Institut Pasteur in Ho Chi Minh City

Human-to-Human Coronavirus Transmission in Vietnam The authors describe transmission of 2019-nCoV from a father, who had flown with his wife from Wuhan to Hanoi, to the son, who met his father and ...

BACKGROUND: A candidate tetravalent dengue vaccine is being assessed in three clinical trials involving more than 35,000 children between the ages of 2 and 16 years in Asian-Pacific and Latin American countries. We report the results of long-term follow-up interim analyses and integrated efficacy analyses. METHODS: We are assessing the incidence of hospitalization for virologically confirmed dengue as a surrogate safety end point during follow-up in years 3 to 6 of two phase 3 trials, CYD14 and CYD15, and a phase 2b trial, CYD23/57. We estimated vaccine efficacy using pooled data from the first 25 months of CYD14 and CYD15. RESULTS: Follow-up data were available for 10,165 of 10,275 participants (99%) in CYD14 and 19,898 of 20,869 participants (95%) in CYD15. Data were available for 3203 of the 4002 participants (80%) in the CYD23 trial included in CYD57. During year 3 in the CYD14, CYD15, and CYD57 trials combined, hospitalization for virologically confirmed dengue occurred in 65 of 22,177 participants in the vaccine group and 39 of 11,089 participants in the control group. Pooled relative risks of hospitalization for dengue were 0.84 (95% confidence interval [CI], 0.56 to 1.24) among all participants, 1.58 (95% CI, 0.83 to 3.02) among those under the age of 9 years, and 0.50 (95% CI, 0.29 to 0.86) among those 9 years of age or older. During year 3, hospitalization for severe dengue, as defined by the independent data monitoring committee criteria, occurred in 18 of 22,177 participants in the vaccine group and 6 of 11,089 participants in the control group. Pooled rates of efficacy for symptomatic dengue during the first 25 months were 60.3% (95% CI, 55.7 to 64.5) for all participants, 65.6% (95% CI, 60.7 to 69.9) for those 9 years of age or older, and 44.6% (95% CI, 31.6 to 55.0) for those younger than 9 years of age. CONCLUSIONS: Although the unexplained higher incidence of hospitalization for dengue in year 3 among children younger than 9 years of age needs to be carefully monitored during long-term follow-up, the risk among children 2 to 16 years of age was lower in the vaccine group than in the control group. (Funded by Sanofi Pasteur; ClinicalTrials.gov numbers, NCT00842530, NCT01983553, NCT01373281, and NCT01374516.).

BACKGROUND: Typhoid fever is common in developing countries. The licensed typhoid vaccines confer only about 70 percent immunity, do not protect young children, and are not used for routine vaccination. A newly devised conjugate of the capsular polysaccharide of Salmonella typhi, Vi, bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has enhanced immunogenicity in adults and in children 5 to 14 years old and has elicited a booster response in children 2 to 4 years old. METHODS: In a double-blind, randomized trial, we evaluated the safety, immunogenicity, and efficacy of the Vi-rEPA vaccine in children two to five years old in 16 communes in Dong Thap Province, Vietnam. Each of the 11,091 children received two injections six weeks apart of either Vi-rEPA or a saline placebo. Cases of typhoid, diagnosed by the isolation of S. typhi from blood cultures after 3 or more days of fever (a temperature of 37.5 degrees C or higher), were identified by active surveillance over a period of 27 months. We estimated efficacy by comparing the attack rate of typhoid in the vaccine group with that in the placebo group. RESULTS: S. typhi was isolated from 4 of the 5525 children who were fully vaccinated with Vi-rEPA and from 47 of the 5566 children who received both injections of placebo (efficacy, 91.5 percent; 95 percent confidence interval, 77.1 to 96.6; P<0.001). Among the 771 children who received only one injection, there was 1 case of typhoid in the vaccine group and 8 cases in the placebo group. Cases were distributed evenly among all age groups and throughout the study period. No serious adverse reactions were observed. In all 36 children studied four weeks after the second injection of the vaccine, levels of serum IgG Vi antibodies had increased by a factor of 10 or more. CONCLUSIONS: The Vi-rEPA conjugate typhoid vaccine is safe and immunogenic and has more than 90 percent efficacy in children two to five years old. The antibody responses and the efficacy suggest that this vaccine should be at least as protective in persons who are more than five years old.

