Multiple polymerase gene mutations for human adaptation occurring in Asian H5N1 influenza virus clinical isolates.

The role of the influenza virus polymerase complex in host range restriction has been well-studied and several host range determinants, such as the polymerase PB2-E627K and PB2-D701N mutations, have been identified. However, there may be additional, currently unknown, human adaptation polymerase mutations. Here, we used a database search of influenza virus H5N1 clade 1.1, clade 2.3.2.1 and clade 2.3.4 strains isolated from 2008-2012 in Southern China, Vietnam and Cambodia to identify polymerase adaptation mutations that had been selected in infected patients. Several of these mutations acted either alone or together to increase viral polymerase activity in human airway cells to levels similar to the PB2-D701N and PB2-E627K single mutations and to increase progeny virus yields in infected mouse lungs to levels similar to the PB2-D701N single mutation. In particular, specific mutations acted synergistically with the PB2-D701N mutation and showed synergistic effects on viral replication both in human airway cells and mice compared with the corresponding single mutations. Thus, H5N1 viruses in infected patients were able to acquire multiple polymerase mutations that acted cooperatively for human adaptation. Our findings give new insight into the human adaptation of AI viruses and help in avian influenza virus risk assessment.

Surveillance of Severe Acute Respiratory Infection (SARI) for Hospitalized Patients in Northern Vietnam, 2011-2014.

Severe acute respiratory infections (SARI) are leading causes of hospitalization, morbidity, and mortality in children worldwide. The aim of this study was to identify viral pathogens responsible for SARI in northern Vietnam in the period from 2011 to 2014. Throat swabs and tracheal aspirates were collected from SARI patients according to WHO guidelines. The presence of 13 different viral pathogens (influenza A[H1N1]pdm09; A/H3N2; A/H5; A/H7 and B; para influenza 1,2,3; RSV; HMPV; adeno; severe acute respiratory syndrome-CoV and rhino) was tested by conventional/real-time reverse transcription-polymerase chain reaction. During the study period, 975 samples were collected and tested. More than 30% (32.1%, 313 samples) of the samples showed evidence of infection with influenza viruses, including A/H3N2 (48 samples), A (H1N1) pdm09 (221 samples), influenza B (42 samples), and co-infection of A (H1N1) pdm09 or A/H3N2 and influenza B (2 samples). Other respiratory pathogens were detected in 101 samples, including rhinovirus (73 samples), adenovirus (10 samples), hMPV (9 samples), parainfluenza 3 (5 samples), parainfluenza 2 (3 samples), and RSV (1 sample). Influenza A/H5, A/H7, or SARS-CoV were not detected. Respiratory viral infection, particularly infection of influenza and rhinoviruses, were associated with high rates of SARI hospitalization, and future studies correlating the clinical aspects are needed to design interventions, including targeted vaccination.

Viral co-infections among children with confirmed measles at hospitals in Hanoi, Vietnam, 2014.

OBJECTIVE: To characterize viral co-infections among representative hospitalized measles cases during the 2014 Hanoi outbreak. METHODS: Throat swabs were collected from 54 pediatric patients with confirmed measles, and molecular diagnostics performed for 10 additional viral respiratory pathogens (Influenza A/H1N1pdm09; A/H3N2 and influenza B; Parainfluenza 1, 2, 3; Respiratory Synctial Virus, RSV; human Metapneumovirus, hMPV; Adenovirus and Picornavirus). RESULTS: Twenty-one cases (38.9%) showed evidence of infection with other respiratory viruses: 15 samples contained measles plus one additional virus, and 6 samples contained measles plus 2 additional viruses. Adenovirus was detected as a predominant cause of co-infections (13 cases; 24.1%), followed by RSV (6 cases; 11.1%), A/H1N1pdm09 (3 cases; 5.6%), PIV3 (3 cases; 3.7%), Rhinovirus (3 cases; 3.7%) and hMPV (1 case; 1.96%). CONCLUSIONS: Viral co-infections identified from pediatric measles cases may have contributed to increased disease severity and high rate of fatal outcomes. Optimal treatment of measles cases may require control of multiple viral respiratory pathogens.

An Updated Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of H5N1 Avian Influenza Viruses.

We designed a new set of primers for reverse transcriptase loop-mediated isothermal amplification (RTLAMP) to specifically amplify the HA gene of avian influenza viruses subtype H5N1. By testing nine H5N1 virus strains and 41 clinical samples collected in Northern Vietnam, we found that the new primers showed higher sensitivity and specificity than the previously published RT-LAMP primers and were comparable to the RT-PCR method currently recommended by WHO. These results suggest that our RT-LAMP assay may be a better choice as a diagnostic tool for current H5N1 influenza virus infection.

Pathogenicity of highly pathogenic avian H5N1 influenza A viruses isolated from humans between 2003 and 2008 in northern Vietnam.

Vietnam is one of the countries most affected by highly pathogenic H5N1 influenza A viruses. To evaluate the potential pathogenicity in mammals of H5N1 viruses isolated from humans in Vietnam, we determined the sequences of all eight genes of 22 human isolates collected between 2003 and 2008 and compared their virulence in mice. The isolates were classified into clade 1 and clade 2.3.4 and differed in pathogenicity for mice. Whilst lysine at position 627 of PB2 (PB2-627K) is a critical virulence determinant for clade 2.3.4 viruses, asparagine at position 701 of PB2 and other unknown virulence determinants appear to be involved in the high pathogenicity of clade 1 viruses, warranting further studies to determine the factors responsible for the high virulence of H5N1 viruses in mammals.