mosquitoes in 2010. Genotype shift, i.e. a change of genotype from genotype 3 to genotype 1, has occurred in some countries, namely Japan, South Korea, Chinese Taipei and Vietnam. Laboratory methods are available for confirming the causative agent of the disease. There are control measures to prevent or minimise infection and, among them, vaccination is one of the most important and one which should be adopted in endemic and epidemic areas.

Association of increased platelet-associated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections.

Severe thrombocytopenia and increased vascular permeability are two major characteristics of dengue haemorrhagic fever (DHF). To develop a better understanding of the roles of platelet-associated IgG (PAIgG) and IgM (PAIgM) in inducing thrombocytopenia and its severity of disease in patients with secondary dengue virus infection, the relationship between the PAIgG or PAIgM levels and disease severity as well as thrombocytopenia was examined in 78 patients with acute phase secondary infection in a prospective hospital-based study. The decrease in platelet count during the acute phase recovered significantly during the convalescent phase. In contrast, the increased levels of PAIgG or PAIgM that occurred during the acute phase of these patients decreased significantly during the convalescent phase. An inverse correlation between platelet count and PAIgG or PAIgM levels was found in these patients. Anti-dengue virus IgG and IgM activity was found in platelet eluates from 10 patients in an acute phase of secondary infection. Increased levels of PAIgG or PAIgM were significantly higher in DHF than those in dengue fever (DF). An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with a high specificity. Our present data suggest that platelet-associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary dengue virus infections.

IgM-capture ELISA of serum samples collected from Filipino dengue patients.

Viral antigens for 4 dengue serotypes were produced in C6/36 Aedes albopictus cells. These were used as assay antigens for IgM-capture ELISA to detect IgM antibodies in sera of dengue patients from 3 hospitals in Metro Manila, Philippines. A total of 378 serum samples came from National Children's Hospital (NCH), San Lazaro Hospital (SLH), and St Luke's Medical Center (SLMC), from January to November 1995. Three hundred and four (304) out of 378 serum samples, or 80.42% showed positive IgM ELISA titer against at least one of the 4 assay antigens. Dengue type 4 (D4) antigen detected antibodies in 61.90% (234/378) of these serum samples, whereas type 1 (D1), type 3 (D3), and type 2 (D2) had detection rates of 60.05% (227/378), 50.79% (192/378) and 49.47% (187/378) respectively. Although the results show that both D1 and D4 are the most effective antigens in identifying dengue infections for this batch of samples, the use of a cocktail of antigens is still recommended. The results of this study are the basis for the IgM-capture ELISA protocol presently applied for the laboratory confirmation of dengue cases in the Philippines.

Degradation of vitellogenins by 170 kDa trypsin-like protease in the plasma of the tilapia, Oreochromis niloticus.

Proteolytic degradation of plasma vitellogenins during purification procedure has been noted in several teleost fishes. We have characterized here a trypsin-like serine protease in the plasma of the tilapia, Oreochromis niloticus, which degrades vitellogenins. The molecular mass of the protease was estimated as 230 kDa by gel filtration and as 170 kDa both by nondenaturing and by SDS-polyacrylamide gel electrophoresis. The protease efficiently hydrolyzed the synthetic peptide substrates for trypsin-like proteases but not the substrates for chymotrypsin-like proteases nor aminopeptidases. Hydrolysis of the peptide substrates was strongly inhibited by leupeptin, aprotinin and N-tosyl-L-lysine chloromethyl ketone and to certain extent by chymostatin, 3,4-dichloroisocoumarin, phenylmethanesulfonyl fluoride, and soybean trypsin inhibitor. Leupeptin and aprotinin also inhibited the degradation of a vitellogenin in the plasma. Although the physiological functions of the 170 kDa protease in vivo have not been elucidated, the results on exzymatic properties of this protease will be useful for the isolation and characterization of vitellogenin not only in tilapia but also in other organisms.

Vitellogenins of Oreochromis niloticus: identification, isolation, and biochemical and immunochemical characterization.

Two native forms of vitellogenin (EIP1 and EIP2) were identified in the plasma of Oreochromis niloticus. They were present in females and were estrogen-inducible in males. Both were phosphoglycolipoproteins and both immunoreacted with the antiserum raised against egg proteins. Two prominent bands (EIpp1 and EIpp2; corresponding to 185 and 120 kDa, respectively), observed on sodium dodecyl sulfate-polyacrylamide gels, were induced by estradiol treatment of males and immunoreacted with the antiserum against egg proteins. EIP1 and EIP2 were isolated by precipitation with Mg2+ ions and ethylenediamine tetraacetic acid, preparative polyacrylamide gel electrophoresis, and electroelution. During purification the fractions containing EIP1 and EIP2 also retained EIpp2 and EIpp1, respectively. Immunoblot analyses using affinity-purified antibodies against EIP1, EIP2, EIpp1, and EIpp2 confirmed that EIP1 and EIP2 were at least composed of EIpp2 and EIpp1, respectively. These results suggest that at least two immunochemically different proteins are induced and are secreted into the blood to serve as vitellogenin and to provide a source of nutrient for the developing embryo of Oreochromis niloticus.