During 2005, 764 children were brought to a large children's hospital in Ho Chi Minh City, Vietnam, with a diagnosis of hand, foot, and mouth disease. All enrolled children had specimens (vesicle fluid, stool, throat swab) collected for enterovirus isolation by cell culture. An enterovirus was isolated from 411 (53.8%) of the specimens: 173 (42.1%) isolates were identified as human enterovirus 71 (HEV71) and 214 (52.1%) as coxsackievirus A16. Of the identified HEV71 infections, 51 (29.5%) were complicated by acute neurologic disease and 3 (1.7%) were fatal. HEV71 was isolated throughout the year, with a period of higher prevalence in October-November. Phylogenetic analysis of 23 HEV71 isolates showed that during the first half of 2005, viruses belonging to 3 subgenogroups, C1, C4, and a previously undescribed subgenogroup, C5, cocirculated in southern Vietnam. In the second half of the year, viruses belonging to subgenogroup C5 predominated during a period of higher HEV71 activity.

A prospective study of clinical and cytokine profiles of 107 infants with dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) was conducted. Fever, petechiae on the skin, and hepatomegaly were the most common clinical findings associated with DHF/DSS in infants. DSS occurred in 20.5% of the patients. Hemoconcentration and thrombocytopenia were observed in 91.5% and 92.5% of the patients, respectively. Serologic testing revealed that almost all of the patients (95.3%) had primary dengue virus infections. These data demonstrate that clinical and laboratory findings of DHF/DSS in infants are compatible with the World Health Organization's clinical diagnostic criteria for pediatric DHF. The present study is the first to report evidence of production of cytokines in infants with DHF/DSS and to describe the difference between the cytokine profile of infants with primary dengue virus infections and children with secondary infections. Overproduction of both proinflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) and anti-inflammatory cytokines (interleukin-10 and -6) may play a role in the pathogenesis of DHF/DSS in infants.

Rabies is a progressively fatal and incurable viral encephalitis caused by a lyssavirus infection. Almost all of the 55 000 annual rabies deaths in humans result from infection with dog rabies viruses (RABV). Despite the importance of rabies for human health, little is known about the spread of RABV in dog populations, and patterns of biodiversity have only been studied in limited geographical space. To address these questions on a global scale, we sequenced 62 new isolates and performed an extensive comparative analysis of RABV gene sequence data, representing 192 isolates sampled from 55 countries. From this, we identified six clades of RABV in non-flying mammals, each of which has a distinct geographical distribution, most likely reflecting major physical barriers to gene flow. Indeed, a detailed analysis of phylogeographic structure revealed only limited viral movement among geographical localities. Using Bayesian coalescent methods we also reveal that the sampled lineages of canid RABV derive from a common ancestor that originated within the past 1500 years. Additionally, we found no evidence for either positive selection or widespread population bottlenecks during the global expansion of canid RABV. Overall, our study reveals that the stochastic processes of genetic drift and population subdivision are the most important factors shaping the global phylogeography of canid RABV.

Anti-dengue virus immunoglobulin M kits were evaluated. Test sensitivities were 21%-99% and specificities were 77%-98% compared with reference ELISAs. False-positive results were found for patients with malaria or past dengue infections. Three ELISAs showing strong agreement with reference ELISAs will be included in the World Health Organization Bulk Procurement Scheme.

We addressed the role of innate immunity in the protection against HIV-1 infection by studying NK cell function in 37 Vietnamese intravascular drug users (IDUs), who appeared to remain HIV-1 uninfected despite many years of high-risk exposure (exposed uninfected, EU), 10 IDUs who underwent seroconversion and 28 unexposed blood donors. Main results were: NK cell lytic activities against both the NK-susceptible K562 cell line and the NK-resistant Daudi cell line were significantly augmented in EU IDUs compared with either controls or seroconverters before or after seroconversion; NK cells producing the cytokines IFN-gamma and TNF-alpha and the beta chemokines CCL3, CCL4, and CCL5 were also increased in the EU IDUs, either after in vitro activation or without stimulation. The finding of an enhanced NK cell function in EU IDUs, especially compared with IDUs who became HIV-1 infected, supports the hypothesis that NK cells contribute to the protection against HIV-1 infection.

