Influenza Virus Inactivation for Studies of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling

Abstract

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and ␤-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and ␤-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin-and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

Influenza virus replication in macrophages: balancing protection and pathogenesis

“…The induction of proinflammatory cytokines in response to HPAI H5 infection of human MDMs was inhibited by b-propiolactone-inactivation of the virus [36]. Although this result suggests that replication is required for the induction of proinflammatory cytokines following H5N1 IAV infection of macrophages, b-propiolactone was shown to cause a 16-fold reduction in viral HA activity [61]. Thus, decreased cytokine expression may have resulted from decreased virus attachment/entry rather than from decreased productive replication per se.…”

Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges

“…In vitro infection assays and western blot analysis showed that in the case of PRRSV the entry-associated domains were indeed preserved after treatment at 37°C and that the virus did not replicate anymore [41]. However, heat-inactivated influenza virus (>55°C) lost hemagglutinin and neuraminidase activity [62].…”

“…However, BPL-inactivated influenza showed reduced hemagglutinin and neuraminidase activity [62]. Vaccines based on BPL-inactivated virus against SARS-CoV [64], hematopoietic necrosis virus (HNV) [67] and influenza [65], were able to protect on challenge.…”

“…In fact, acylation and crosslinking reactions also occur, to a significant extent, with proteins (cysteine, methionine and histidine as most reactive residues) being modified more extensively than originally agreed from earlier literature, which can have serious implications for vaccine production. Indeed, modifications on amino acids by BPL treatment can, for example, lead to the disability of influenza viruses to enter cells by virus-membrane fusion and modification of hemagglutinin [61,62].…”

Emergence of the Virulence-Associated PB2 E627K Substitution in a Fatal Human Case of Highly Pathogenic Avian Influenza Virus A(H7N7) Infection as Determined by Illumina Ultra-Deep Sequencing

“…RNA was extracted from the FFPE lower lung tissue using the RNeasy FFPE kit (Qiagen) according to the manufacturer's protocol. Total RNA was eluted in a volume of 50 l. All A(H7N7)-positive RNA extracts were subjected to a semiquantitative RT-PCR targeting the influenza virus matrix gene segment to prevent possible resequencing due to low-copy-number input in the reverse transcription (RT)-PCR (24).…”