Rotavirus remains the most common cause of severe, dehydrating diarrhea among children worldwide. Several rotavirus vaccines are under development. Decisions about new vaccine introduction will require reliable data on disease impact. The Asian Rotavirus Surveillance Network, begun in 2000 to facilitate collection of these data, is a regional collaboration of 36 hospitals in nine countries or areas that conduct surveillance for rotavirus hospitalizations using a uniform World Health Organization protocol. We summarize the Network's organization and experience from August 2001 through July 2002. During this period, 45% of acute diarrheal hospitalizations among children 0-5 years were attributable to rotavirus, higher than previous estimates. Rotavirus was detected in all sites year-round. This network is a novel, regional approach to surveillance for vaccine-preventable diseases. Such a network should provide increased visibility and advocacy, enable more efficient data collection, facilitate training, and serve as the paradigm for rotavirus surveillance activities in other regions.

We have previously associated high natural killer (NK)-cell activity and protection against HIV-1 infection in Vietnamese exposed uninfected intravascular drug users (EUs). Considering that activating and inhibitory signals sensed by NK-cell receptors regulate NK-cell activation, we performed phenotypic and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcript analyses of the NK-cell receptor (NKR) repertoire in 25 EUs, 19 HIV(+) intravenous drug users, and 26 uninfected blood donors. Although NK-cell activation was not linked to a unique NKR repertoire in EUs, various patterns consistent with NK-cell activation were detected in EUs: high KIR3DS1/KIR3DL1 ratio associated with down-regulated KIR3DL1 transcript levels, KIR2DL3(+) low-affinity receptor expansion associated to group HLA-C1 ligand in 2DS2(-)/2DL2(-) EUs, enhanced NKG2C/NKG2A ratio, and increased CD69 expression. Remarkably, EUs exhibited high constitutive degranulation activity in the absence of exogenous stimulation, as shown by the CD107a assay. Furthermore, CD161 expression was increased within the CD107a(+) NK-cell compartment. Our results suggest that in response to viral exposition, particular genetic or regulated features of the NKR repertoire of EUs contribute to their high constitutive NK-cell potential. This might allow NK cells to generate a more rapid and effective immune response to HIV-1, thereby contributing to prevention toward infection.

Hepatitis C viruses (HCVs) display a high level of sequence diversity and are currently classified into six genotypes and an increasing number of subtypes. Most likely, this heterogeneity is caused by genetic drift; evidence for recombination is scarce. To study the molecular heterogeneity of HCV in Vietnam, we analyzed 58 HCV RNA-positive sera from Vietnamese blood donors by sequence analysis of the CORE and NS5B regions. Phylogenetic analyses revealed the presence of genotype 1 (38%), genotype 2 (10.3%), and genotype 6 viruses (51.7%). All samples showed concordant results except for two (D3 and D54). Sample D54 was a mixed infection of genotype 2i and 6h viruses. Whole-genome analysis and bootscan analysis of sample D3, on the other hand, revealed a recombinant virus with genotype 2i and genotype 6p sequences at the 5' and 3' ends, respectively. The crossover point was located between nucleotide positions 3405 to 3464 (numbering according to prototype strain HCV-H, M67463) at the NS2/NS3 junction. The identification of this naturally occurring recombinant virus strengthens the concept that recombination may play a role in HCV epidemiology and evolution. Furthermore, the location of the recombination breakpoint may be relevant for constructing infectious chimeric viruses.

BACKGROUND: Resistance of Aedes aegypti to photostable pyrethroid insecticides is a major problem for disease-vector control programs. Pyrethroids target the voltage-gated sodium channel on the insects' neurons. Single amino acid substitutions in this channel associated with pyrethroid resistance are one of the main factors that cause knockdown resistance in insects. Although kdr has been observed in several mosquito species, point mutations in the para gene have not been fully characterized in Ae. aegypti populations in Vietnam. The aim of this study was to determine the types and frequencies of mutations in the para gene in Ae. aegypti collected from used tires in Vietnam. METHODS AND FINDINGS: Several point mutations were examined that cause insensitivity of the voltage-gated sodium channel in the insect nervous system due to the replacement of the amino acids L1014F, the most commonly found point mutation in several mosquitoes; I1011M (or V) and V1016G (or I), which have been reported to be associated to knockdown resistance in Ae. aegypti located in segment 6, domain II; and a recently found amino acid replacement in F1269 in Ae. aegypti, located in segment 6, domain III. Among 756 larvae from 70 locations, no I1011M or I1011V nor L1014F mutations were found, and only two heterozygous V1016G mosquitoes were detected. However, F1269C mutations on domain III were distributed widely and with high frequency in 269 individuals among 757 larvae (53 collection sites among 70 locations surveyed). F1269C frequencies were low in the middle to north part of Vietnam but were high in the areas neighboring big cities and in the south of Vietnam, with the exception of the southern mountainous areas located at an elevation of 500-1000 m. CONCLUSIONS: The overall percentage of homozygous F1269C seems to remain low (7.4%) in the present situation. However, extensive and uncontrolled frequent use of photostable pyrethroids might be a strong selection pressure for this mutation to cause serious problems in the control of dengue fever in Vietnam.

The capsular polysaccharide of Salmonella typhi, Vi, is an essential virulence factor and a protective vaccine for people older than 5 years. The safety and immunogenicity of two investigational Vi conjugate vaccines were evaluated in adults, 5- to 14-year-old children, and 2- to 4-year-old children in Vietnam. The conjugates were prepared with Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier, using either N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; Vi-rEPA(1)) or adipic acid dihydrazide (ADH; Vi-rEPA(2)) as linkers. None of the recipients experienced a temperature of >38.5 degrees C or significant local reactions. One injection of Vi-rEPA(2) into adults elicited a geometric mean (GM) increase in anti-Vi immunoglobulin G (IgG) from 9.62 enzyme-linked immunosorbent assay units/ml (EU) to 465 EU at 6 weeks; this level fell to 119 EU after 26 weeks. In the 5- to 14-year-old children, anti-Vi IgG levels at 6 weeks elicited by Vi-rEPA(2), Vi-rEPA(1), and Vi were 169, 22.8, and 18.9 EU, respectively (P = 0.0001 for Vi-rEPA(1) and Vi with respect to Vi-rEPA(2)). At 26 weeks, the anti-Vi IgG levels for recipients of Vi-rEPA(2), Vi-rEPA(1), and Vi were 30.0, 10.8, and 13.4 EU, respectively (P < 0.001 for Vi-rEPA(1) and Vi with respect to Vi-rEPA(2)); all were higher than the preinjection levels (P = 0. 0001). Vi-rEPA(2) also elicited the highest anti-Vi IgM and IgA levels of the three vaccines. In the 2- to 4-year-old children at 6 weeks following the first injection, Vi-rEPA(2) elicited an anti-Vi IgG level of 69.9 EU compared to 28.9 EU for Vi-rEPA(1) (P = 0.0001). Reinjection increased Vi antibody levels from 69.9 to 95.4 EU for Vi-rEPA(2) and from 28.9 to 83.0 EU for Vi-rEPA(1). At 26 weeks, anti-Vi IgG levels remained higher than those at preinjection (30.6 versus 0.18 for Vi-rEPA(2) and 12.8 versus 0.33 for Vi-rEPA(1); P = 0.0001 for both). Vi vaccine is recommended for individuals of 5 years of age or older. In the present study, the GM level of anti-Vi IgG elicited by two injections of Vi-rEPA(2) in the 2- to 4-year-old children was higher than that elicited by Vi in the 5- to 14-year-old children (30.6 versus 13.4; P = 0.0001). The safety and immunogenicity of the Vi-rEPA(2) conjugate warrant further investigation.

BACKGROUND: Dengue virus (DV) infection is one of the most important mosquito-borne diseases in the tropics. Recently, the severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have become the leading cause of death among children in Southern Vietnam. Protective and/or pathogenic T cell immunity is supposed to be important in the pathogenesis of DHF and DSS. METHODOLOGY/PRINCIPAL FINDINGS: To identify HLA alleles controlling T cell immunity against dengue virus (DV), we performed a hospital-based case control study at Children's Hospital No.2, Ho Chi Minh City (HCMC), and Vinh Long Province Hospital (VL) in Southern Vietnam from 2002 to 2005. A total of 211 and 418 patients with DHF and DSS, respectively, diagnosed according to the World Health Organization (WHO) criteria, were analyzed for their characteristic HLA-A, -B and -DRB1 alleles. Four hundred fifty healthy children (250 from HCMC and 200 from VL) of the same Kinh ethnicity were also analyzed as population background. In HLA class I, frequency of the HLA-A*24 showed increased tendency in both DHF and DSS patients, which reproduced a previous study. The frequency of A*24 with histidine at codon 70 (A*2402/03/10), based on main anchor binding site specificity analysis in DSS and DHF patients, was significantly higher than that in the population background groups (HCMC 02-03 DSS: OR = 1.89, P = 0.008, DHF: OR = 1.75, P = 0.033; VL 02-03 DSS: OR = 1.70, P = 0.03, DHF: OR = 1.46, P = 0.38; VL 04-05 DSS: OR = 2.09, P = 0.0075, DHF: OR = 2.02, P = 0.038). In HLA class II, the HLA-DRB1*0901 frequency was significantly decreased in secondary infection of DSS in VL 04-05 (OR = 0.35, P = 0.0025, Pc = 0.03). Moreover, the frequency of HLA-DRB1*0901 in particular was significantly decreased in DSS when compared with DHF in DEN-2 infection (P = 0.02). CONCLUSION: This study improves our understanding of the risk of HLA-class I for severe outcome of DV infection in the light of peptide anchor binding site and provides novel evidence that HLA-class II may control disease severity (DHF to DSS) in DV infection.

BACKGROUND: Common causes of acute febrile illness in tropical countries have similar symptoms, which often mimic those of dengue. Accurate clinical diagnosis can be difficult without laboratory confirmation and disease burden is generally under-reported. Accurate, population-based, laboratory-confirmed incidence data on dengue and other causes of acute fever in dengue-endemic Asian countries are needed. METHODS AND PRINCIPAL FINDINGS: This prospective, multicenter, active fever surveillance, cohort study was conducted in selected centers in Indonesia, Malaysia, Philippines, Thailand and Vietnam to determine the incidence density of acute febrile episodes (≥ 38 °C for ≥ 2 days) in 1,500 healthy children aged 2-14 years, followed for a mean 237 days. Causes of fever were assessed by testing acute and convalescent sera from febrile participants for dengue, chikungunya, hepatitis A, influenza A, leptospirosis, rickettsia, and Salmonella Typhi. Overall, 289 participants had acute fever, an incidence density of 33.6 per 100 person-years (95% CI: 30.0; 37.8); 57% were IgM-positive for at least one of these diseases. The most common causes of fever by IgM ELISA were chikungunya (in 35.0% of in febrile participants) and S. Typhi (in 29.4%). The overall incidence density of dengue per 100 person-years was 3.4 by nonstructural protein 1 (NS1) antigen positivity (95% CI: 2.4; 4.8) and 7.3 (95% CI: 5.7; 9.2) by serology. Dengue was diagnosed in 11.4% (95% CI: 8.0; 15.7) and 23.9% (95% CI: 19.1; 29.2) of febrile participants by NS1 positivity and serology, respectively. Of the febrile episodes not clinically diagnosed as dengue, 5.3% were dengue-positive by NS1 antigen testing and 16.0% were dengue-positive by serology. CONCLUSIONS: During the study period, the most common identified causes of pediatric acute febrile illness among the seven tested for were chikungunya, S. Typhi and dengue. Not all dengue cases were clinically diagnosed; laboratory confirmation is essential to refine disease burden estimates.

BACKGROUND: Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. CONCLUSIONS: As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

Among 730 Escherichia coli, 438 Klebsiella pneumoniae, and 141 Proteus mirabilis isolates obtained between September 2000 and September 2001 in seven hospitals in Ho Chi Minh City, Vietnam, 26.6% were resistant to ceftazidime, 30% were resistant to cefotaxime, 31.5% were resistant to ceftriaxone, 15.9% were resistant to cefoperazone, and 6% were resistant to cefepime. Resistance to imipenem was found in 5.6% of the isolates. In 55 strains producing extended-spectrum beta-lactamases (32 E. coli isolates, 13 K. pneumoniae isolates, and 10 P. mirabilis isolates), structural genes for VEB-1 (25.5%), CTX-M (25.5%), SHV (38.1%), and TEM (76.3%) enzymes were detected alone or in combination. Sequencing of the PCR products obtained from the K. pneumoniae isolates revealed the presence of bla(VEB-1), bla(CTX-M-14), bla(CTX-M-17), bla(SHV-2), and bla(TEM-1). Molecular typing of the strains with a similar resistance phenotype to broad-spectrum cephalosporins indicated polyclonal spread. ISEcp1 was presumably responsible for dissemination of the bla(CTX-M-like) gene.

The Asian tiger mosquito Aedes albopictus is one of the most significant pathogen vectors of the twenty-first century. Originating from Asia, it has invaded a wide range of eco-climatic regions worldwide. The insect-associated microbiota is now recognized to play a significant role in host biology. While genetic diversity bottlenecks are known to result from biological invasions, the resulting shifts in host-associated microbiota diversity has not been thoroughly investigated. To address this subject, we compared four autochthonous Ae. albopictus populations in Vietnam, the native area of Ae. albopictus, and three populations recently introduced to Metropolitan France, with the aim of documenting whether these populations display differences in host genotype and bacterial microbiota. Population-level genetic diversity (microsatellite markers and COI haplotype) and bacterial diversity (16S rDNA metabarcoding) were compared between field-caught mosquitoes. Bacterial microbiota from the whole insect bodies were largely dominated by Wolbachia pipientis. Targeted analysis of the gut microbiota revealed a greater bacterial diversity in which a fraction was common between French and Vietnamese populations. The genus Dysgonomonas was the most prevalent and abundant across all studied populations. Overall genetic diversities of both hosts and bacterial microbiota were significantly reduced in recently established populations of France compared to the autochthonous populations of Vietnam. These results open up many important avenues of investigation in order to link the process of geographical invasion to shifts in commensal and symbiotic microbiome communities, as such shifts may have dramatic impacts on the biology and/or vector competence of invading hematophagous insects.

The association between sex, nutritional status, and the severity of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), and immune status was investigated in 245 Vietnamese infants with predominantly primary infections with dengue virus. Male and female infants were at equal risk of developing DHF/DSS. However, infants of low height and weight for age were under-represented among DHF/DSS cases compared with 533 healthy baby clinic infant controls. Acute illness phase blood levels of selected cytokines (interferon-gamma and tumor necrosis factor-alpha) and serum levels of antibodies to dengue virus were elevated in the same range in male and female infants with DHF/DSS, as well as in infants with and without malnutrition.

To the Editor: A highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), which affected >2 million pigs, emerged in early 2006 in the People’s Republic of China. The disease was characterized by high fever (41°C), high illness rates (50%–100%), and high death rates (20%–100%) for pigs of all ages (1). A number of HP-PRRSVs have been isolated from 2006 through 2009 from infected pigs in different provinces of China and confirmed to be the causative agent of the new outbreaks (1,2). These HP-PRRSVs have a deletion of 30 amino acids in the nonstructural 2 (NSP-2). However, the evolutionary origin and path of the HP-PRRSV remain unknown. We analyzed the full-length sequences of 67 PRRSVs: 35 HP-PRRSVs (HuN4 and LNSY-08-1 isolated in our laboratory and 33 viruses isolated in other laboratories), 28 classic PRRSVs (18 viruses isolated from China and 10 viruses representing other Asian countries and North America), and 4 commercially available attenuated live PRRSV vaccine viruses. Except for the 2 viruses we isolated (HuN4 and LNSY-08-1), the full-length sequences of the other 65 viruses were obtained from GenBank. Nucleotide and deduced amino acid sequences of these PRRSVs were aligned and compared by using previous methods (3,4). Whole genome–based phylogenetic analysis showed that these 67 PRRSVs could be divided into 4 subgroups (Appendix Figure). Ten classic PRRSVs from China, together with the North American prototype virus VR-2332 and the vaccine virus RespPRRS/Repro modified live vaccine, were classified into subgroup 1. The first Chinese isolate, CH-1a, and its 3 derivatives (CH2002, CH2003, and CH2004) were classified into subgroup 2. All 35 HP-PRRSVs were classified into subgroup 4, and they shared high homology (>99%) in their genomic sequences. The other 4 Chinese PRRSVs, including HB-1(sh)/2002, HB-2(sh)/2002, Em2007, and SHB, belonged to subgroup 3, an intermediate subgroup between subgroups 2 and 4. Phylogenetically, HP-PRRSVs had a close relationship with subgroups 2 and 3. Four conserved deletions were shown among all HP-PRRSVs, including an adenosine deletion at position 122 in the 5′-untranslated region, a guanosine deletion at position 15,278 in the 3′-untranslated region, and 2 discontinuous deletions in the NSP-2, including a single amino acid deletion at position 482 (L482) and a second deletion of 29 amino acids between positions 533 and 561 (S533–A561). The presence of these 4 deletions among subgroup 4 viruses is a unique phenomenon, which may be used as a distinctive molecular marker for HP-PRRSVs. The occurrence of these 4 deletions might be explained as a stepwise accumulation from subgroup 2 to subgroup 4. None of the 4 deletions were found in subgroup 2. Among viruses in subgroup 3, one, 2, or 3 of the 4 deletions occurred. For example, a single deletion was present at 122 nt in Em2007, double deletions at 122 nt and 15,278 nt in HB-1(sh)/2002 and SHB, and triple deletions at 122 nt, 15,278 nt, and 482 aa in GD3-2005 (this sequence was not submitted to GenBank until now). In 2008, Ma et al. compared GD3-2005 with several PRRSVs and reported the homology within them, pointing out that the 2 deletions in NSP-2 were identical to the HP-PRRSV (5). After careful analysis, we found the GD3-2005 more interesting than what was reported in Ma et al.; it belongs to an intermediate group, and shares the characters of gradual evolution. Eventually, all 4 deletions occurred in subgroup 4. This obvious pattern suggests that these 4 conserved deletions might have evolved step by step. The primary neutralizing epitope (PNE), which is located on glycoprotein 5 and composed of the residues S37H(F/L)QLIYN with F/L39 as the binding site for the neutralizing antibody (6,7), also displayed similar changes at the 39 position among the 4 subgroups. The PNE residues in subgroups 1 (SHL39QLIYN) and 2 (SHF39QLIYN) were considerably conservative. Subgroup 3 contained either F39 or I39 (F39 in Em2007 and HB-2(sh)/2002, and I39 in both HB-1(sh)/2002 and SHB); subgroup 4 contained I39 only. The existence of either F39 or I39 in subgroup 3 PNE indicates its intermediate position between subgroups 2 and 4 in the evolution of HP-PRRSVs. Pairwise comparison of subgroups 2, 3, and 4 did not find recombination or large fragment replacement, which suggests that all HP-PRRSVs originated from the same ancestor by gradual evolution. Notably, the recently isolated intermediate PRRSVs mentioned above (SHB, Em2007, and GD3-2005) were isolated in the region of South China where the outbreak of HP-PRRS initially occurred. Furthermore, the epidemiologic data show that the outbreak of HP-PRRSV emerged from 1 particular place and then spread widely. This evidence indicates that all HP-PRRSVs isolated in China likely originated from the same source. In summary, our findings suggest that the newly emerged HP-PRRSVs originated from the Chinese CH-1a-like PRRSV. Further study is needed to determine what contributes to the increased pathogenicity of HP-PRRSV. Although the 4 deletions are conserved in all HP-PRRVs, the increased pathogenicity of HP-PRRSV may not merely be caused by the deletions; pathogenicity is affected by multigenetic factors.